Handbook of Isolation and Characterization of Impurities in Pharmaceuticals (SST) (Separation Science and Technology)

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Author: Satinder Ahuja | Karen Mills Alsante

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HANDBOOK OF ISOLATION AND CHARACTERIZATION OF IMPURITIES IN PHARMACEUTICALS

This is Volume 5 of SEPARATION SCIENCE AND TECHNOLOGY A reference series edited by Satinder Ahuja

HANDBOOK OF ISOLATION AND CHARACTERIZATION OF IMPURITIES IN PHARMACEUTICALS Edited by

Satinder Ahuja Ahuja Consulting Calabash, NC

Karen Mills Alsante Pfizer, Inc. Groton, CT

Amsterdam Boston London New York Oxford San Francisco Singapore Sydney Tokyo

Paris

San Diego

Academic Press An imprint of Elsevier Science 525 B Street, Suite 1900 San Diego, California 92101-4495

ß 2003 Elsevier Science (USA) All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-proﬁt educational classroom use. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: permissions@elsevier. com. You may also complete your request on-line via the Elsevier Science homepage (http://www.elsevier.com), by selecting ‘Customer Support’ and then ‘Obtaining Permissions’. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (+1) (978) 7508400, fax: (+1) (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London W1P 0LP, UK; phone: (+44) 207 631 5555; fax: (+44) 207 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier’s Science & Technology Rights Department, at the phone, fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent veriﬁcation of diagnoses and drug dosages should be made. British Library Cataloguing in Publication Data Handbook of isolation and characterization of impurities in pharmaceuticals. – (Separation science and technology; v. 5) 1. Drugs – Analysis 2. Contamination (Technology) I. Ahuja, Satinder, 1933 – II. Alsante, Karen Mills 615.10 901 ISBN 012044982X Library of Congress Cataloging-in-Publication Data Handbook of isolation and characterization of impurities in pharmaceuticals / edited by Satinder Ahuja, Karen Mills Alsante. p. ; cm – (Separation science and technology; v. 5) Includes index. ISBN 0-12-044982-X (alk. paper) 1. Drugs–Puriﬁcation–Handbooks, manuals, etc. 2. Drugs–Separation–Handbooks, manuals, etc. I. Ahuja, Satinder, 1933 – II. Alsante, Karen Mills. III. Separation science and technology (San Diego, Calif.); v. 5. [DNLM: 1. Pharmaceutical Preparations–analysis–Handbooks. 2. Pharmaceutical Preparations–analysis–Laboratory Manuals. 3. Drug Contamination–prevention & control–Handbooks. 4. Drug Contamination–prevention & control– Laboratory Manuals. 5. Pharmaceutical Preparations–standards–Handbooks. 6. Pharmaceutical Preparations– standards–Laboratory Manuals. 7. Technology, Pharmaceutical–methods–Handbooks. 8. Technology, Pharmaceutical– methods–Laboratory Manuals. QV 25 H2364 2003] RS404.5 H355 2003 6150 .19–dc21 2003040309 First edition 2003 ISBN: 0-12-044982-X The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). Printed in The United Kingdom.

CONTENTS

PREFACE xi CONTRIBUTORS

1.

xv

Overview: Isolation and Characterization of Impurities SATINDER AHUJA I. II. III. IV. V. VI. VII. VIII. IX.

2.

Introduction 1 Designations of Impurities 4 Regulatory Requirements 7 Sources of Impurities 8 Analytical Method Development Isolation Methods 18 Characterization Methods 20 Case Studies 22 Summary 24 References 24

14

Review of Regulatory Guidance on Impurities RADHIKA RAJAGOPALAN I. II. III. IV.

Introduction 27 Types of Impurities—Drug Substance 28 Role of Compendia 30 Role of Drug Master Files (DMF)—Type II and Impurities Evaluation 31 V. Reference Standards for the Quantitation of Impurities and Analytical Procedures 32

v

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CONTENTS

VI. VII. VIII. IX. X. XI.

3.

Qualiﬁcation of Impurities and New Impurities 32 Impurities in Drug Products 33 Analytical Methodology for Impurities in Drug Product Impurities Quantitation Post-Approval 36 Role of Sponsors 36 Summary 36 References 37

33

Polymorphic and Solvatomorphic Impurities HARRYG. BRITTAIN AND ALES MEDEK I. II. III. IV. V.

4.

Introduction 39 X-ray Diffraction 40 Thermal Methods of Analysis 44 Vibrational Spectroscopy 49 Solid-State Nuclear Magnetic Resonance Spectrometry References 69

57

Impurities in Drug Products KENNETH C.WATERMAN, ROGER C. ADAMI, AND JINYANG HONG I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.

5.

Introduction 75 Water 76 Peroxides 78 Aldehydes 79 Metal Impurities 80 Small Molecule Carboxylic Acids Leachables/Extractables 82 Alcohols as Impurities 83 Biological Impurities 83 Additives in Excipients 84 Final Observations 85 Summary 85 References 85

82

Strategies for Investigation and Control of Process- and Degradation-Related Impurities BERNARD A. OLSEN AND STEVEN W. BAERTSCHI I. II. III. IV. V.

Introduction 89 Goals and Strategies 91 Process-Related Impurities 95 Degradation-Related Impurities 102 Summary and Conclusions 115 References 116

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CONTENTS

6.

Reference Standards PAUL A. CULBERTAND BRUCE D. JOHNSON I. II. III. IV. V. VI.

7.

Introduction 119 Deﬁnitions 120 Life Cycle 121 Governance 125 Qualiﬁcation Process Summary 139 References 139

127

Sample Selection for Analytical Method Development HUGH J. CLARKE AND KENNETH J. NORRIS I. Introduction 145 II. Components of the Key Predictive Sample Set (KPSS) 147 III. Stereoisomers 147 IV. Matrix Components 150 V. Process-Related Impurities (PRIs) 150 VI. Purposeful Degradation Samples 152 VII. Stability Samples 155 VIII. Phase-Solubility Analysis 156 IX. Sample Selection Strategies 159 X. Summary 162 Glossary 162 References 163

8.

Sample Preparation Methods for the Analysis of Pharmaceutical Materials DAVID T. ROSSI AND KENNETH G. MILLER I. II. III. IV. V. VI. VII. VIII.

Introduction 166 Solid-Phase Extraction (SPE) 166 Liquid–Liquid Extraction (LLE) 174 Supercritical Fluid Extraction (SFE) 181 Accelerated Solvent Extraction (ASE) 189 Centrifugation 194 Filtration 195 Summary 199 References 199

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CONTENTS

9.

Isolation Methods I: Thin-Layer Chromatography PAMELA M.GORMAN AND HONG JIANG I. II. III. IV.

Introduction to Thin-Layer Chromatography (TLC) 203 TLC Applications in Pharmaceutical Industry 206 TLC Method Development and Validation 207 Impurity Isolation and Characterization by TLC 221 References 228

10. Isolation Methods II: Column Chromatography MARKGUINN, RONALD BATES, BENJAMIN HRITZKO, TERI SHANKLIN, GLENN WILCOX, AND SAM GUHAN I. II. III. IV. V. VI. VII.

Introduction 231 Background 232 Stationary Phases 233 Equipment 237 Screening 240 Development of Preparative Method 244 Scaleup of Preparative Method 246 Summary 248 References 248

11. Mass Spectral Characterization DAVID J. BURINSKYAND FENG WANG I. Introduction 249 II. Relevance of Impurity Characterization 252 III. The coupling of Liquid-Phase Separations and Mass Spectrometers 259 IV. Ion Formation 264 V. Analyzers 273 VI. Ion Structure Interrogation 277 VII. Data Acquisition and Interpretation 282 VIII. Applications 286 IX. Conclusions 288 X. Summary 289 References 290

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12. NMR Characterization of Impurities LINDA L. LOHR, ANDREW J. JENSEN, AND THOMAS R. SHARP I. II. III. IV. V. VI. VII. VIII. IX. X.

Introduction to Nuclear Magnetic Resonance (NMR) Information Gathering 304 Sample Preparation for NMR 305 Sample Preparation for LC-NMR 307 NMR Instrumentation 309 NMR Experiments 314 Choosing an Experiment Set 324 Data Interpretation 325 Final Steps 334 Summary 336 References 337

301

13. Hyphenated CharacterizationTechniques THOMAS N. FEINBERG I. Introduction 341 II. Experimental Examples III. Conclusions 356 References 357

350

14. Solving Impurity/Degradation Problems: Case Studies KAREN M. ALSANTE, TODD D. HATAJIK, LINDA L. LOHR, DINOS SANTAFIANOS, AND THOMAS R. SHARP I. Introduction and Background 361 II. Case Studies 368 III. Summary and Conclusions 398 Appendix—Lessons Learned 398 References 399

Index 401

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PREFACE

The pharmaceutical industry is required by the Food, Drug, and Cosmetic Act to establish the identity and purity of all marketed drug products. The United States Food and Drug Administration (FDA) and other regulatory bodies around the world require that impurities in drug substance and drug product when present at threshold levels recommended by the International Conference on Harmonisation (ICH) be isolated and characterized. This book ﬁlls the need for a text on the complex process of isolation and characterization of process-related (synthesis and formulation) impurities and degradation products to meet critical regulatory requirements. The identiﬁcation of process-related impurities and degradation products can provide an understanding on production of impurities and deﬁne degradation mechanisms. When this process is performed at an early stage of drug development, there is ample time to address various aspects of drug development to prevent or control the production of impurities and degradation products well before the regulatory ﬁling and thus assure production of a high-quality drug product. The chapters in this book have been organized in a logical sequence to reﬂect the process used for the isolation and characterization of impurities. Chapter 1 points out that there are ethical, economic, and persuasive regulatory reasons to isolate and characterize impurities and degradation products. It provides an understanding of various sources of impurities and degradation products, the process and methodologies involved in separation, isolation, and characterization of impurities. Chiral impurities are also discussed from the standpoint of their origin, analytical methodology, and regulatory perspective for controlling them. Regulatory guidance is provided in Chapter 2, which includes a signiﬁcant discussion on ICH guidelines. Thresholds for identiﬁcation, safely qualiﬁcation, and reporting impurities have been set in these guidelines with

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PREFACE

speciﬁc action levels reﬂective of the proposed daily dose of drugs under development. The rule of thumb for identifying impurities in drug substance and drug product is 0.1% (depending on the daily dose). Identiﬁcation and control of toxic impurities present even well below the 0.1% level may be required. Chapter 3 discusses polymorphic and solvatomorphic impurities. Since a major pharmaceutical manufacturing goal is to produce drug substance that is phase-pure and stable, the question of small amounts of polymorphic and solvatomorphic impurities in a bulk solid is of great importance. The drug substance should also remain phase-pure during drug product formulation and shelf life. Chapter 4 speciﬁcally addresses impurities in drug product originating from formulation ingredients and processing that are likely to cause stability or performance issues in the drug product. The sources of the impurities as well as resulting drug stability issues are outlined in this chapter. Chapter 5 describes strategies for investigating process-related and degradation-related impurities in drug substances and drug products, with emphasis on a ‘‘chemistry-guided approach.’’ A critical component in the analytical quantiﬁcation of impurity levels is reference standard materials. Chapter 6 provides useful information on the role of reference standards in monitoring impurities. It includes the qualiﬁcation process and governance of reference standards. Chapter 7 reviews analytical method development for the quantiﬁcation of impurities and degradation products present in drug substance and drug product. This process involves selecting the key impurities/degradation product sample set, screening of chromatographic conditions, and optimizing method parameters. To assure the high quality of pharmaceutical products, it is of critical importance to carry out elucidation of structure of impurities and degradation products present in the drug substance and drug product throughout the drug development process. For low-level impurities/ degradation products, this quite often involves isolation. The next three chapters detail the isolation of impurities and degradation products. Chapter 8 provides guidance on extraction and isolation techniques for successful sample preparation including speciﬁcity for the targeted material, homogeneity, and good recovery. An excellent isolation technique is exempliﬁed by thin-layer chromatography (TLC), discussed in Chapter 9. TLC is particularly useful when highperformance liquid chromatography (HPLC) fails to yield useful information because of retention on the head of column, early elution, or poor detection issues. It can be easily scaled up for preparative work. The resolving power of HPLC is frequently needed for challenging isolation problems. Chapter 10 details the use of HPLC for the isolation of impurities and covers the various options that are available for stationary phases, detectors, and the preparative scaleup process. Structure elucidation of impurities and degradation products at trace levels in complex matrices requires advanced instrumental techniques and collaborative efforts of scientists from various disciplines. Chapter 11 describes the fundamental of mass spectrometry-based techniques for ion

PREFACE

xiii structure analysis, including aspects of ion formation, attributes of various mass analyzers, and scan modes used for collision-induced dissociation experiments and interpretation of mass spectra. It also discusses at some length LC-MS, the powerful combination of a versatile separation technique like HPLC with the universal detectability of MS. Chapter 12 focuses on nuclear magnetic resonance (NMR) spectroscopy, which provides key structural information and intramolecular interactions not readily available from other analytical methods. Vast improvements in NMR sensitivity limits have been made to assist with structural elucidation of impurities at low levels. Nondestructive NMR analysis allows additional characterization experiments to be performed with the same sample. Chapter 13 explains how hyphenated techniques have improved efﬁciency in structure elucidation of impurities and degradation products. Techniques discussed include HPLCDAD, LC-MS, GC-MS, LC-IR, and LC-NMR. Chapter 14 provides practical guidance with case studies on isolating and characterizing process-related impurities and degradation products for pharmaceutical drug candidates. The case studies utilize isolation or synthesis in conjunction with mass spectral and NMR characterizations. A collaborative multiple disciplinary strategy has been found to be the most efﬁcient way to solve impurity/degradation product problems. We sincerely believe that the detailed information provided by all authors, actively working with the pharmaceutical industry and FDA, will be of great value to various readers who are interested in the isolation and characterization of impurities and degradation products. Satinder Ahuja Karen Mills Alsante

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CONTRIBUTORS

Numbers in parentheses indicate the page on which the authors’ contributions begin.

Roger C. Adami (75) Pﬁzer Global Research and Development Division, Pharmaceutical Research and Development Department, Groton, CT 06340 Satinder Ahuja (1) Ahuja Consulting, 1061 Rutledge Court, Calabash, NC 28467 Karen M. Alsante (361) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton, CT 06340 Steven W. Baertschi (89) Eli Lilly and Company, Indianapolis, IN 46285 Ronald Bates (231) Pﬁzer Global Research and Development Division, Bioprocess Research and Development Department, Groton, CT 06340 Harry G. Brittain (39) Center for Pharmaceutical Physics, 10 Charles Road, Milford, NJ 08848 David J. Burinsky (249) Chemical Development Division, GlaxoSmithKline, Five Moore Drive, PO Box 13398, Mail stop 2-4075-4A, Research Triangle Park, NC 27709-3398 Hugh J. Clarke (145) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton, CT 06340 Paul A. Culbert (119) Lilly Analytical Research Laboratories, Eli Lilly Canada Inc., 3650 Danforth Ave., 3rd Floor, Bldg. 600, Scarborough, ON, MIN 2E8 Thomas N. Feinberg (341) Cardinal Health, Structural Chemistry Division, Magellan Pharmaceutical Development, P.O. Box 13341, Research Triangle Park, NC 27709 Pamela M. Gorman (203) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton, CT 06340

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CONTRIBUTORS

Sam Guhan (231) Pﬁzer Global Research and Development Division, Bioprocess Research & Development Department, Groton, CT 06340 Mark Guinn (231) Pﬁzer Global Research and Development Division, Bioprocess Research & Development Department, Groton, CT 06340 Todd D. Hatajik (361) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton, CT 06340 Jinyang Hong (75) Pﬁzer Global Research and Development Division, Pharmaceutical Research and Development Department, Groton, CT 06340 Benjamin Hritzko (231) Pﬁzer Global Research and Development Division, Bioprocess Research and Development Department, Gorton, CT 06340 Andrew J. Jensen (301) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton, CT 06340 Hong Jiang (203) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton CT 06340 Bruce D. Johnson (119) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Ann Arbor, MI 48105 Linda L. Lohr (301, 361) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton, CT 06340 Ales Medek (39) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton, CT 06340 Kenneth G. Miller (165) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Ann Arbor, MI 48105 Kenneth J. Norris (145) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton, CT 06340 Bernard A. Olsen (89) Eli Lilly and Company, 1650 Lilly Road, DC TL12 Lafayette, IN 47909 Radhika Rajagopalan (27) Center for Drug Evaluation and Research, FDA, 7500 Standish Place, Rockville, MD 20855 David T. Rossi (165) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Ann Arbor, MI 48105 Dinos Santaﬁanos (361) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton, CT 06340 Teri Shanklin (231) Pﬁzer Global Research and Development Division, Bioprocess Research & Development Department, Groton, CT 06340 Thomas R. Sharp (301, 361) Pﬁzer Global Research and Development Division, Analytical Research and Development Department, Groton, CT 06340 Feng Wang (249) Chemical Development Division, GlaxoSmithKline, Five Moore Drive, PO Box 13398, Mail stop 2-4075-4A, Research Triangle Park, NC 27709-3398 Kenneth C. Waterman (75) Pﬁzer Global Research and Development Division, Pharmaceutical Research and Development Department, Groton, CT 06340 Glenn Wilcox (231) Pﬁzer Global Research and Development Division, Bioprocess Research and Development Department, Groton, CT 06340

1 OVERVIEW: ISOLATION AND CHARACTERIZATION OF IMPURITIES SATINDER AHUJA Ahuja Consulting,Calabash, NC 28467

I. INTRODUCTION A. Definitions B. The Need to Isolate and Characterize Impurities II. DESIGNATIONS OF IMPURITIES A. Common Names B. United States PharmacopeiaTerminology C. ICH Terminology III. REGULATORY REQUIREMENTS IV. SOURCES OF IMPURITIES A. Crystallization-Related Impurities B. Stereochemistry-Related Impurities C. Residual Solvents D. Synthetic Intermediates and By-products E. Formulation-Related Impurities F. Impurities Arising During Storage V. ANALYTICAL METHOD DEVELOPMENT A. The Role of Reference Standards B. Spectroscopic Methods C. Separation Methods VI. ISOLATION METHODS VII. CHARACTERIZATION METHODS VIII. CASE STUDIES IX. SUMMARY REFERENCES

I. INTRODUCTION Pharmaceutical analysts play a major role in isolation and characterization of impurities. The success in this endeavor requires a broad knowledge of a variety of ﬁelds of chemistry and excellent interactions with experts in various other disciplines.1,2

1

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S. AHUJA

A. Definitions Let us ﬁrst brieﬂy review what constitutes an impurity in a drug substance (a new chemical entity of therapeutic interest) or drug product (a drug substance formulated into a suitable product for administration to patients) to enable us to better understand the need to isolate and characterize impurities. As recently as the 1990s, there was no precise deﬁnition for ‘‘impurity’’ in the pharmaceutical world, because of the apparent negativity attached to this word.3 The deﬁnition for impurity in Webster’s dictionary is ‘‘something that is impure or makes something else impure.’’ In the pharmaceutical industry, it is the latter meaning that is frequently attached to the meaning of an impurity. An impure substance is sometimes described as a substance of interest mixed or impregnated with an extraneous or usually inferior substance. A simple deﬁnition of impurity has been offered:2 An impurity is any material that affects the purity of the material of interest, viz., drug substance or drug product. The following deﬁnition of impurity is currently under consideration by the regulatory bodies, which is likely to be included in the future guidance:4 Impurity: any entity of the drug substance (bulk material) or drug product (ﬁnal container product) that is not the chemical entity deﬁned as the drug substance, an excipient, or other additives to the drug product.

This deﬁnition of impurity is broad enough to include degradation products as impurities. The term degradation product is deﬁned in International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH; the American spelling for harmonization will be used from here on in this chapter) as follows:5 Degradation product: a molecule resulting from a change in the drug substance (bulk material) brought about over time. For the purpose of stability testing of the products in this guidance, such changes could occur as a result of processing or storage (e.g., deamidation, oxidation, aggregation, and proteolysis).

These deﬁnitions can be used as a guide in the search for a number of inorganic or organic compounds that can be found in a drug substance or a drug product until a ﬁnalized deﬁnition is available. At present, the impurities are given various names (see Section II); some of the terms, such as related compounds, may tend to soft-pedal them. In the pharmaceutical world, an impurity is generally considered as any other organic material besides the drug substance or active pharmaceutical ingredient (API) that arises out of synthesis. Most of the time, the inorganic contaminants are not given adequate consideration as impurities unless they are toxic, such as heavy metals or arsenic. Organic volatile impurities (OVI, which are generally made up of residual solvents as well as other organic volatile impurities used in the synthesis) are often considered virtual impurities. Interaction products produced during formulation processes and degradation products (frequently referred to colloquially as degradants in the

1 ISOLATION AND CHARACTERIZATION OF IMPURITIES

3

pharmaceutical industry; the terms have been used interchangeably in this text) that can be produced prior to use by the patient are additional sources of impurities. Section IV lists various sources of impurities in API or drug products. It is important to recognize at this stage that any material that leads to a decrease in the purity value of the API should be considered an impurity. Therefore, for all intents and purposes, various contaminants mentioned here can be called impurities and should be labeled as such because they decrease the purity of API.

B. The Need to Isolate and Characterize Impurities Impurities are generally assumed to be inferior to API because they might not have the same level of pharmacologic activity. However, they are not necessarily always inferior. From the standpoint of its usage, the drug substance is compromised in terms of purity even if it contains another material with superior pharmacologic or toxicologic properties. At ﬁrst pass this may not be readily apparent; however, on further thought it will become clear that if we are to ensure that the accurate amount of the drug substance is being administered to the patient, we must assess its purity independent of the extraneous materials. Therefore, any extraneous material present in the drug substance or active ingredient must be considered an impurity even if it is totally inert or has superior pharmacologic properties, so that an appropriate evaluation of its content in the drug product can be made. The control of low-level impurities is of great importance when a drug is taken in large quantities; for example, the use of methotrexate (10–20 g) to treat neoplasia or the use of vitamins as a fad, notably vitamin C. Penicillins and cephalosporins have been known to sustain facile cleavage of the -lactam bond in aqueous solution. This is particularly interesting since some studies on penicillins have shown that their lack of stability may inﬂuence possible reactions involved in penicillin allergy.6 Special attention should be paid to the detection of DNA in all ﬁnished biotechnology products because DNA can be incorporated in the human genome and become a potential oncogene. It is expedient to exhibit the absence of DNA at the picogram-per-dose level to warrant the safety of biotechnology products.7 This book addresses isolation and characterization of a variety of materials mentioned in Section II, which can be simply called impurities that can affect the purity of API or can be harmful to patients. It is necessary to isolate and characterize a number of impurities and degradation products mentioned in Section II because it is not always possible to unambiguously characterize them with the widely used hyphenated methods that are frequently the ﬁrst line of defense. These methods utilize detectors such as diode array UV detector (DAD), nuclear magnetic resonance spectrometer (NMR), and mass spectrometer (MS) with separation methods such as gas chromatography (GC), high-pressure or high-performance liquid chromatography (HPLC), supercritical ﬂuid chromatography (SFC), and capillary electrophoresis (CE). Methods such as gas chromatography-mass spectrometry (GC/MS), high-pressure or high-performance liquid chromatography–mass spectrometry (HPLC/MS, or simply

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LC/MS), GC or LC/MS/MS, LC/DAD/MS, LC/NMR, LC/DAD/MS/NMR, SFC/MS, or CE/MS and various other combinations are extremely useful and can shorten the time needed for characterization of impurities. These methods have been discussed at length (Chapters 11–14 in this book; also see references 1 and 2). It should be apparent from the preceding discussion that complete characterization of the impurities or evaluation of their toxicologic properties generally requires that the impurities be isolated in sufﬁcient quantities or be synthesized.

II. DESIGNATIONS OF IMPURITIES Impurities have been named differently by various groups of scientists who deal with them. Described here are commonly used terms and those terms that are used by ofﬁcial bodies such as compendia or that have been found acceptable by ICH and various regulatory bodies.

A. Common Names Various terms that have been commonly used to describe impurities are listed alphabetically below. . . . . . . .

By-product Degradation product Interaction product Intermediate Penultimate intermediate Related product Transformation product

Some of these terms indicate potential sources of impurities; e.g., intermediates; others tend to downplay the negativity, as exempliﬁed by the use of the term ‘‘related product.’’ By-products: The unplanned compounds generated in the reaction to produce API are generally called by-products. Because it might not be possible to theorize all of them, they present a signiﬁcant challenge to the analytical chemist. Degradation products: The compounds produced as a result of decomposition of the material of interest or API are often called degradation products (or degradants). It is necessary to be concerned with these products as well as those brought about by degradation of other compounds that may also be present as impurities in the drug substance. Interaction products: This term is slightly more inclusive and more difﬁcult to evaluate than the two previously described, i.e., by-products and degradation products, in that it takes into account interactions that could possibly occur between various involved chemicals—purposely or inadvertently.

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Intermediates: The planned compounds produced during synthesis of the desired substance are called intermediates, especially if they have been isolated and characterized. The most important requirements are isolation and characterization, i.e., they cannot be just potential reaction products that may be produced theoretically (see by-products). The theorized products are best designated as ‘‘potential intermediates.’’ Penultimate intermediate: As the name implies, this is the last compound in the synthetic chain just preceding the production of the ultimate desired compound. Confusion sometimes occurs when the desired material is a salt of a free base or acid. It is not appropriate to label the free acid or base as the penultimate intermediate if the drug substance is a salt. Related products: As suggested previously, the term ‘‘related products’’ tends to imply that the impurity is similar to the drug substance, and it thus tends to downplay the negativity frequently attached to the term ‘‘impurity.’’ These products may have similar chemical structures and potentially similar biological activities; however, we know that the structure alone does not provide any surety about biological activity. Transformation products: This is a relatively commonplace term that relates to theorized and non-theorized products that may be produced in the reaction. Transformation products are comparable to by-products, except that this term tends to imply that more is known about the reaction products.

B. United States Pharmacopeia Terminology The United States Pharmacopeia (USP)8 discusses impurities in various sections: . . .

Impurities in Official Articles Ordinary Impurities Organic Volatile Impurities

The pharmacopeia states that our notions about purity are likely to change with time and that purity is closely related to current developments in analytical chemistry. Therefore, what we regard as pure today may be considered impure at some future time if methods are found that can determine other components in a particular compound. Inorganic, organic, biochemical, isomeric, or polymeric components may all be considered impurities. The following terms have been used to describe impurities: . . . . . .

Concomitant components Foreign substances Ordinary impurities Organic volatile impurities Signal impurities Toxic impurities

Concomitant components: Bulk pharmaceutical chemicals (frequently referred to as API in the pharmaceutical industry) may have concomitant

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components, e.g., geometric and optical isomers and antibiotics that are mixtures. Foreign substances: These are materials that are introduced by contamination or adulteration, and not as a result of formation or preparation; they are classiﬁed as foreign substances, e.g., pesticides in oral analgesics. Ordinary impurities: The types of impurities in bulk pharmaceutical chemicals that are harmless by virtue of having no serious undesirable biological activity in the amounts present are speciﬁed as ordinary impurities. Organic volatile impurities: This term describes the residual solvents that may be detected in the drug substance. Signal impurities: These are differentiated from ordinary impurities discussed earlier in that they call for individual identiﬁcation and quantiﬁcation by speciﬁc tests. These impurities include some process-related impurities or degradation products that provide a signiﬁcant knowledge about the process. Toxic impurities: These impurities have important undesirable biological activity, even as minor constituents, and demand speciﬁc identiﬁcation and quantiﬁcation by explicit tests. Compendial articles may contain additional inorganic impurities. These impurities may be as common as table salt or a variety of other compounds that are regulated at low levels, such as heavy metals, arsenic, and so forth.

C. ICH Terminology According to ICH guidelines (on the Internet, see http://www.fda.gov/ cder/guidance), impurities can be broadly classiﬁed into the following three categories for the drug substance produced by chemical synthesis: . . .

Organic impurities (starting materials, process-related products, intermediates, and degradation products). Inorganic impurities (salts, catalysts, ligands, and heavy metals or other residual metals). Residual solvents (organic and inorganic liquids used during production and/or recrystallization).

The International Conference on Harmonization addresses the questions relating to impurities as follows: . . . . .

Q1A(R) Stability testing of new drug substances and products Q3A(R) Impurities in drug substances Q3B Impurities in drug products Q3C Impurities: residual solvents Q6A Specifications: test procedures and acceptance criteria for new drug substances and new drug products: chemical substances

It should be noted that none of the terminologies given above adequately highlights polymorphic, solvatomorphic, and chiral impurities (see Section III).

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III. REGULATORY REQUIREMENTS Ethical, economic, and competitive reasons, as well as those of safety and efﬁcacy, support the need to monitor impurities in drug products.9 However, monitoring impurities and controlling these impurities mean different things to different people or to the same people at different times, even those in the pharmaceutical sciences and industry.2 A uniﬁed terminology is necessary to assure that everyone uses the same vocabulary when addressing questions related to impurities. In this context, the leadership provided by ICH is very helpful. A number of requirements have an effect on monitoring impurities (see Chapter 2). For example, a country’s pharmacopeia or the one accepted by that country often provides the primary guidance as to how impurities are to be monitored and regulated. In a majority of countries these pharmacopeias are run under the auspices of the government. The USP is a notable exception to this case. If a product is considered a pharmacopeial item, it must meet the compendial requirements. The United States Food and Drug Administration (FDA) has endorsed the guidance prepared under the auspices of the ICH. The guidance, developed with the joint efforts of regulators and industry representatives from the European Union, Japan, and the United States, has helped ensure that the different regions have consistent requirements for the data that should be submitted to the various regulatory agencies. For the threshold of various impurities allowed in drug substances and drug products, see Chapter 2. The guidelines not only aid the sponsors of New Drug Applications (NDA) or Abbreviated New Drug Applications (ANDA) with the type of information that should be submitted with their applications, but also assist the FDA reviewers and ﬁeld investigators in their consistent interpretation and implementation of regulations. In the United States, the federal FD&C Act and its amendments require that a manufacturer demonstrate the safety and efﬁcacy of a new drug prior to introducing it into interstate commerce. The requirements are clearly spelled out in the Notice of Claimed Investigational Exemption for a New Drug (IND) and the NDA. The FDA also provides the following guidance on impurities: . .

NDAs: Impurities in drug substances ANDAs: Impurities in drug substances

INDs require ‘‘a statement of the methods, facilities and controls used for the manufacturing, processing, and packing of the new drug to establish and maintain appropriate standards of identity, strength, quality, and purity as needed for safety and give signiﬁcance to clinical investigation made with the drug.’’ NDAs demand more speciﬁc and explicit information, including stability studies to guarantee that the identity, quality, and purity of the drug product is maintained until its expiration date.

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The FDA may initiate action under the Food and Drug Act to bring about removal of a product from the market, or, as is granted in the law, a manufacturer may voluntarily withdraw from the marketplace any batches that do not meet the approved speciﬁcations. In each country, regulatory authorities use their own standards for conducting clinical studies on a new drug product, i.e., a drug substance that has been formulated or the product that is approved for commerce. Speciﬁc comments may be found in Chapter 2 in Reference 2 as to how regulations vary from country to country and the subsequent effect on international trade. Efforts are being made to unify these approaches, as demonstrated by ICH guidelines; nonetheless, there is still much to be done. ICH Guidelines on Impurities in New Drug Substances have the following requirements: The studies conducted to characterize the structure of actual impurities present in the new drug substance at a level greater than 0.1% (depending on the daily dose, calculated using the response factor of the drug substance) should be described. Similarly, ICH Guidelines on Impurities in New Drug Products require that the degradation products observed in stability studies conducted at recommended storage conditions be identiﬁed when present at a level greater than the identiﬁcation thresholds (1% for a maximum daily dose of < 1 mg to 0.1% for a maximum daily dose of > 2 g).

IV. SOURCES OF IMPURITIES From the preceding discussion, it should be clear that impurities can originate from various sources. The most obvious source of impurities is the synthesis, where intermediates and by-products may be carried into the API as impurities or become a source of other impurities resulting from them. Any impurity that may be present in the starting material has the potential to be carried into the active ingredient of interest. Furthermore, the impurities that relate to inert ingredients (excipients) and solvents used during synthesis must also be considered. Impurities can be produced during various drug product formulation steps. These impurities have the possibility of being present in the ﬁnal drug product. Potential reaction products relating to these impurities must also be evaluated. The impurities in drug products can be attributed not only to the drug substance or inert ingredients used for formulating a drug product; they can be brought into the drug product through the formulation process or by contact with packaging (see Chapter 4). Of the various impurities that can be found in drug products, there are relatively few that can inﬂuence the stability or performance of the dosage form. It is important to give greater consideration to these detrimental impurities. In general, most of these impurities are small molecules. This is especially true in solid dosage forms where the limited mobility restricts the reactivity of larger molecules. For most drugs, the reactive species consists of water (which can hydrolyze some drugs or affect the dosage form performance), small electrophiles (e.g., aldehydes and carboxylic acid derivatives), peroxides (which can oxidize

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some drugs), and metals (which can catalyze oxidation and other drug degradation pathways). All of these impurities are discussed in Chapter 4, which focuses on how these impurities are likely to alter dosage forms through chemical reactivity and physical changes to the systems. Additionally, some impurities can cause toxicological problems. These impurities may not directly affect the performance or stability of a dosage form, but must be controlled to make a safe drug product.

A. Crystallization-Related Impurities Polymorphism is the term used to denote crystal systems where a substance can exist in different crystal packing arrangements, all of which have the same elemental composition (see Chapter 3). It is also possible to have crystal systems where the substance exists in different crystal packing arrangements, each of which has a different elemental composition; this phenomenon is known as solvatomorphism. Based on the realization that the nature of the structure adopted by a given compound upon crystallization could exert a profound effect on the solid-state properties of that system, the pharmaceutical industry is required by regulatory authorities to take a strong interest in polymorphism and solvatomorphism. The nature of the crystal structure of a given material can inﬂuence the following properties: . . . . . . . . . . . . . . . . . . . .

Conductivity Crystal hardness Crystal shape and color Density Diffusivity Dissolution rate Electrolytic conductivity Enthalpy of transitions Heat capacity Heat of solution Hygroscopicity Latent heat of fusion Melting or sublimation properties Phase diagrams Rates of reactions Refractive index Solubility Surface tension Viscosity Volume

It is usually the goal in pharmaceutical manufacturing to produce a drug substance that is phase-pure and remains in that state as long as the bulk material is stored. Another goal is to formulate the drug substance in a manner so that it remains in the same phase-pure state during the manufacture of the drug product and during any subsequent storage. This requires the

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development and validation of assay methodology for the determination of phase composition. In Chapter 3, the question of small amounts of polymorphic and solvatomorphic impurities in a bulk solid is addressed. Methods such as X-ray diffraction, thermal methods, vibrational spectroscopy, and solid-state NMR are useful for performing the required studies. These methods are outlined and illustrated with appropriate examples.

B. Stereochemistry-Related Impurities It is of paramount importance to look for stereochemistry-related compounds, i.e., those compounds that have similar chemical structure but different spatial orientation. These compounds can be considered impurities in the API. Included in this group are various stereoisomers. The simplest case of chirality can be seen in a molecule that has one or more tetrahedral carbons with four different substituents (asymmetric carbon atom) such that its mirror image is not superimposable. Chiral molecules may also occur for a number of other reasons and must be factored into any evaluation of impurities.10–12 Stereoisomerism is possible in molecules that have any of the following characteristics: . . . . . .

One or more center of chirality Helicity (e.g., helical nature of tertiary structure of proteins, polysaccharides, and nucleotides) Planar chirality (e.g., polycyclophanes) Axial chirality (e.g., spiranes with cyclic skeleton) Torsional chirality (e.g., torsion about double or single bonds like cis and trans isomers and rotomers) Topological asymmetry (e.g., catenanes)

Chiral molecules are frequently called enantiomers. Enantiomers are optical isomers that have the same chemical structure but different spatial arrangement, which leads to different optical rotation. It is important not to overlook them because the d-isomer of a compound can have different pharmacologic or toxicologic activity from that of the l-isomer.11 Therefore, the undesired optical isomer is considered a chiral impurity of the API. Furthermore, it is important to remember that the number of chiral impurities increases with the increasing number of asymmetric carbon atoms in a molecule. The ﬁrst set of guidelines regarding this issue was issued by the FDA in 1987 where the question of stereochemistry was approached directly on the manufacture of drug substances.13 The FD&C Act requires a full description of the methods used in the manufacture of the drug, which includes testing to demonstrate its identity, strength, quality, and purity. For chiral compounds, this includes identiﬁcation of all chiral centers. The enantiomer ratio, although 50:50 by deﬁnition for a racemate, should be deﬁned for any other admixture of stereoisomers. It is expected that the toxicity of impurities, degradation products, and residues from the manufacturing process be investigated as the development of the drug is pursued. The same standards

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should, therefore, be applied to the racemates. For all practical purposes, optical isomers are considered impurities.

C. Residual Solvents Water is commonly present in drug products. As a result, water is by far the most commonly found volatile impurity in drug products, and most of the time it is not even considered an impurity (see Chapter 4). It is prevalent both in drug substances and excipients (water is present in a number of excipients as supplied by the vendor) and is used in dosage form preparations as well. Moisture content can be important when a dosage form is packaged such that equilibration with the environment does not occur. Under these conditions, the moisture brought into the system through the excipients can be sufﬁcient to bring about hydrolysis. In addition, water from the environment can affect drug products and can very often be detrimental to their chemical stability or dosage form performance. The chemical stability problems relating to water are generally caused by hydrolysis, particularly of carboxylic acid derivatives (esters, amides), acetals, and other susceptible functionalities. Even in nonaqueous liquid formulations, water can be present in amounts sufﬁcient to lead to drug degradation. For some drugs, the moisture level will determine if a hydrate can form. In some cases, hydrate formation or generation of an anhydrous form (by having a sufﬁciently low humidity level) can lead to a loss in crystallinity, which in turn may bring about greater chemical instability of the drug. In addition, changes in crystal morphology may affect changes in drug dissolution rates (see Chapter 3). A number of solvents that are used for the synthesis of the API or formulation of the drug product can be present in the drug product. The content of these solvents, which are commonly called organic volatile impurities (OVI), is generally determined by the OVI methods speciﬁed in the compendia. Their content is controlled by the guidelines offered by various bodies (see Chapter 2). Residual solvents can affect the stability of drug product (see Sections IV. D and E).

D. Synthetic Intermediates and By-products In addition to the residual solvents, polymorphic, solvatomorphic, and chiral impurities mentioned previously, impurities in a pharmaceutical compound or a new chemical entity (NCE) can originate during the synthetic process from raw materials, intermediates, and/or by-products. Raw materials are usually produced to lesser purity requirements than a drug substance. Therefore, it is easy to understand why they could include a number of components that in turn could have an impact on the purity of the drug substance. The solvents used in synthesis are also likely to involve a number of impurities that may extend from trace levels to critical quantities that can react with various chemicals used in the synthesis, to give rise to other

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impurities. Intermediates are also not usually held to the same purity level as that of the drug substance—hence the observations made for the raw materials apply. By-products are often unknown and are very seldom controlled. So they too are a cause of concern in terms of monitoring impurities. ‘‘Pot reactions’’ (reactions where the intermediates are not isolated) are convenient, economical, and time saving; however, they raise havoc in terms of the generation of impurities because a number of reactions can occur simultaneously. The penultimate intermediate in the pharmaceutical synthesis is generally required to meet certain preset speciﬁcations. However, the more demanding standards of purity for the drug substance are very rarely exercised at this stage. It is important to bear in mind that this step is the last possible source of impurities from the synthesis. The methods used for analysis at this stage should be rigorous, and the tightest economically and practically feasible speciﬁcations should be applied.

E. Formulation-Related Impurities As mentioned before, many impurities in a drug product can originate from excipients used to formulate a drug substance. In addition, a drug substance is subjected to a variety of conditions in the process of formulation that can cause its degradation or have other undesirable reactions. For example, if heat is used for drying purposes or for some other reason, it can hasten degradation. Solutions and suspensions are inherently prone to degradation due to hydrolysis or solvolysis.2 These reactions may also take place in the dosage form in a solid state, such as in the case of capsules and tablets, when water or another solvent has been employed for granulation. The water used in the formulation can contribute not only its own impurities, but can also afford a perfect situation for hydrolysis and metal catalysis. Comparable reactions are conceivable in other solvents that may be used. If no precautions are taken, oxidation is quite possible for freely oxidized materials. In the same way, light-sensitive materials may sustain photochemical reactions. Details concerning the ways in which varied excipients may contribute to degradation and the resulting impurities may be found in Chapter 6 of Reference 2. In a number of cases, chemical stability issues associated with an excipient are due to impurities in the excipient rather than the excipient itself (see Chapter 4). The most reactive impurities tend to be small molecules. In addition to chemical stability, impurities in drug products can cause problems associated with toxicity or dosage form performance. It is often valuable to quantitatively determine the level of important impurities in drug products and to trace the origin of those impurities to their source. If the source is from an excipient, variability from lot to lot may make a marginal product unacceptable for reliability. If the source is related to the dosage form preparation process, it may be worthwhile considering technologies and processes that render less of the undesirable impurity.

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F. Impurities Arising During Storage A number of impurities can originate during storage (shelf life) or shipment of drug products. It is essential to carry out stability studies to predict, evaluate, and ensure drug product safety.9 Stability, however, can have different meanings to different people, based on their discipline in the pharmaceutical sciences and industry. A variety of terms are currently used to encompass the what, how, and why of stability: kinetic study, compatibility study, stability evaluation, stability-indicating assay, expiration dating, outdating, shelf life, storage legend, preformulation study, failure of a batch to meet speciﬁcations, microbiological stability, stability of the active ingredient, stability of the formulation, stability in the marketed package, stability in the sample package, stability in the dispensing package, and stability in the hands of the consumer. All of these are considerations in stability of a drug product, and it is important to ensure that everyone understands the importance of stability studies. Chapter 8 in Reference 2 reviews the many facets of stability studies and describes what a modern stability program includes. Efforts have been made to relate scientiﬁc considerations with regulatory requirements. Degradation kinetics is discussed later in this section to highlight their importance in monitoring and evaluating impurities.2 The common goal for investigation of both process- and degradationrelated impurities is to determine which of the many potential impurities are, in fact, produced in the manufacturing process and which occur under a given set of storage conditions (see Chapter 5). During the development phase, this knowledge can be used to modify the process in an effort to eliminate or minimize levels of impurities. Similarly, the knowledge of stability characteristics can be used to store the drug substance and drug product in appropriate packaging and environmental conditions to minimize or eliminate unacceptable degradation. As mentioned previously, a better understanding of the degradation behavior of the drug and its interactions with excipients is also useful in developing the best formulation. The information obtained from impurity investigations can help establish speciﬁcation tests and acceptance limits at various control points in the manufacturing process. 1. Degradation Kinetics The majority of the degradation reactions of pharmaceuticals take place at ﬁnite rates and are chemical in nature. Solvent, concentration of reactants, temperature, pH of the medium, radiation energy, and the presence of catalysts are important factors that affect these reactions. The order of the reaction is characterized by the manner in which the reaction rate depends on the reactant concentration. The degradation of most pharmaceuticals is classiﬁed as zero order, ﬁrst order, or pseudo-ﬁrst order, although the compounds may degrade by complicated mechanisms, and the true expression may be of higher order or be complex and noninteger. To ensure better stability predictions, an understanding of the limitations of experimentally obtained heat of activation values is critical. For instance,

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the apparent heat of activation of a pH value where two or more mechanisms of degradation are involved is not inexorably constant with temperature. Furthermore, the ion product of water, pKw, is temperature-dependent, and Ha is approximately 12 kcal, an often overlooked factor that must be considered when calculating hydroxide concentration. Therefore, the heat of activation for all bimolecular rate constants involved in a rate–pH proﬁle to predict degradation rates at all pH values for different temperatures must be obtained. Pharmaceutical scientists generally perform the desired kinetic studies to predict stability of a drug substance. It is important, however, to recognize the qualifying factors of such predictions. The signiﬁcance of kinetic studies and the effect of a variety of additives on the reaction rates have been discussed at some length.2

V. ANALYTICAL METHOD DEVELOPMENT New drug development requires that meaningful and reliable analytical data be produced at various stages of the development.1,2,12,14,15 Assuring the safety of a new pharmaceutical compound or drug substance demands that the new drug substance meet the established purity standards as a chemical entity or when admixed with animal feeds for toxicity studies or when formulated with or without pharmaceutical excipients for human use. Furthermore, it should exhibit excellent stability throughout its shelf life. These requirements mandate that the analytical method(s) employed for this purpose should be sufﬁciently sensitive to measure low levels of impurities. This has resulted in development of analytical techniques that are appropriate for measurement of trace/ultratrace levels, i.e., sub-microgram quantities of a variety of chemical entities.15 Analytical method development for the quantiﬁcation of low-level impurities present in pharmaceuticals can be thought of as a three-step process (see Chapter 7): 1. Sample set selection for analytical method development 2. Screening of chromatographic conditions and phases, typically using the linear-solvent-strength model of gradient elution 3. Optimization of the method to fine-tune parameters related to ruggedness and robustness. This can be accomplished using a factorial optimization approach. The key sample set selection for analytical method development has been discussed at length in Chapter 7. There are a great variety of methods used for monitoring impurities.1,2 The primary requirement for such techniques is the capacity to differentiate between the compounds of interest. This requirement frequently necessitates utilization of separation methods (covered in Section V. C) in combination with a variety of detectors (Section V. B). For gas chromatography, ﬂame ionization and electron capture detectors are commonly used. However, these detectors are not suitable for isolation and characterization of impurities, which require

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nondestructive detectors. Thermal conductivity detector ﬁlls that bill, but is generally not sufﬁciently sensitive. HPLC is the technique of choice for separation and analysis of pharmaceutical compounds because the compounds do not have to be volatile (or made volatile by derivatization), in contrast with gas chromatography, and thus they are not subjected to excessive heat. A number of spectroscopic detectors may be used with HPLC (see Section V. B). The spectroscopic methods may also be used independently for monitoring impurities; however, the combination of separation power of chromatography with spectroscopic detection provides very useful methods that provide high resolution of impurities. Electrochemical and other detectors are used where spectroscopic detection is not possible. Chiral Compounds Spectroscopy or chromatography can be used to monitor chiral compounds. Uniquely selective chromatographic approaches are often needed10–12; therefore, they have been highlighted here. The separations of chiral compounds discussed in various chapters of this book are often necessary because chiral pharmaceutically active moieties are increasingly common. It is imperative to determine the enantiomeric purity of chiral pharmaceutical compounds, including reference standards (see Chapter 6). In general, the minor enantiomer is considered an impurity. A chiral pair can be distinguished spectrally or chromatographically through the interaction with other chiral molecules to form either long-lived or transient diastereomers. The exploitation of the latter mechanism is the basis for the majority of enantiomeric chromatographic separations. The formation of a transient diastereomer is ordinarily performed either by using a chiral stationary phase or by adding a chiral selector to the mobile phase. Numerous chromatographic techniques, including those discussed in this book, have been demonstrated to achieve chiral separations through one of these routes. Chiral separation technology has now matured to the extent that quantiﬁcation down to 0.1% of the minor enantiomer is commonly attained.

A. The Role of Reference Standards Practical guidance on pharmaceutical reference standards, which are important in all phases of drug discovery, development, and commercialization is provided in Chapter 6. The key objective of the chapter is to provide clarity to the overall life cycle and qualiﬁcation and governance of reference standards used in the development and control of new drugs. Reference standards serve as the basis of evaluation of both process and product performance and are the benchmarks for assessment of drug safety for patient consumption. These standards are needed not only for the active ingredients in dosage forms but also for impurities, degradation products, starting materials, process intermediates, and excipients. The FDA has provided some guidance on the topic of reference standards (Chapter 2); some information also exists in the form of best practice documents from a variety of sources. However, the availability of reference standards and the degree to which

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they are characterized and governed is often contingent on the stage of the drug development process. At any phase of drug development a reference standard should be assessed versus its intended use, and a balance should be struck between resource commitments, scientiﬁc judgment, and regulatory requirements.

B. Spectroscopic Methods The following spectroscopic measurement techniques have been used for characterizing impurities; most of these are very useful as detectors for chromatographic methods: . . . . .

Ultraviolet (UV) Infrared (IR) Raman spectroscopy Mass spectrometry (MS) Nuclear magnetic resonance (NMR)

Ultraviolet spectrophotometry (UV) at a single wavelength furnishes minimum selectivity of analysis; however, with the current accessibility of diode array detectors, it is conceivable to obtain sufﬁcient simultaneous information at various wavelengths to assure greater reliability. Infrared spectrophotometry (IR) affords speciﬁc information on some functional groups that offer selectivity and allow quantiﬁcation. However, low-level detectability is difﬁcult. This requires more complex approaches, which are generally a deterrent to pharmaceutical analysts. Raman spectroscopy is based on the measurement of scattered electromagnetic radiation resulting from the irradiation of matter. Speciﬁcally, when a material is irradiated with a strong monochromatic light source (e.g., laser), a small amount of radiation is inelastically scattered at a wavelength different from the original incoming wavelength. It is this difference in vibrational energy between the scattered beam and incident beam that is measured. Raman spectroscopy is considered complementary to IR spectroscopy, as the two techniques provide a complete vibrational picture of a material. Raman spectroscopy is not as widely used for identiﬁcation purposes as IR spectroscopy because of the relative complexity and the cost of instrumentation. However, it should be noted that Raman spectroscopy is an extremely powerful tool in characterizing the presence of polymorphs (see Chapter 3). Mass spectrometry (MS) provides excellent structural information, and, based on the resolution of the instrument, it may be an effective tool for differentiating molecules with small differences in molecular weight (see discussions in Section V. C and in Chapter 11). However, it has ﬁnite uses as a quantitative procedure. Nuclear magnetic resonance spectroscopy (NMR) provides reasonably detailed structural information on a molecule and is an extremely useful method for characterization of impurities (see discussions in Section VII and in Chapter 12); however, its use as a quantitative method is limited.

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To summarize, UV detectors are commonly used for analyzing most pharmaceutical samples with HPLC. IR, NMR, and MS are excellent techniques for characterization of impurities that have been isolated by any of the techniques discussed here. The combination of MS and NMR with separation methods (discussed in Section VII and Chapters 11 and 12) provide excellent tools for characterization of impurities.

C. Separation Methods The following methods (listed in alphabetic order) can be used for separation of impurities and degradation products: . . . . . .

Capillary electrophoresis (CE) Chiral separations Gas chromatography (GC) High-pressure liquid chromatography (HPLC) Supercritical fluid chromatography (SFC) Thin-layer chromatography (TLC)

The nature and complexity of the separation problem determines which method should be used. The primary goal of a good separation method is resolution of all impurities of interest. A brief account of the abovelisted methods is given here to provide a quick review of their potential use. Except for CE, all these methods are chromatographic methods. CE is an electrophoretic method that is frequently lumped with the chromatographic methods because it shares with chromatography many of the common requirements. However, it is not strictly a two-phase separation system— a primary requisite for chromatography. Capillary electrochromatography meets this requirement. Hyphenated methods such as GC-MS, LC-MS, GC-LC-MS, LC-MS-MS, etc. are all discussed throughout this book. Also, Chapter 13 has been especially planned for this purpose. Capillary electrophoresis is an effective technique in situations where very low quantities of samples are available and high resolution is essential. Its relatively lower reproducibility is the principal difﬁculty of this procedure. Gas chromatography is an extremely useful technique for quantiﬁcation. It can afford the desired resolution, selectivity, and ease of quantiﬁcation. The chief limitation, however, is that the sample must be volatile or must be made volatile by derivatization. This technique is very practical for organic volatile impurities (OVI). High-pressure liquid chromatography is often referred to as highperformance liquid chromatography today. Both terms can be abbreviated as HPLC, and the terms are used interchangeably by chromatographers. The applications of this very effective technique have been signiﬁcantly expanded for the pharmaceutical chemist by the use of a variety of detectors such as ﬂuorescence, electrometric, MS, and so forth. Supercritical ﬂuid chromatography (SFC) offers some of the advantages of GC in terms of detection and of HPLC in terms of separations, in that volatility of the sample is not of paramount importance. The greatest

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application of this technique has been found in the extraction of samples. SFC is generally performed in the normal phase (NP) mode, and often NP-TLC or NP-HPLC methods can be readily adapted to SFC methods. SFC generally provides an orthogonal separation method to traditional reversed phase HPLC. Because of the similarity to HPLC in the chromatographic measurement process, this technique can be used to accurately quantify nonpolar impurities of the sample of interest. Thin-layer chromatography coupled with densitometric detection is a highly sensitive method for quick assessment of the purity of various compounds including reference standards (see Chapter 6). High-performance TLC (HPTLC) is an improved version of TLC that uses stationary phases of decreased thickness and lower particle size, providing improved resolution over shorter elution distances. TLC can resolve a large range of compounds by employing a variety of different plates and mobile phases. Limited resolution, detection, and ease of quantiﬁcation are the main problems associated with this method. The foremost advantages are ease of use and low cost.

VI. ISOLATION METHODS It is often necessary to isolate impurities because the instrumental methods that were mentioned earlier for directly characterizing impurities without isolating them are not available or when the authentic material is needed for further conﬁrmation of the structure or its toxicity. Isolation entails removal of the compound of interest from the other compounds present in a mixture. Further puriﬁcation is achieved based on the compound’s intended use. Chapter 8 provides practical guidance on the use of widely used extraction and isolation techniques from the sample preparation perspective. The ﬁrst two sections, solid-phase extraction and liquid–liquid extraction deal with liquid samples. The sections on supercritical ﬂuid extraction and accelerated solvent extraction focus mainly on solid samples while the centrifugation and ﬁltration sections handle suspensions. A successful sample preparation protocol accounts for speciﬁcity and homogeneity as well as recovery and ﬁnal physical state of the targeted material. The ultimate aim is to produce a sample that is compatible with the desired analytical technique to assure generation of maximum information. Isolation methods include both chromatographic and nonchromatographic methods. Simple methods should be tried ﬁrst, as they can lead to considerable savings in time and can produce a larger quantity of materials with greater ease. For isolation of a given compound from a complex mixture, the chromatographic methods utilized for separation of impurities in analytical determinations are the methods of ﬁrst choice that are suitably modiﬁed for the purpose of isolation of impurities where an appropriate fraction is collected. A list of the methods that can be used for isolation of impurities is given below. . .

Solid-phase extraction methods Liquid–liquid extraction methods

1 ISOLATION AND CHARACTERIZATION OF IMPURITIES . . . . . . . . .

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Accelerated solvent extraction methods Supercritical fluid extraction Column chromatography Flash chromatography Thin-layer chromatography (TLC) Gas chromatography (GC) High-pressure liquid chromatography (HPLC) Capillary electrophoresis (CE) Supercritical fluid chromatography (SFC)

As mentioned earlier, it is desirable to initiate the isolation process on the basis of simple extraction or partition methods as exempliﬁed by the ﬁrst four methods listed above (see Chapter 8). Frequently, it is possible to extract impurities selectively on the basis of acidity, basicity, or neutrality of the compounds in question.2 The extraction process usually involves liquid– liquid extraction, where one phase is an aqueous solution and the other is a nonpolar organic phase. By appropriate adjustment of the pH of the aqueous solution, one can extract acidic, basic, or neutral impurities. These methods work well when a few impurities are present and their polarity or pKa is sufﬁciently different from that of API. If necessary, further separations can be achieved by chromatographic methods. It is well known that techniques such as chromatography can provide separation of compounds from each other and from the main compound. Separations are based on properties such as adsorption, partition, ion exchange, or molecular size. The choice of isolation method should be based on all available separation methods (see those listed here and in Section V. C). However, it should be pointed out that scaleup to preparative scale might be necessary. Also, special steps may have to be taken in some cases to circumvent particular problems. For example, it is not possible to utilize a destructive detector, such as a ﬂame ionization detector, to isolate a component in GC. It would be necessary to replace it with a nondestructive detector, such as a thermal conductivity detector, or to use a split stream with accurate timing control that would allow collection of the desired separated component. The selection of an optimum method for isolation of impurities is dependent on a number of considerations, the foremost being the method that was initially used to ﬁnd the impuritiy. If the method is not too cumbersome to carry out and the projected amount of the isolated impurity can be handled by the methodology in place, that method is obviously the preferred technique. If the amount of impurity needed for characterization, pharmacologic studies, or toxicologic studies is much greater, it may be necessary to rely on simple separation methods such as column chromatography, ﬂash chromatography, or TLC. In this book special chapters have been included on TLC (see Chapter 9) and on column chromatographic methods including HPLC (see Chapter 10), as these methods are more commonly used. Because of its simplicity, ﬂexibility, speed of analysis, and unique detection methods for both qualitative and quantitative analysis, TLC is

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a valuable technique that is commonly used for separations of pharmaceutical compounds (see Chapter 9). Furthermore, it is complementary to commonly used HPLC methods; the majority of TLC separations are carried out in normal phase mode, whereas the method of choice in HPLC is frequently reversed phase. This generally entails selection of a suitable solvent system (mobile phase) for resolution of impurities on a silica gel plate. If the nature of impurities is not known, systems that are acidic, neutral, or basic are investigated. Universal detectors (UV or spray reagents) are preferred. Details on these methods and examples of sophisticated preparative separations are discussed in Chapter 9. Analytical chemists generally run a series of different assays (orthogonal assays) to conﬁrm that they have sufﬁciently characterized the sample and have identiﬁed all impurity peaks found in the sample. Furthermore, they ensure that peaks which may co-elute using one technique can be detected with an orthogonal method (see Chapter 10). Analytical HPLC is the most common technology employed for assay and impurity proﬁling of pharmaceuticals. When the sample has been analyzed, the impurity may need to be isolated (for structural characterization, for instance). While techniques such as LC-MS may give an indication of the compound identiﬁcation from the bulk assay, the deﬁnitive proof is always obtained from an independent analysis of the isolated compound. Because of the relatively low levels of the impurities (and also in many cases, their close resemblance to the main compound), conventional separation techniques are usually not successful. The resolving power of chromatography (and in many cases, HPLC) is needed for this isolation. Chapter 10 details the use of column chromatography for the isolation of impurities. It also discusses the various options that are available for stationary phases and analytical detectors, as well as the current equipment available for this work. The choice of puriﬁcation techniques (prep HPLC, low-pressure silica columns, etc.) is also discussed. Finally, a procedure is described for both analytical methods development as well as scale-up to preparative columns. Capillary electrophoresis can provide only limited quantities of substances. However, this technique has been used for micro-preparative applications.16 Supercritical ﬂuid chromatography as an isolation method is useful in those cases where it has been used as an analytical method for resolving impurities. It offers a great advantage in terms of removal of the mobile phase from the isolated fractions, since the mobile phase is mostly gaseous.

VII. CHARACTERIZATION METHODS When an impurity has been detected, it becomes necessary to estimate its content. Adequate detectability frequently means that a given component provides a signal at least twice that of background noise or baseline noise. At times, the multiple is set higher for greater assurance of detectability. Initial estimations are generally done against the parent compound because

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in most cases the authentic sample of impurity is not available. It is important that the authentic sample be used for estimations when it is available. If the estimations indicate that a given impurity content is greater than 0.1%, it must be characterized according to FDA requirements. The ability of NMR to provide information regarding the speciﬁc bonding structure and stereochemistry of molecules of pharmaceutical interest has made it a powerful analytical tool for structure elucidation (see Chapter 12). Unfortunately, NMR has traditionally been sensitivity-limited compared to other analytical techniques. Conventional sample requirements for NMR are on the order of 10 mg, as compared with mass spectroscopy, for example, which requires less than 1 mg. Therefore, NMR spectroscopy historically has not been the ﬁrst choice for an analytical chemist when identifying an unknown compound. Technological advancements in the ﬁeld of magnetic resonance have made signiﬁcant strides in improving sensitivity levels. This is particularly important in the structural characterization of drug impurities and degradation products, which often are available only in extremely limited quantities. The nondestructive, noninvasive nature of NMR spectroscopy makes it a valuable tool for the characterization of low-level impurities and degradation products. In addition, NMR can be considered close to a universal detector for hydrogen and carbon, as well as for other magnetically active nuclei. This is both good and bad because all signals are detected, those arising from the compound of interest and all other components in the sample, such as solvents and starting materials. Quantiﬁcation is accurate over a dynamic range of nominally four orders of magnitude, although not as precisely as with other analytical tools, particularly at low levels. This poses a challenge for studying mixtures containing low-level components. It is therefore preferred, if possible, to isolate a given impurity prior to study by NMR rather than to analyze a mixture. This technique also will simplify spectral interpretation to a great extent. Mass spectrometry has had an increasingly signiﬁcant impact on the pharmaceutical development process over the past several decades (see Chapter 11). Advances in the design and efﬁciency of the interfaces that directly connect separation techniques with mass spectrometers have afforded new opportunities for monitoring, characterizing, and quantifying drugrelated substances in active pharmaceutical ingredients and pharmaceutical formulations. Possessing exceptional analytical speciﬁcity and sensitivity, mass spectrometry signiﬁcantly reduces the cycle time of chromatographic method development, validation, and sample analysis. The popularity of LC-MS-MS systems for complex mixture analysis of thermally labile, biologically relevant molecules is largely attributed to the ‘‘soft’’ nature of atmospheric pressure ionization techniques such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI). Chapter 11 describes the fundamentals of LC-MS-based techniques for ion structure analysis, including aspects of ion formation in API sources, attributes of various mass analyzers and scan modes used for collision-induced dissociation experiments, and issues

22

S. AHUJA

surrounding the interpretation of the resulting mass spectra. Although the determination of chemical identity or molecular structure for related substances in pharmaceutical products has continuously beneﬁted from the availability and evolution of modern instrumentation, fundamental knowledge about solution phase chemistry, ionization, and gas phase processes is still vitally important for achieving success in this endeavor. There is economic pressure to devise instrumentation methods that can deliver many data sets at once, thus shortening the time-to-market of drug products (see Chapter 13). Coupling of instruments has become easier with expanded capabilities, e.g., HPLC-DAD-MS (HPLC coupled with a diode array UV detector and a mass spectrometer), is almost routinely used. Nuclear magnetic resonance spectrometry has now been added to this combination to provide HPLC-DAD-NMR-MS capabilities in a commercial instrument. Another pressure on pharmaceutical scientists is the promise of biopharmaceuticals and high-potency active pharmaceutical ingredients. These compounds often have complicated impurity and degradation proﬁles at low absolute concentrations. Fortunately, instrument manufacturers have been quick to attempt to satisfy both these needs. The challenge now to the pharmaceutical scientist is the organization of the many data sets into presentation-quality formats so that scientists and managers can make correct decisions quickly. Software has been developed to speed the collation, analysis, and presentation of the many spectroscopic characterization techniques necessary. Detection limits for mass spectrometers are now approaching the zeptomole level, and NMR spectrometers have recently seen dramatic increases in sensitivity down to the nanogram level. Hyphenated methods such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) and a number of other chromatographic and spectroscopic conﬁgurations are perfectly suitable for initial characterization of the impurities. Of course, these methods are not always applicable, especially when the authentic material is needed for purposes of structure conﬁrmation, synthesis, or toxicity studies (this subject is discussed in various chapters of this book; also see reference 2).

VIII. CASE STUDIES Diverse applications have been sought in the areas of monitoring quality, stability, and safety of pharmaceutical compounds, whether made synthetically, extracted from natural products, or produced by recombinant methods. Many of these applications are included throughout this book to afford ready reference to various methods. In addition, the reader may want to refer to the scientiﬁc literature where various applications of monitoring impurities are generally classiﬁed in the following categories:2 . . .

Alkaloids Amines Amino acids

1 ISOLATION AND CHARACTERIZATION OF IMPURITIES . . . . . . . .

23

Analgesics Antibacterials Anticonvulsants, antidepressants, and tranquilizers Antineoplastic agents Local anesthetics Macromolecules Steroids Miscellaneous

This classiﬁcation scheme has been preferably used over strictly chemistrybased schemes; however, the latter approach may be better suited for method development purposes. A number of case studies are included in this book. A brief review of the approach used for these studies follows (see Chapter 14). The process of identiﬁcation of impurities and/or degradation products should begin early in drug development. It is imperative to seek participation of all parties familiar with the project. The project group should meet to assess the timelines for completion and to gather all pertinent information. The initial planning and discussions can save signiﬁcant time in the experimental stage. The following questions should be raised at this early stage: . . . .

Is the compound of interest arising out of synthesis, formulation, or degradation? Is the impurity or degradation product present at significant levels? Is this a process- or degradation-related impurity, and under what conditions is it produced? Are enriched samples with the unknown impurity/degradation products available?

The next step is to determine at what level the unknown impurities are present. Identiﬁcation of impurities below the 0.1% level is generally not necessary unless the potential impurities are expected to be unusually potent or toxic. Therefore, it is imperative to determine the level of the unknown impurity and/or degradation product early in the process. If the unknown is at or above the 0.1% limit, effort should be made to identify it. However, if the unknown is below the 0.1% threshold, a discussion should be conducted with the project team members to determine if isolation and identiﬁcation are necessary. After a decision has been made to identify an unknown, the next logical step is to review all known process-related impurities, precursors, intermediates, and degradation products. A critical analysis of all available data can save considerable time and energy. By reviewing the HPLC retention data of all known process-related impurities, precursors, and intermediates (if available), it can quickly be determined whether the impurity of interest is truly unknown. If the retention time of the unknown impurity matches that of a standard, the unknown can be identiﬁed by using HPLC-DAD-MS. The identity can be conﬁrmed by correlating the retention time, UV spectra, and mass spectra of the unknown impurity with that of the reference standard. Identifying an unknown by using a reference

24

S. AHUJA

standard as in the preceding description is a quick and easy process. However, other steps must be taken when the retention time of an unknown does not match that of a standard. The next step generally is to obtain molecular mass and fragmentation data via HPLC-MS. It is essential to determine the molecular mass of the unknown. Not only does the molecular mass help in the identiﬁcation of the unknown, it also allows tracking the correct peak by HPLC when isolation becomes necessary. To run LC-MS, a mass spectrometry-compatible HPLC method is necessary. If such a method is not available, it has to be developed, which adds considerable time to the identiﬁcation process. The decisions regarding the best approach to characterizing impurities and degradation products should be based on sound reasoning in order to minimize the time and cost of new drug development.

IX. SUMMARY A new drug development program should include a series of physicochemical tests to fully deﬁne the impurity proﬁle of a pharmaceutical compound prior to performance of extensive pharmacologic and toxicologic studies. This is necessary to assure that observed toxicologic or pharmacologic proﬁles are in fact due to the compound of interest and not to impurities. ICH guidelines should be used to control impurities. Most importantly, the degree to which any impurity should be controlled ought to be dictated principally by its pharmacologic and toxicologic effects. Here it is necessary to include all impurities, those originating out of synthesis and those from other origins, such as degradation. The hyphenated methods should be used as the ﬁrst line of defense for characterization of impurities. To obtain absolute conﬁrmation of the structure of impurities or to conduct pharmacologic or toxicologic studies on impurities, it is frequently necessary to isolate and characterize them.

REFERENCES 1. Ahuja, S. and Scypinski, S., Handbook of Modern Pharmaceutical Analysis, Academic Press, NY, 2001. 2. Ahuja, S., Impurities Evaluation of Pharmaceuticals, Marcel Dekker, NY, 1998. 3. Ahuja, S., Eastern Analytical Symposium, November 16, 1995. 4. Ahuja, S., Personal communication, August 30, 2002. 5. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, see on the Internet, http://www.fda.gov/cder/guidance. 6. Yamana, T. and Tsuji, A., J. Pharm. Sci., 65:1563, 1976. 7. Bogdansky, F. M., Pharm. Technol., 72 Sept. 1987. 8. United States Pharmacopeia, Rockville, MD, 2000. 9. Mollica, J. A., Ahuja, S. and Cohen, J. J. Pharm. Sci., 67:443, 1978. 10. Ahuja, S., Chiral Separations by Liquid Chromatography, Am. Chem. Soc., Washington DC, 1991.

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11. Ahuja, S., Chiral Separations: Applications and Technology, Am. Chem. Soc., Washington, DC, 1997. 12. Ahuja, S., Chiral Separations by Chromatography, Oxford University Press, NY, 2000. 13. Guidelines for Submitting Supporting Documentation in Drug Applications for the Manufacture of Drug Substances: Office of Drug Evaluation and Research (HFD-100), Food and Drug Administration, Rockville, MD, 1987. 14. Ahuja, S., Chromatography of Pharmaceuticals. Natural, Synthetic and Recombinant Products, ACS Symposium Series #512, Am. Chem. Soc., Washington, DC, 1992. 15. Ahuja, S., Trace and Ultratrace Analysis by HPLC, Wiley, New York, NY, 1992. 16. Altria, K. D., Isolation and Purification, 2:113, 1996.

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2 REVIEW OF REGULATORY GUIDANCE ON IMPURITIES RADHIKA RAJAGOPALAN* Center for Drug Evaluation and Research, FDA, Rockville, MD 20855

I. INTRODUCTION II. TYPES OF IMPURITIESDRUG SUBSTANCE A. Foreign Substances and Other Impurities B. Toxic Impurities C. Ordinary Impurities III. ROLE OF COMPENDIA IV. ROLE OF DRUG MASTER FILES (DMF)TYPE II AND IMPURITIES EVALUATION V. REFERENCE STANDARDS FOR THE QUANTITATION OF IMPURITIES AND ANALYTICAL PROCEDURES VI. QUALIFICATION OF IMPURITIES AND NEW IMPURITIES VII. IMPURITIES IN DRUG PRODUCTS VIII. ANALYTICAL METHODOLOGY FOR IMPURITIES IN DRUG PRODUCT IX. IMPURITIES QUANTITATION POST-APPROVAL X. ROLE OF SPONSORS SUMMARY REFERENCES

I. INTRODUCTION The United States Food and Drug Administration (FDA) has endorsed the guidance prepared under the auspices of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). The guidance, developed through the joint efforts of regulators and industry representatives from the European Union, Japan, and the United States, helps ensure that the different regions have consistent requirements for the data that should be submitted to the various

*The author is writing this article on her personal time, and the views expressed do not necessarily represent the views of the Agency or the United States.

27

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regulatory agencies. They not only aid the sponsors of New Drug Applications (NDA) with what information should be submitted with their applications, but also assist the FDA reviewers, Abbreviated New Drug Applications (ANDA) and ﬁeld investigators in their consistent interpretation and implementation of regulations. In the area of impurities, both through ICH efforts, and by the efforts of the Agency in publishing Guidance, a tremendous amount of information is available. Technology advances have mandated that the global regulatory community, pharmacopeia, and the multinational sponsors of regulated products ﬁnd common ground on implementing tighter limits and stringent controls on the production of drug substances and drug products. These trends have created a need to adopt consistent quality standards. This chapter will discuss the existing Guidance on drug substances and drug products, the role of compendia, Drug Master File requirements, and the role of sponsors in detail. The scope of this article is also limited to chemically synthesized materials and does not apply to biological/ biotechnological substances.

II. TYPES OF IMPURITIESDRUG SUBSTANCE Increasing the purity of a drug substance depends on lowering the level of impurities, not only at the time of release, but also maintaining low levels of degradants during the shelf life of the drug substance. Procedures are usually proposed to control synthetic precursors, synthesis-related materials, intermediates, heavy metals, moisture, and volatile solvents. Stabilityindicating methodology that can differentiate between active ingredient and degradation products, process impurities or other potential impurities is necessary to monitor the degradation proﬁle over a long period of time. Starting the synthesis with high purity chemicals certainly adds to the purity proﬁle of the ﬁnished product. Table 1 is a summary of the current Guidance that pertain to impurity information for NDA and ANDA products. In addition, a number of other Guidances are also in the draft stage.1 These Guidances are available on the Internet at http://www.fda.gov/cder/ guidance. In addition to the above, ICH Q6B, and Q5C are also available at the Web site, although they are not discussed here. Q6B and Q5C relate to biological and biotechnological products. ICH Guidance addresses impurities in drug substances to accomplish the following: . .

Classify, identify, qualify, set specifications, and discuss analytical procedures for impurities. Discuss the stability evaluation of the drug substance under long-term and accelerated conditions while taking into account the type of packaging materials used for storage and distribution.

It should be noted that the methodology used for identifying and quantifying impurities is covered by another ICH guidance2 for the validation

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2 REVIEW OF REGULATORYGUIDANCE ON IMPURITIES

TABLE 1 Guidances on Impurities Title

Authorship

Q1A(R) Stability Testing of New Drug Substances and Products

ICH

Q3A(R) Impurities in Drug Substances

ICH

Q3B Impurities in Drug Products

ICH

Q3C Impurities: Residual Solvents

ICH

Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances

ICH

NDAs: Impurities in Drug Substances

FDA

ANDAs: Impurities in Drug Substances

FDA

of chromatographic methods. Impurities can be broadly classiﬁed into the following three categories3 when the drug substance is chemically synthesized: . . .

Organic impurities (starting materials, process-related products, intermediates, and degradants) Inorganic impurities (salts, catalysts, ligands, and heavy metals or other residual metals) Residual solvents (organic and inorganic liquids used during production and/or recrystallization)

It is therefore necessary to evaluate impurities utilizing a variety of techniques and instruments, prior to assigning purity values to the drug substance. In addition to the above, the United States Pharmacopeia (USP) addresses impurities using additional terminology:4 foreign substances and other impurities, toxic impurities, and ordinary impurities.

A. Foreign Substances and Other Impurities Many USP monographs contain tests for speciﬁc impurities. However, USP recognizes that other impurities may come from a variety of situations, such as a change in processing or extraneous sources, and tests should be employed to detect these other impurities in addition to the speciﬁc tests provided in the individual monograph. The USP Committee of Revision recommends that whenever a new impurity is present consistently in quantities >0.1% and the monograph HPLC procedure does not detect the new impurity, a revision be proposed to include this substance in the monograph. USP further recommends that the sum of all impurities including the monograph-speciﬁed impurities should not exceed 2.0%.

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B. Toxic Impurities Substances known to be toxic can arise from synthesis or degradation of compendial articles and must not be listed under this category. The manufacturer should provide data supporting the classiﬁcation and the limit for such impurities (refer to Section VI).

C. Ordinary Impurities These arise from the synthesis, preparation, or degradation of compendial articles. In general, USP provides tests to limit these impurities. However, since process-related impurities are different for varying processes, holders of Type II Drug Master File(s) and NDA/ANDA sponsors should address the impurities that are unique to their processes. The identiﬁcation of impurities present at a level greater than the threshold is usually recommended for organic impurities. Normally 0.1% or 0.05% (calculated using the response factor of the drug substance, Table 3) is considered an acceptable threshold. The inorganic impurities usually can be quantitated using pharmacopeial or other appropriate procedures. ICH Q3C guides in determining, on a safety basis, acceptable residual solvent levels for intake by use of the term ‘‘permitted daily exposure’’ (PDE). This Guidance classiﬁes residual solvents used in the synthesis and processing into four categories. The Guidance recommends that Class I solvents be avoided. These include benzene, carbon tetrachloride, 1,2-dichloromethane, 1,1-dichloroethane, and 1,1,1-trichloroethane. Table 2 is an example from the list of Class II solvents that should be limited because of their inherent toxicity either by calculation of concentration (PPM) or by PDE. This guidance deﬁnes class III solvents as having low toxic potential and a PDE of 50 mg or more per day and describes solvents for which no adequate toxicological data are found as class IV solvents.

III. ROLE OF COMPENDIA The USP is recognized as the ofﬁcial compendium by the FDA (21 U.S.C € 321(j)). Under General Notices in the USP 25, both foreign substances and TABLE 2 Common Organic Volatile Impurities (Class II) Organic volatile impurity

Limit (PPM)

PDE (mg/day)

Acetonitrile

410

4.1

Chloroform

60

0.6

600

6.0

80

0.8

1,4-Dioxane

380

3.8

Pyridine

200

2.0

Methylene chloride 1,1,2-Trichloroethene

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31

other impurities are controlled. However, many USP monographs do not take into account multiple synthetic pathways or sources for drug substance and varying excipients or manufacturing processes for drug products. Nevertheless, for any drug article to meet identity, strength, quality, purity and potency attributes, the evaluation of impurities is a must. Often, there are no USP monographs for drug substances and drug products covered by INDs and NDAs. The sponsors of NDAs or ANDAs initiate USP monographs through articles in the Pharmacopeial Forum (PF), after approval of the application. The draft PF goes through a public comment period where FDA, industry sponsors and any other interested party may offer suggestions before the monograph is ﬁnalized for incorporation into the compendium. It should be recognized that despite this mechanism, many older monographs might remain unrevised for a long period despite the presence of test procedures using updated technology. An example of the lack of adequate control (for a drug substance) is an assay test by titration combined with a purity test by thin-layer chromatography (TLC). Updated procedures using, for example, high-performance liquid chromatography (HPLC) or other superior technology should be incorporated in the monograph. Regardless of the age of the USP monograph, the requirements in the ‘‘General Notices’’ section of the USP apply to the possibility of the presence of ordinary, toxic, and foreign substances or impurities. In general, evaluation beyond what is prescribed in the USP is needed by the sponsors of NDA/ANDA, and Type II Drug Master File (DMF) holders. In many cases, drug articles may be covered by a monograph in a foreign compendium (European Pharmacopeia, British Pharmacopeia, Japanese Pharmacopeia, etc.). Until such time as pharmacopeial harmonization across the regions occurs, the FDA does not accept the items covered by foreign pharmacopeia as ofﬁcial. However, the speciﬁcations (test procedures and acceptance criteria) cited in these monographs can be used as a secondary source of information.

IV. ROLE OF DRUG MASTER FILES (DMF)TYPE II AND IMPURITIES EVALUATION A Type II DMF may be ﬁled for a drug substance (DS) or for a DS intermediate. For NDAs, drug substance information is usually ﬁled in the NDA itself, instead of a DMF. The Agency reviews Drug Master Files only in connection with the evaluation of a ﬁled IND, NDA, ANDA, or supplemental application when a letter authorizing such review is provided. There is no regulatory requirement that a DMF be submitted to support any of the above applications. The agency does not approve or deny approval to a DMF. The DMF holder is expected to follow the ICH and FDA guidance and ensuring that the different classes of impurities due to synthesis and degradation are addressed and adequately controlled in the drug substance. In addition, the DMF holder should address the stability of the drug substance and document it as per ICH guidance. Type II DMFs are to follow ICH conditions for ambient and accelerated testing and for the use of stability-indicating methods while testing. The amount of information

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expected in a DMF is the same as what is usually ﬁled in a NDA for drug substance synthesis and characterization. The regulatory responsibilities of DMF holders are detailed in 21 CFR 314.420.

V. REFERENCE STANDARDS FOR THE QUANTITATION OF IMPURITIES AND ANALYTICAL PROCEDURES While compendial standards are available for some monographed article impurities, it may be difﬁcult at times to obtain pure standards of impurities. Manufacturers of pharmaceuticals function as a potential source for obtaining reference standards of impurities, which may be synthesis precursors, process intermediates, or degradation products. The characterization and evaluation of these impurities reference standards should be constant with their intended use. In many cases, analytical procedures are developed and validated, where the response of an impurity is compared to that of the new drug substance itself. Response factor evaluation of impurities at the chosen detection wavelength is necessary to determine if a correction factor is needed (when the responses differ). Potentiometric detection, ﬂuorescence/ chemiluminescence detection, and refractive index detection are some examples of detection modes available for compounds that may not be suitable for UV detection.

VI. QUALIFICATION OF IMPURITIES AND NEW IMPURITIES The proﬁle of impurities in a new drug substance may change for a variety of reasons, such as process scale-up changes, synthetic route changes, and changes made to key intermediates. ICH decision trees help classify, qualify, and select limits for new molecular entities (NMEs). If an impurity exceeds the qualiﬁcation threshold listed below in Table 3 (ICH Q3A(R)), studies are needed to qualify that impurity in drug substances. In many cases, studies performed to qualify an impurity will depend not only on the daily dose intake, but also on the patient population, route of administration, and duration of drug administration. In some cases,

TABLE 3 Thresholds Maximum daily dose1

Reporting threshold2,3

2 g/day

> 2 g/day 1

Identification threshold3

Qualification threshold3

0.05%

0.10% or 1.0 mg per day intake (whichever is lower)

0.15% or 1.0 mg per day intake (whichever is lower)

0.03%

0.05%

0.05%

The amount of drug substance administered per day. Higher reporting thresholds should be scientifically justified. 3 Lower thresholds can be appropriate if the impurity is unusually toxic. 2

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33

decreasing the level of impurity below the threshold (by optimizing the synthesis or manufacturing conditions), rather than providing additional studies and data, may be the simplest course of action. For ANDAs,5 comparison with innovator products, and evaluation using Quantitative Structure Activity Relationship (QSAR)6 models, may also be an acceptable way of evaluating impurities, in addition to in vitro genotoxicity studies. Adequate data may be available in the scientiﬁc literature to qualify an impurity. Additional safety testing becomes necessary when safety data from the literature is not available and decreasing the level of the impurity below the threshold is not possible. Figure 1 is an illustration of the decision tree for consideration of Safety Studies for drug substances in NDAs.

VII. IMPURITIES IN DRUG PRODUCTS ICH Guidance covers the degradation product(s) of drug substances (New Molecular Entities, NMEs) in the ﬁnished drug product and reaction products of the drug substance with an excipient and/or immediate container/ closure system. Excluded are extraneous contaminants (these are covered by good manufacturing practices, or GMP), and process impurities present in the drug substance. In general, sponsors who conduct stability studies should propose limits for observed impurities/degradation products based on their experience in analyzing representative pilot batches (at least two) and the third one can be smaller if justiﬁed. Figure 2 is a summary of identiﬁcation and qualiﬁcation thresholds for impurities in drug products, from the ICH Guidance on how to ﬁle information necessary to support the application. During NDA ﬁling, the proposed limits should address . . .

Each specified degradation product Any unspecified degradation product Total degradation products

Adequacy of the proposed limits should be, where possible, evaluated using the principle of mass balance or material balance.

VIII. ANALYTICAL METHODOLOGY FOR IMPURITIES IN DRUG PRODUCT Many NDAs and ANDAs contain one assay method for the quantitation of the drug product and its impurities/degradation products. While consolidating the two tests can be advantageous to a Quality Control Laboratory, it should be noted that regulations do not limit the number of test methods that can be proposed for supporting an application. Indeed, the selection of one stability-indicating method for the drug product analysis with impurity quantitation capabilities can be challenging. The presence of excipients, the interaction of excipients with drug substance, and the drug substance’s degradation tendency make it difﬁcult to devise an optimum

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FIGURE 1 Decision tree for identification and qualification. (a) If considered desirable, a minimum screen (e.g., genotoxic potential) should be conducted. A study to detect point mutations and one to detect chromosomal aberrations, both in vitro, are considered an appropriate minimum screen. (b) If general toxicity studies are desirable, one or more studies should be designed to allow comparison of unqualified to qualified material. The study duration should be based on available relevant information and performed in the species most likely to maximize the potential to detect the toxicity of an impurity. On a case-by-case basis, single-dose studies can be appropriate, especially for single-dose drugs. In general, a minimum duration of 14 days and a maximum duration of 90 days would be considered appropriate. (c) Lower thresholds can be appropriate if the impurity is unusually toxic. For example, do known safety data for this impurity or its structural class preclude human exposure at the concentration present?

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THRESHOLDS FOR REPORTING OF DEGRADATION PRODUCTS IN NEW DRUG PRODUCTS Maximum Daily Dose1 1g >1g

Threshold3 0.1 % 0.05 %

THRESHOLDS FOR IDENTIFICATION OF DEGRADATION PRODUCTS IN NEW DRUG PRODUCTS Maximum Daily Dose1 < 1 mg 1 mg–10 mg 10 mg–2 g >2g

Threshold3 1.0% or 5 mg TDI2 whichever is lower 0.5% or 20 mg TDI whichever is lower 0.2% or 2 mg TDI whichever is lower 0.1%

THRESHOLDS FOR QUALIFICATION OF DEGRADATION PRODUCTS IN NEW DRUG PRODUCTS Maximum Daily Dose1 < 10 mg 10 mg–100 mg 100 mg–2 g >2g

Threshold3 1.0% or 5 mg TDI whichever is lower 0.5% or 20 mg TDI whichever is lower 0.2% or 2 mg TDI whichever is lower 0.1%

FIGURE 2 Description of different thresholds. 1The amount of drug substance administered per day. 2Total daily intake. 3Threshold is based on percent of the drug substance.

method, as is evidenced by the sheer number of high-performance liquid chromatography (HPLC) methods. HPLC is still the leading candidate for assay and impurity quantitation. Often, literature examples from other researchers may help in identifying suitable HPLC methods for drug product method development. When compendial methods or otherwise published methods are chosen for regulatory purposes, the method must still be shown to be suitable for the proposed use through validation. ICH Q2A and Q2B documents should be followed while validation is performed for quantitative impurities’ procedures. Often the inactive ingredients in dosage forms such as solutions, suspensions, and emulsions are themselves prone to degradation due to solvolysis and can contribute to impurities during shelf life evaluations. Impurity analytical proﬁles from scaled-up and laboratory scale batches should be compared with each other while ﬁnalizing NDA or ANDA speciﬁcations.

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IX. IMPURITIES QUANTITATION POST-APPROVAL Due to changes in the source of the drug substance, in the manufacturing process of the drug substance or drug product, impurity proﬁles can change for a marketed pharmaceutical product. NDA/ANDA impurity limits and test procedures should be reviewed and/or revised based on new ﬁndings in the form of supplemental applications to the Agency. Incorporation of the NDA speciﬁcations in a USP monograph can occur after the NDA is approved, to create a public standard for the drug substance and drug product. When this happens, the USP test methods and criteria will become ofﬁcial.

X. ROLE OF SPONSORS Pharmaceutical manufacturers ensure the safety and efﬁcacy of drug products by coupling the tight control of raw materials and manufacturing processes with well-executed toxicological and clinical trials for NDAs. Setting the right speciﬁcations for impurities is an evolving process in any drug product’s life cycle. When an Investigational New Drug Application (IND) is ﬁled, the proposed set of speciﬁcations for impurities is relatively tentative in nature. However, throughout Agency evaluation prior to NDA approval, the situation changes and additional well-deﬁned controls and limits may be added both by the sponsors and as a result of regulatory review of the process. The establishment of a USP monograph also affects the limits set on the impurities. Safety and efﬁcacy objectives regarding drug products are well recognized by pharmaceutical manufacturers. The manufacturers have been active in the development of the ICH Guidance and participate readily in the FDA’s Guidance development process. By providing valuable comments on draft Guidance (FDA and ICH), when solicited through Federal Register publications, the industry has assisted the Agency. Their interactions with the Agency to ensure the approval of new drug products in shorter time frames, and with the USP to develop ofﬁcial monographs, have beneﬁted the American Public. It is hoped that regulators, compendia, and industry will continue to stay on course to bring high-quality drug products to market.

XI. SUMMARY The United States Food and Drug Administration (FDA) has been involved with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). The guidance, developed through the joint efforts of regulators and industry representatives from the European Union, Japan and the United States, helps ensure that the different regions have consistent requirements for the data that should be submitted to the various agencies. This chapter reviews the existing information from the ICH Guidance, and compendia requirements with emphasis on impurities for the drug substance and the drug product.

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REFERENCES 1. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Steering Committee, July 2000, Q3B(R) Impurities in New Drug Products, and ANDAs: Impurities in Drug Products, Federal Register, 64, 2 (1/5/ 1999) 516 2. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Steering Committee, March 30, 1995, Q2A-Text on Validation of Analytical Procedures, and Q2B- Validation of Analytical Procedures: Methodology 3. This classification does not apply to biological/biotechnological, peptide, oligonucleotide, radiopharmaceutical, fermentation and semisynthetic products derived therefrom, herbal products, and crude products of animal or plant origin. 4. United States Pharmacopeia, 25, US Pharmacopeial Convention, Rockville, MD 2000, 7, General Notices 5. ANDAs: Impurities in Drug Substances, Federal Register, 64, 232, (12/3/1999), 67917 6. Gombar, V.K. and Enslein, K. Quant. Struct.-Act. Relat. 9:321–325, 1990.

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3 POLYMORPHIC AND SOLVATOMORPHIC IMPURITIES HARRYG. BRITTAINa AND ALES MEDEKb a

Center for Pharmaceutical Physics, Milford, NJ 08848

b

Pfizer, Inc.,Groton,CT 06340

1. INTRODUCTION II. X-RAY DIFFRACTION A. Single Crystal X-ray Diffraction B. X-ray Powder Diffraction III. THERMAL METHODS OF ANALYSIS A. Thermogravimetry B. Differential Scanning Calorimetry IV. VIBRATIONAL SPECTROSCOPY A. Infrared Absorption Spectroscopy B. Raman Spectroscopy C. Near-Infrared Spectroscopy V. SOLID-STATE NUCLEAR MAGNETIC RESONANCE (SS-NMR) SPECTROMETRY A. Solid-State NMR Techniques B. SS-NMR Studies of Polymorphism C. Quantitative Analysis of Polymorphs REFERENCES

1. INTRODUCTION It had been known since the eighteenth century that many substances could be obtained in more than one crystal form, and so the properties of these solids were studied to the fullest extent possible with the characterization tools (e.g., crystal morphology and melting phenomena) available at that time.1 Eventually the science of crystallography caught up with these studies, and quickly became the methodology of choice to study the various crystal forms of materials. The term polymorphism is the term used to denote crystal systems where a substance can exist in different crystal packing arrangements, all of which have the same elemental composition.2 It is also possible to have crystal systems where the substance exists in different crystal

39

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H.G. BRITTAIN AND A. MEDEK

packing arrangements, each of which has a different elemental composition, and this phenomenon is known as solvatomorphism.3 These phenomena have been discussed in detail.4,5 The pharmaceutical industry has been required by regulatory authorities to take a strong interest in polymorphism and solvatomorphism once it was realized that the nature of the structure adopted by a given compound upon crystallization would then exert a profound effect on the solid-state properties of that system. For a given material, the heat capacity, conductivity, volume, density, viscosity, surface tension, diffusivity, crystal hardness, crystal shape and color, refractive index, electrolytic conductivity, melting or sublimation properties, latent heat of fusion, heat of solution, solubility, dissolution rate, enthalpy of transitions, phase diagrams, stability, hygroscopicity, and rates of reactions, were all affected by the nature of the crystal structure. It is usually the goal in pharmaceutical manufacturing to produce a drug substance that is phase-pure and which remains in that state as long as the bulk material is stored. It is also a goal to formulate the drug substance in a manner so that it remains in the same phase-pure state during the manufacture of the drug product and during any subsequent storage. This requires the development and validation of assay methodology for the determination of phase composition. The use of suitable techniques for the physical characterization of polymorphic and solvatomorphic solids has been discussed before,6–10 but these works usually focused on the study of the phase present in the largest extent. In this chapter, the question of small amounts of polymorphic and solvatomorphic impurities in a bulk solid will be addressed. The most useful methods for performing such work will be outlined, and illustrated with appropriate examples.

II. X-RAY DIFFRACTION Since polymorphism and solvatomorphism are crystallographic phenomena, it follows that the most important techniques for their detection and any quantiﬁcation must be crystallography based. The technique of X-ray diffraction is such a method since it represents direct methodology whereby fundamental structural information can be obtained on the structure of a crystalline substance. For example, it is only by pure coincidence that two compounds might form crystals in which the three-dimensional spacing of planes is identical in all directions. One such example is provided by the trihydrate phases of ampicillin and amoxicillin,11 but such instances are uncommon. Typical applications of X-ray diffraction methodology include the determination of crystal structures, evaluation of polymorphism and solvate structures, evaluation of degrees of crystallinity, and the study of phase transitions.12 Bragg13 explained the diffraction of X-rays by crystals using a model where the atoms of a crystal are regularly arranged in space, and that they can be regarded as lying in parallel sheets separated by a deﬁnite and deﬁned distance. Then he showed that scattering centers arranged in a plane act like

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3 POLYMORPHIC AND SOLVATOMORPHIC IMPURITIES

a mirror to X-rays incident on them, so that constructive interference would occur for the direction of specular reﬂection. Within a given family of planes, deﬁned by a Miller index of (h k l) and each plane being separated by the distance (d), each plane produces a specular reﬂectance of the incident beam. If the incident X-rays are monochromatic (having wavelength equal to ), then for an arbitrary glancing angle of , the reﬂections from successive planes are out of phase with one another. This yields destructive interference in the scattered beams. However, by varying , a set of values for can be found so that the path difference between X-rays reﬂected by successive planes will be an integral number (n) of wavelengths, and then constructive interference will occur. Hence one ultimately obtains an expression known as Bragg’s law that explains the phenomenon: 2d sin ¼ n

ð1Þ

Unlike the case of diffraction of light by a ruled grating, the diffraction of X-rays by a crystalline solid leads to observation of constructive interference (i.e., reﬂection), which occurs only for the critical Bragg angles. When reﬂection does occur, it is stated that the plane in question is reﬂecting in the nth order, or that one observes nth order diffraction for that particular crystal plane. Therefore one will observe an X-ray scattering response for every plane deﬁned by a unique Miller index of (h k l).

A. Single Crystal X-ray Diffraction The analysis of single crystal X-ray diffraction data is divided into three parts.14,15 The ﬁrst of these is the geometrical analysis, where one measures the exact spatial distribution of X-ray reﬂections and uses them to compute the size and shape of a unit cell. The second phase entails a study of the intensities of the various reﬂections; this information is used to determine the atomic distribution within the unit cell. Finally, one looks at the X-ray diagram to deduce qualitative information about the quality of the crystal or the degree of order within the solid. This latter analysis may permit the adoption of certain assumptions that may aid in solving of the crystalline structure. The phenomenon of X-ray diffraction has found widespread use as a means to determine the structures of single crystals and represents the most powerful and direct method to obtain bond lengths and bond angles for molecules in the solid state. This information is of extreme importance to workers in pharmaceutics when they encounter the existence of polymorphism or solvatomorphism. There is no doubt that single crystal X-ray diffraction is a powerful technique for the study of polymorphs and solvatomorphs, but it is equally apparent that this methodology is not well suited for the routine evaluation of the crystalline state of powdered solids. It is deﬁnitely not a technique that one would want to use in the determination of polymorphic phase impurities in a sample, since by deﬁnition only a single crystal is studied at one time. That crystal would certainly be phase-pure, and its quantitative analysis not

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H.G. BRITTAIN AND A. MEDEK

subject to analysis. However, the powder pattern calculated from the single crystal structure would be of great use in interpreting the powder patterns of real samples.

B. X-ray Powder Diffraction For the crystallographic analysis of phase impurities, X-ray powder diffraction (XRPD) is deﬁnitely the method of choice.12 Since a powdered sample will present all possible crystal faces of all crystalline components at a given interface, the diffraction off this powdered surface will therefore provide information on all possible atomic spacings (i.e., deﬁned by the crystal lattice). The powder pattern consists of a series of peaks having varying intensities detected at various scattering angles. These angles, and their relative intensities, are correlated with computed d-spacings to provide a full crystallographic characterization of the powdered sample.16,17 To measure a powder pattern, a randomly oriented powdered sample is prepared so as to expose all the planes of a sample. The scattering angle is determined by slowly moving the sample and measuring the angle of diffracted X-rays (typically using a scintillation detector) with respect to the angle of the incident beam. Alternatively, the angle between sample and source can be kept ﬁxed while moving the detector to determine the angles of the scattered radiation. Knowing the wavelength of the incident beam, the spacing between the planes (identiﬁed as the d-spacings) is calculated using Bragg’s Law. In a mixture of crystalline materials, each species will be characterized by its distinctive series of scattering peaks, and the observed powder pattern will consist of a sum of the individual components. The intensities of the peaks associated with each component are proportional to its weight fraction in the mixture, so the performance of a quantitative analysis is based on the accurate and reproducible measurement of peak intensities. The analysis can be performed either with or without an international standard, and the theory associated with each approach has been set forth by Suryanarayanan12 and others.18–21 Quantitative XRPD was used to determine the relative component amounts of carbamazepine anhydrate and its dihydrate phase in powdered mixtures of these.22 Each form was characterized by a unique d-spacing (6.78 A˚ or 13.05 degrees 2- for the anhydrate, and 9.93 A˚ or 8.90 degrees 2- for the dihydrate), as shown in Figure 1, and the ratio of the integrated intensities of these scattering peaks could be correlated to the amount of Form-I in the mixture. In this work, the effects on the results that could be caused by preferred orientation, extinction, and particle size were discussed. A proﬁle analysis method of ﬁtting has been used to determine the relative amounts of prazosin hydrochloride polymorphs (the and forms) in powdered mixtures.23 The method was calibrated over -prazosin levels of 0.5–10% using a series of calibration samples, and it was found that the limit of detection was equal to 0.5%. The favorable limit was attainable owing to signiﬁcant regions of non-overlap in the powder patterns of the two forms.

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43

FIGURE 1 Portions of the X-ray powder patterns of carbamazepine anhydrate (upper trace) and dihydrate (lower trace), illustrating the strong scattering peak associated with the dihydrate phase at 8.90 degrees 2-h (9.93 —).The figure was adapted from data presented in reference 22.

The quantitation of cefepine dihydrochloride dihydrate in samples of cefepine dihydrochloride monohydrate has been performed using quantitative XRPD.24 In this study, the working range was established as 1.0–8.0% w/w, with a detection limit of 0.75% and a quantitation limit of 2.5%. In this work, these workers overcame the difﬁcult problem of mixing reference materials whose component crystals were of very different morphologies, which represents a problem not often recognized in work of this type. Since the compound was insoluble in acetone, the reference materials were slurried in the solvent, mixed thoroughly, and allowed to dry. It is evident from the electron microscopic images shown in the paper that very homogeneous powder blends were obtained using the slurry approach.

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H.G. BRITTAIN AND A. MEDEK

In a system having attained great notoriety owing to patent litigations, excellent sensitivity has been obtained in the use of quantitative XRPD for the quantitation of the polymorphs of ranitidine.25 Owing to signiﬁcant nonoverlap, diffraction peaks suitable for the determination of either Form-I in bulk Form-II, or for Form-II in bulk Form-I, are easily identiﬁed. Quantitative XRPD has been used to study the effects of temperature on the phase transformation of two forms of chlorpropamide during tableting.26 Although these authors did not use an internal standard, they were able to generate calibration curves by taking ratios of a characteristic scattering peak for each form. It was learned that the stability of Form A was affected by compression temperature, while that of Form C was independent of temperature. The pathways of polymorphic transitions for cephalexin, chloramphenicol palmitate, and indomethacin have been studied, and the composition of partially transformed samples determined by XRPD.27 The metastable forms of chloramphenicol palmitate were found to transform into the stable phase when found at room temperature, conﬁrming the results of a previous study.28 In contrast, cephalexin became amorphous upon grinding. Indomethacin exhibited the interesting behavior of becoming amorphous when ground at 4 C, but could be transformed into a metastable phase when ground at 30 C. The kinetics associated with the thermally induced phase transformations of phenanthrene and caffeine monohydrate were studied using hotstate quantitative XRPD.29 Using a single non-isothermal experiment conducted at a constant heating rate, it was possible to obtain the activation parameters for the solid-state reactions. In another study, quantitative XRPD was used to study the tetrahydrate to monohydrate transition of the sodium salt of 5-(4-oxo-phenoxy-4H-quinolizine-3-carboxamide)-tetrazolate.30 Although not a polymorphic phase in the formal sense, the amorphous state can represent an important quantity of measure in a crystalline sample. A general XRPD procedure for such work has been described,31 and similar approaches have been reported for cilastatin sodium,32 caffeine,33 imipenem,34 acetaminophen,35 and a Lumaxis analog.36 It is well known that resolved enantiomers and the racemic mixtures of dissymmetric substances ordinarily crystallize in different space groups,37 so it is natural to use quantitative XRPD as a means to determine the enantiomeric composition. This approach was demonstrated in the case of ibuprofen, taking advantage of the differing powder patterns of the resolved enantiomers and the racemate.38 The detection limits for either form (in the presence of the other form) were very similar, being approximately 3.3% w/w.

III. THERMAL METHODS OF ANALYSIS Thermal analysis methods are deﬁned as those techniques for which one or more properties of a sample are determined as a function of an externally

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45

applied temperature. Regardless of the observable parameter measured, the usual practice requires that the physical property and the sample temperature be recorded continually and automatically, and that the sample temperature is altered at a predetermined rate. Measurements of thermal analysis are conducted for the purpose of evaluating the physical and chemical changes that may take place in a heated sample, requiring that the operator interpret the events noted in a thermogram in terms of plausible reaction processes. Thermal reactions can be identiﬁed as being either endothermic (melting, boiling, sublimation, vaporization, desolvation, solid–solid phase transitions, chemical degradation, etc.) or exothermic (crystallization, oxidative decomposition, etc.) in nature. The various thermal methods in common use have been discussed at great detail elsewhere.39–40 Such methodology has found widespread use in the pharmaceutical industry for the characterization of compound purity, polymorphism, solvation, degradation, and excipient compatibility.66,67 However, given the utility which thermal microscopy has shown for the characterization of polymorphic systems, it is not surprising that the quantitative applications of thermal analysis have proven to be even more useful. Although a large number of techniques have been developed, the most commonly applied are those of thermogravimetry (TG), differential thermal analysis (DTA), and differential scanning calorimetry (DSC).

A. Thermogravimetry Thermogravimetry is a measure of the thermally induced weight loss of a material as a function of the applied temperature.44–46 TG analysis is restricted to transitions that involve either a gain or loss of mass and is most commonly used to study desolvation processes and compound decomposition. TG analysis is a very useful method for the quantitative determination of the total volatile content of a solid and can be used as an adjunct to Karl Fischer titrations for the determination of moisture. As such, it readily permits the distinction between solvates and the anhydrous forms of a given compound. When performed in conjunction with an auxiliary technique (such as gas chromatography or infrared spectroscopy), one may also obtain compound-speciﬁc decomposition information. Thermogravimetry analysis also represents a powerful adjunct to the other methods of thermal analysis, since a combination of either a DTA or DSC study with a TG determination can be used in the assignment of observed thermal events. Desolvation processes or decomposition reactions must be accompanied by weight changes, and can be identiﬁed by a TG weight loss over the same temperature range. On the other hand, solid–liquid or solid–solid phase transformations are not accompanied by any loss of sample mass and would not register in a TG thermogram. When a solid is capable of decomposing by means of several discrete, sequential reactions, the magnitude of each step can be separately evaluated. The TG analysis of compound decomposition can also be used to compare the stability of similar compounds. In general, the higher the decomposition temperature of a given compound, the greater would be its stability.

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H.G. BRITTAIN AND A. MEDEK

The measurement of thermogravimetry is simple in principle and consists of the continual recording of the mass of the sample as it is heated in a furnace. The weighing device used in most instruments is a microbalance, which permits the characterization of milligram quantities of sample. The balance chamber itself is constructed so that the atmosphere may be controlled, which is normally accomplished by means of a ﬂowing gas stream. The furnace must be capable of being totally programmable in a reproducible fashion, whose inside surfaces are resistant to the gases evolved during the TG study. It is most essential in TG design that the temperature readout be that of the sample, and not to that of the furnace. To achieve this end, the thermocouple or resistance thermometer must be mounted as close to the sample pan as possible. Although the TG methodology is conceptually simple, the accuracy and precision associated with the results are dependent on both instrumental and sample factors. The furnace heating rate used for the determination will greatly affect the transition temperatures, while the atmosphere within the furnace can inﬂuence the nature of the thermal reactions. The sample itself can play a role in governing the quality of data obtained, with factors such as sample size, nature of evolved gases, particle size, heats of reaction, sample packing, and thermal conductivity all inﬂuencing the observed thermogram. Other than its ability to demonstrate the anhydrous nature of genuine polymorphic materials, one extremely useful aspect of TG analysis is in the differentiation and characterization of solvatomorphs. The methodology is particularly useful in the determination of a solvate phase in the presence of its anhydrate phase, or vice versa. The most commonly encountered form of lactose is its -anomer, which is obtained in the form of a monohydrate phase. The anhydrous form of the -anomer is known to be very hygroscopic and difﬁcult to either obtain or handle. The -anomer of lactose is obtained as an anhydrate phase, which apparently has no tendency to form any hydrate phases. The thermal properties of these materials have been discussed.47 The theoretical water content for the -monohydrate phase is 5.0% w/w, so in principle one can use thermogravimetry to determine either the anhydrate phase content in a largely monohydrate sample or the monohydrate phase content in a largely anhydrous sample. To illustrate the utility of TG analysis for such determinations, a series of calibration samples were prepared at levels approximately 90% or 10% in the monohydrate phase, using anhydrous -lactose as a diluent in bulk -monohydrate.48 These were subjected to a standard TG evaluation, and the results of the analysis are shown in Table 1. The results indicate that the TG method yields reasonable estimations of the phase composition, but that the difference between measured and found compositions could be unacceptably large for some applications. The data appear to indicate the existence of a small positive bias, which was found to be worse in the 10% monohydrate samples. In a properly controlled and validated system, however, it is highly likely that method limitations could be overcome and TG analysis appropriately used for quantitative analysis of phase composition.

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TABLE 1 Thermogravimetric Analysis of Lactose Calibration Standards 48

Actual weighed composition of sample, expressed as monohydrate (%)

Anticipated TG weight loss of sample (%)

Experimentally measured TG weight loss (%)

Calculated monohydrate content, based on weight loss of 5.0% for the monohydrate (%)

(a) Approximately 90% monohydrate phase 89.8

4.490

4.58

91.60

88.4

4.420

4.34

86.80

92.3

4.615

4.72

94.40

91.1

4.555

4.63

92.60

92.6

4.630

4.71

94.20

(b) Approximately 10% monohydrate phase 9.8

0.490

0.68

13.60

11.4

0.570

0.73

14.60

12.8

0.640

0.88

17.60

10.9

0.545

0.75

15.00

8.7

0.435

0.59

11.80

B. Differential Scanning Calorimetry Differential thermal analysis was the ﬁrst major improvement developed over simple melting point analysis, and in countless studies was used to determine the characteristic temperature ranges associated with a variety of thermally induced reactions. Differential scanning calorimetry subsequently effectively replaces the DTA method, primarily because of its ability to yield quantitative information regarding the magnitude of the heat associated with the thermal reaction. For this reason, DSC has become accepted as the most widely used method of thermal analysis for the pharmaceutical industry. In the DSC method, the sample and reference materials are maintained at the same temperature, and the heat ﬂow required to keep the equality in temperature is measured. DSC plots are therefore obtained as the differential rate of heating (in units of watts/second calories/second or joules/second) against temperature.39,40 The area under a DSC peak is directly proportional to the heat absorbed or evolved by the thermal event, and integration of these peak areas yields the heat of reaction (in units of calories/secondgram or joules/secondgram). Two types of DSC measurements are possible, which are usually identiﬁed as power-compensation DSC and heat-ﬂux DSC, and the details of each conﬁguration have been fully described.39,40 In power-compensated DSC, the sample and reference materials are kept at the same temperature by the use of individualized heating elements, and the observable parameter recorded is the difference in power inputs to the two heaters. In heat-ﬂux

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H.G. BRITTAIN AND A. MEDEK

DSC, one simply monitors the heat differential between the sample and reference materials, with the methodology not being terribly different from that used for DTA. In the DTA measurement, an exothermic reaction is plotted as a positive thermal event, while an endothermic reaction is usually displayed as a negative event. Unfortunately, the use of power-compensation DSC results in endothermic reactions being displayed as positive events, a situation which is counter to the IUPAC recommendations.49 When the heat-ﬂux method is used to detect the thermal phenomena, the signs of the DSC events concur with those obtained using DTA, and also agrees with the IUPAC recommendations. The calibration of DSC instruments is normally accomplished through the use of compounds having accurately known transition temperatures and heats of fusion, and an extraordinarily extensive list of suitable reference materials is available.50 Once the DSC system is properly calibrated, it is trivial to obtain the melting point and enthalpy of fusion data for any compound upon integration of its empirically determined endotherm and application of the calibration parameters. The current state of methodology is such, however, that unless a determination is repeated a large number of times, the deduced enthalpies must be regarded as being accurate only to within approximately 5%. The DSC thermograms of a number of lactose monohydrate samples were determined, and it was ultimately found that the dehydration endotherm was characterized by an enthalpy of desolvation equal to 150.5 J/g.48 This value for the pure monohydrate phase can then be used in conjunction with the measured desolvation enthalpies of actual samples to determine either the anhydrate phase content in a monohydrate sample or the monohydrate phase content in an anhydrate sample. To illustrate the utility of DSC analysis for such determinations, a series of calibration samples were prepared at levels approximately 90% or 10% in the monohydrate phase, using anhydrous -lactose as a diluent in bulk -monohydrate.48 These were subjected to a standard DSC evaluation, and the results of this analysis are shown in Table 2. The results indicate that the DSC method was able to provide very good estimations of the phase composition. It is concluded that the magnitude of the error associated with the use of DSC analysis would be acceptable for quantitative analysis of mixtures of lactose monohydrate and anhydrate. As long as the two forms of the analyte do not interconvert during acquisition of the DSC thermogram, and values for the transition enthalpies have been determined for reference materials, one can always use DSC as a quantitative method for the determination of phase composition. The method will fail, however, when a metastable form converts to a more stable form during the DSC analysis. It is therefore always prudent to establish the phase relationship and order of stability between the various forms51 before undertaking development of a quantitative DSC method. A very interesting procedure has been described where DSC is used to determine the water content in hydrates.52 The method is based on the assumption that the enthalpy of binding associated with n moles of water in

49

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TABLE 2 Differential Scanning Calorimetric Analysis of Lactose Calibration Standards 48 Actual weighed composition of sample, expressed as monohydrate (%)

Anticipated DSC enthalpy of desolvation for sample (J/g)

Experimentally measured enthalpy of desolvation (J/g)

Calculated monohydrate content, based on an enthalpy of desolvation of 150.5 J/g (%)

(a) Approximately 90% monohydrate phase 91.4

137.56

137.9

91.63

90.2

135.75

135.0

89.70

88.7

133.49

133.0

88.37

89.3

134.40

133.4

88.66

87.2

131.24

131.9

87.64

(b) Approximately 10% monohydrate phase 8.7

13.09

12.5

8.31

10.2

15.35

14.9

9.90

11.8

17.76

17.2

11.43

9.6

14.45

15.0

9.97

9.3

14.00

14.7

9.77

the hydrate is the same as that associated with n moles of water in bulk water. Knowing the enthalpy of vaporization for liquid water and the enthalpy of desolvation for the hydrate sample enables a facile calculation of the number of moles of water bound in the hydrate. The method was applied to the hydrates of ampicillin, carbamazepine, caffeine, theophylline, and metal salts of nedocromil with good agreement between known hydration numbers and those calculated using the DSC method.

IV. VIBRATIONAL SPECTROSCOPY The energies associated with the vibrational modes of a chemical compound lie within the range of 400–4000 cm1. These modes can be observed directly through their absorbance in the infrared region of the spectrum or through the observation of the low-energy scattered bands that accompany the passage of an intense beam of light through the sample (the Raman effect). In either case, the use of Fourier transform methodology has vastly improved the quality of data that can be obtained.53 Most workers are familiar with the use of mid-infrared spectra for identity purposes, where the pattern of absorption bands is taken to be diagnostic for a given compound. However, it has come to be recognized that the vibrational spectra of solid materials will reﬂect details of the crystal structure, and hence these methods can be used in the spectroscopic investigation of polymorphs and solvates.54–56 In studies of polymorphic or solvatomorphic systems, the purpose of the vibrational spectroscopic investigation should be to gather information from the observed pattern of vibrational frequencies and to use this data to

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H.G. BRITTAIN AND A. MEDEK

understand the structural aspects that yield crystallographic differences. Once suitable spectral features are identiﬁed from this work, they can be used to develop easily performed methods for the quantitative analysis of one polymorph (or solvatomorph) in the presence of the other. Unfortunately, too many workers are satisﬁed to merely obtain the spectra of the various polymorphs and/or solvatomorphs and to simply display the differences. In doing so, they miss a great opportunity to gain additional understanding about the system they are trying to characterize.

A. Infrared Absorption Spectroscopy The acquisition of high-quality infrared absorption spectra appropriate for the characterization of polymorphs and solvates is best performed using Fourier transform technology (the FTIR method), since this approach minimizes transmission and beam attenuation problems. Essentially all FTIR spectrometers use a Michelson interferometer, where radiation entering the interferometer is split into two beams by means of a beam splitter. One beam follows a path of ﬁxed distance before being reﬂected back into the beam splitter, while the other beam travels a variable distance before being recombined with the ﬁrst beam. The recombination of these two beams yields an interference pattern, where the time-dependent constructive and destructive interferences have the effect of forming a cosine signal. Each component wavelength of the source will yield a unique cosine wave, having a maximum at the zero pathlength difference (ZPD) and which decays with increasing distance from the ZPD. The detector is placed so that radiation in the central image of the interference pattern will be incident upon it, and therefore intensity variations in the recombined beam manifest as phase differences. The observed signal at the detector is a summation of all the cosine waves, having a maximum at the ZPD, and which decays rapidly with increasing distance from the ZPD. If the component cosine waves can be resolved, then the contribution from individual wavelengths can be observed. The frequency domain spectrum is obtained from the interferogram by performing the Fourier transformation mathematical operation. More detailed descriptions of FTIR instrumentation and its methodology is available.57 The acquisition of solid-state FTIR spectra suitable for use in the characterization of polymorphic impurities is performed using either the Nujol mull technique, diffuse reﬂectance (DRIFT), or attenuated total reﬂectance (ATR). One should avoid the use of pelleting techniques to eliminate any spurious effects associated with compaction of the KBr pellet. The simplest approach is to prepare a mull of the sample in mineral oil, sandwich this between salt plates, and measure the spectrum using ordinary transmission techniques. The main drawback of the mull technique is that regions in the IR spectrum overlapping with carbon–hydrogen vibrational modes will be obliterated because of absorbance from the oil. A quantitative IR absorption for the two polymorphs of the developmental compound (2R,3S)-2-({(1R)-1-[3,5-bis(triﬂuoromethyl)phenyl] ethyl}oxy)-3-(4-ﬂuorophenyl) morpholine hydrochloride was reported in which the measurements were made in mineral oil mull preparations.58

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51

The mull technique was used to ensure compatibility with wet cake and slurry samples. A calibration curve was established for binary mixtures of the two forms using only an absorbance ratio of two characteristic absorption bands, and a limit of quantitation of 2.7% w/w was established. The method was then applied to the kinetic investigation of the transformation from the metastable form to the stable form. The measurement of diffuse reﬂectance effectively involves focusing the infrared source beam onto the surface of a powder sample and using an integrating sphere to collect the scattered infrared radiation.59 The technique requires careful attention to sample preparation, and often one must dilute the analyte with KBr powder to reduce the occurrence of anomalous effects.60 In practice, one obtains the spectrum of the ﬁnely ground KBr dispersant, and then ratios this to the spectrum of KBr containing the analyte. The relative reﬂectance spectrum is converted into Kubelka–Munk units using standard equations,61 thus obtaining a diffuse reﬂectance spectrum that resembles a conventional IR absorption spectrum. Guillory and coworkers were among the ﬁrst to use diffuse reﬂectance in conjunction with FTIR technology and the partial least-squares (PLS) regression method.62 The particle size of the reference materials was ﬁrst homogenized through light grinding in an agate mortar, and then calibration samples prepared by thorough mixing. The spectra of these samples, and their dilutions in micronized KCl were obtained under a variety of conditions to investigate the robustness of the process. As shown in Figure 2, good linearity was found between the Kubelka–Munk units and the composition of the calibration samples. When using the PLS method good agreement between the actual and predicted values of the calibration samples was obtained, with the average absolute error being 0.006% in KCl for Form-I and 0.004% in KCl for Form-II. In addition to the XRPD quantitation of cefepine dihydrochloride dihydrate in samples of cefepine dihydrochloride monohydrate, diffuse reﬂectance infrared spectroscopy was used to determine the solvatomorphic composition.24 For the IR assay, a working range of 1.0–8.0% w/w, with a detection limit of 0.3% and a quantitation limit of 1.0%, and these analytical limits were superior to those obtained using the quantitative XRPD method. The validity of both assay methods were limited for samples having a particle size range of 125–590 mm. Counting polymorphs and solvatomorphs, delavirdine mesylate has been found in 12 different crystal forms, therefore presenting considerable difﬁculty in the determination of phase composition.63 Only through the combination of factor analysis with the quantitative IR technique could one develop a method useable in the characterization of research and production lots. The composition of drug lots consisting of mixtures of forms was identiﬁed through qualitative factor analysis, and quantitated using principal component analysis. Calibration models were developed for the determination of Form-VIII or Form-XII in Form-XI, and a standard error of prediction of 2.0% of either form was reported, with detection limits of 3–5%. Diffuse reﬂectance IR spectroscopy has been used, in conjunction with XRPD analysis, to determine the levels of ranitidine Form-II in samples of

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FIGURE 2 Infrared absorption intensity (expressed in terms of Kubelka^Munk units) of

sulfamethoxazole Form-I (absorbance at 3468 cm1) and Form-II (absorbance at 3476 cm1) as a function of concentration in a KCl matrix.The figure was adapted from data presented in reference 62.

bulk Form-I.25 Three artiﬁcial neural network models were trained and used to enhance the analysis. Although the model constructed from the XRPD data was superior in predictions at lower concentrations of Form-II (1% and 2%), the model constructed using diffuse IR reﬂectance had better overall average prediction. The ATR method of detection is emerging as a highly useful approach to obtaining IR absorption spectra with minimal sample preparation.64 If an IRtransparent crystal is sandwiched by the sample and if the refractive index of the crystal exceeds that of the sample, at certain orientations an infrared beam entering the crystal will undergo multiple internal reﬂections. At each reﬂection, some of the incident energy is absorbed by the vibrational modes of the sample, and the degree of this absorption builds with the number of internal reﬂections. When the beam ﬁnally emerges from the crystal, it can be processed in the usual way to obtain a pattern of the IR absorption bands of the sample in contact with the crystal. The feasibility of using FTIR–ATR spectroscopy for qualitative and quantitative analysis was investigated using the three known polymorphs of ganciclovir as a model system.65 Deﬁnitive identiﬁcation of each polymorph was obtained from materials that did not need to be subjected to any sample handling or preparation. Quantitation of mixtures was carried out using a partial least-squares procedure, with mean absolute errors of less than 3% being reported for Form-I and Form-II, and about 6% for Form-III.

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B. Raman Spectroscopy The vibrational modes of a compound may also be studied using Raman spectroscopy, where one measures the inelastic scattering of radiation by a nonabsorbing medium.66 When a beam of light is passed through a material, approximately one in every million incident photons is scattered with a loss or gain of energy. The inelastically scattered radiation can occur at lower (Stokes lines) and higher (anti-Stokes lines) frequencies relative to that of the incident (or elastically scattered) light, and the energy displacements relative to the energy of the incident beam correspond to the vibrational transition frequencies of both the medium. The actual intensities of the Stokes and anti-Stokes lines are determined by the Boltzmann factor characterizing the vibrational population. For high-frequency vibrations, the Stokes lines are somewhat intense relative to the anti-Stokes lines, so conventional Raman spectroscopy makes exclusive use of the Stokes component. The Raman effect originates from the interaction of the oscillating induced polarization or dipole moment of the medium with the electric ﬁeld vector of the incident radiation. Raman spectra are measured by passing a laser beam through the sample and observing the scattered light either perpendicular to the incident beam or through back-scatter detection. The scattered light is analyzed at high resolution by a monochromator, and ultimately detected by a suitable device. One key to obtaining good spectra is through the use of a notch ﬁlter, which will eliminate the exciting line, since that is required to obtain acceptable signal-to-noise ratios. Although both infrared absorption and Raman scattering yield information on the energies of the same vibrational bands, the different selection rules governing the band intensities for each type of spectroscopy can yield useful information. For the low-symmetry situations presented by the structures of molecules of pharmaceutical interest, every vibrational band will be active to some degree in both infrared absorption and Raman scattering spectroscopies. The relative intensities of analogous bands will differ, however, when observed by either infrared absorption or Raman spectroscopy. In general, symmetric vibrations and nonpolar groups yield the most intense Raman scattering bands, while antisymmetric vibrations and polar groups yield the most intense infrared absorption bands. A discussion on a large number of practical applications of Raman spectroscopy is available,67 and its application to pharmaceutical analysis has been discussed.68,69 In one study, Raman spectroscopy was used to analyze binary mixtures of the two forms of chlorpropamide.70 A complete series of calibration samples ranging from 0% to 100% in each form were prepared, and characterized between 400–1800 cm1 and 2800–3200 cm1. Through the use of factor analysis, good correlation of predicted versus theoretical polymorphic composition was obtained in studies of instrumental and between-sample reproducibilities. An unidentiﬁed compound of pharmaceutical interest was reported to be able to exist in two polymorphic forms, each of which exhibited distinctive Raman spectra.71 A series of calibration samples were prepared over the Form-A range of 1.8–15.4% w/w in Form-B, and a calibration curve developed using the characteristic scattering bands at 1716 cm1 (Form-A)

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FIGURE 3 Concentration profiles measured by in situ Raman spectroscopy during the

phase transformation of progesterone Form-II () to Form-I () The figure was adapted from data presented in reference 73.

and 1724 cm1 (Form-B). Using spectral subtraction, it was determined that as little as 1.8% of one form could be detected in the presence of the other. The solvent-mediated polymorphic transformation of progesterone Form-II to Form-I was followed in real time using in situ Raman spectroscopy.72 Although Form-I is the thermodynamically stable form, the spectroscopic investigation showed that its appearance is always preceded by the formation of Form-II, which appeared to be kinetically favored. In a kinetic study (plotted in Figure 3), the time dependence of the phase transformation at 45 C was followed and was used to determine the end point of the transformation. The advantage of Raman spectroscopy to provide real-time evaluations of progress of phase transformation was found to be signiﬁcant when compared to the use of other off-line techniques. The in situ Raman spectroscopy method has also been used to study the particle size–dependent molecular rearrangements that take place during the dehydration of trehalose dihydrate.73 Different phases were sieved into fractions < 45-mm and > 425-mm particle size, and the Raman spectra obtained at various times during an isothermal heating at 80 C. After being heated for 210 minutes, the < 45-mm dihydrate material appeared to become amorphous while the > 425-mm dihydrate material transformed into the crystalline anhydrate phase. Ratios of various characteristic scattering peaks were used to follow the kinetics of the phase transformations.

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Raman spectroscopy has been shown to be a useful method for the determination of degrees of crystallinity and can complement other quantitative methods for such work.74 This was demonstrated using indomethacin as an example, with the quantitative analysis being based on the peak intensity ratios of the 1698 cm1 (crystalline) and 1680 cm1 (amorphous) scattering peaks. A linear correlation curve was obtained across the entire 0–100% range of crystallinity, and as little as 1% amorphous or crystalline phase content could be detected. The largest source of error was found to result from inhomogeneous mixing of the calibration standards, a phenomenon commonly encountered during the validation of quantitative assay methods for powdered solids.

C. Near-Infrared Spectroscopy The absorption bands found in the near-infrared (NIR) region of the spectrum (typically 1000–2500 nm) are all due to overtones and combinations of fundamental molecular vibrational modes.75 The NIR spectrum tends to be dominated by vibrational modes of light atoms having strong bond strengths, typically hydrogen bound to nitrogen, oxygen, or carbon. The energies of the overtone bands are more affected by environmental details than are the energies of their fundamentals, so slight perturbations in the bonding can yield drastic frequency and amplitude changes in the nearinfrared. Discussions of the pharmaceutical applications of NIR spectroscopy are available.76–77 Although the molar absorptivities of these bands tend to be rather small, the instrumental state of the art, combined with superior data deconvolution routines, has progressed to the point where their measurement is relatively straightforward.78 The most important experimental requirements are the use of high-intensity stable light sources, low-noise sensitive detectors, and efﬁcient methods for collection of the diffuse reﬂectance of the measurement. NIR spectroscopy became much more useful when the principle of multiple-wavelength spectroscopy was combined with the deconvolution methods of factor and principal component analysis. In typical applications, partial least squares regression is used to model the relation between composition and the NIR spectra of an appropriately chosen series of calibration samples, and an optimal model is ultimately chosen by a procedure of cross-testing. The performance of the optimal model is then evaluated using the normal analytical performance parameters of accuracy, precision, and linearity. Since its inception, NIR spectroscopy has been viewed primarily as a technique of quantitative analysis and has found major use in the determination of water in many pharmaceutical materials. Near-infrared spectroscopy was used to quantitatively determine the phase composition of a compound capable of existing in two polymorphic forms even though the NIR spectra were fairly similar.79 Using a ﬁvewavelength calibration model, nearly 100% recovery was obtained in a series of spiked calibration samples, with relative standard deviation values ranging from 0.1% to 0.9%. The authors concluded that the use of NIR spectroscopy

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FIGURE 4 Near-infrared spectra of a-lactose monohydrate (solid trace) and b-lactose anhydrate (dashed trace), highlighting the strong water combination band at 1940 nm. The figure was adapted from data presented in reference 80.

had a major advantage in that it required large quantities of material (1 g or more) for the measurements, thus minimizing sampling errors. In a wide-ranging study, NIR spectroscopy was used to quantitate the phase composition in various forms of sulfamethoxazole, sulfathiazole, lactose, and ampicillin.80 For instance, as shown in Figure 4, the -monohydrate phase of lactose is easily distinguished from -lactose anhydrate on the basis of the characteristic band at 1940 nm associated with the water combination mode. In all cases, however, properly calibrated NIR methods were able to yield good predictions of phase composition relative to the actual composition of the standards used, and it was concluded that the quantitative NIR was equally effective for such work as other commonly used quantitative methods. Near-infrared spectroscopy has been used to quantitate sulfathiazole Form-I and Form-III in binary physical mixtures in which one form was the dominant component.81 The spectra of each form exhibited sufﬁcient differences that unique wavelengths of absorbance were easily attributable to each form. Excellent linearity in calculated versus actual compositions were obtained over the concentration range of 0–5% for either Form-I in Form-III, or for Form-III in Form-I. After considering appropriate calibration models, a limit of quantitation of approximately 0.3% was ultimately deduced. It is relatively straightforward to apply the NIR method for in situ characterization work, as was reported in the case of the EFGR tyrosine kinase inhibitor 4-(3-ethynylphenylamino)-6,7-bis(2-methoxyethoxy)quinazolinium methanesulfonate.82 NIR spectroscopy was used to monitor the kinetics of transformation between the polymorphs and solvatomorphs, and

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even facilitated the discovery of a new preferred form. It was determined that the in situ NIR method could be generally used in the study of practically all types of two-phase solid–liquid slurries maintained under isothermal conditions. Near-infrared spectroscopy was used to study the solvatomorphic changes and the state of water during the wet granulation of theophylline.83 The anhydrate was granulated in a planetary mixer using water as granulation liquid and characterized by a variety of methods. At a low level of granulation ﬂuid (0.3 moles of water per mole of theophylline anhydrate), water absorption ﬁrst yielded NIR maxima (1475 and 1970 nm) characteristic of theophylline monohydrate. At higher quantities of granulation ﬂuid (1.3–2.7 moles of water per mole of theophylline anhydrate), the absorption maxima (1410 and 1905 nm) of free water became evident. Owing to its greater ease of spectral acquisition, it was determined that NIR spectroscopy was superior for the detection of different states of water during the wet granulation process.

V. SOLID-STATE NUCLEAR MAGNETIC RESONANCE (SS-NMR) SPECTROMETRY Nuclear magnetic resonance (NMR) has become one of the most powerful spectroscopic tools, able to provide detailed information not only about the chemical constitution of compounds but also about their threedimensional structures and dynamics. Most NMR applications have traditionally involved samples dissolved in a solution phase. The two key factors precluding widespread use of solid-state NMR in the past were sensitivity and resolution. A boost in solid-state applications appeared after the introduction of a combination of the line-narrowing technique known as magic angle spinning (MAS)84–86 with the sensitivity enhancing technique termed cross-polarization (CP).87 Cross-polarization magic angle spinning (CP-MAS) brought about the necessary sensitivity enhancement to make solid-state NMR spectroscopy practicable for natural abundance samples.88 Even though pharmaceutical applications of solid-state NMR (SS-NMR) are well documented, the full potential of this technique has not yet been realized, and it is anticipated that solid-state NMR will become a standard technique for characterization of pharmaceutical solids.

A. Solid-State NMR Techniques 1. Proton Solid-State NMR In contrast to proton spectroscopy in solutions, 1H-NMR has found only limited application in the solid state, mainly because of the existence of extensive line broadening originating from strong through-space, dipole– dipole coupling. This interaction is completely averaged out in solution as the molecules rapidly tumble on the NMR time scale. In solids, however, molecules are locked in their crystal lattices. In the absence of extensive molecular motions, the dipole–dipole interaction can be very strong and are

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usually larger than the chemical shielding effect. Moreover, since typical organic molecules contain many protons in close proximity to each other, the through-space dipole–dipole interaction leads to fast exchange of magnetization between the protons, counteracting the MAS line narrowing. Such spin diffusion is fast on the NMR time scale. As a result, a typical proton static line shape of organic molecules consists of a single peak (up to 50 kHz wide), precluding resolution of the chemical shifts (see Figure 5, top trace).89–91 Since magic angle spinning only partially narrows down the proton line shape (Figure 5, middle trace), other line-narrowing techniques have been sought. Combined rotation and multiple pulse spectroscopy (CRAMPS)92 offers a partial solution to this problem (Figure 5, bottom trace). This technique combines conventional magic angle sample spinning with rotation of proton magnetization around the magic angle in spin space.

FIGURE 5 Solid-state proton spectra of (L)-alanine, showing the differences between spectra acquired in static, spinning, and CRAMPS modes. All 500 MHz spectra were acquired at ambient conditions.

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The CRAMPS experiment puts a large demand on the NMR hardware (especially on NMR probes), since high-power radio frequency (rf ) pulses are applied between each acquisition point. High homogeneity of the irradiation ﬁeld, as well as careful setup of experimental variables, is required to avoid distortions of the proton peaks. Typical line widths afforded by the CRAMPS experiment are approximately 1 ppm, limiting the application of the CRAMPS experiment to compounds with a small number of well-resolved protons. 2. Heteronuclear Solid-State NMR Solid-state NMR spectra of heteronuclei such as carbons are largely devoid of the limitations encountered in proton applications. Though a strong dipole–dipole interaction between protons and carbons prevails, it is relatively easy to decouple the heteronuclear interaction by high-power proton irradiation.89–90 In natural abundance samples, the degree of homonuclear 13C–13C dipolar coupling is very small, because of the spin dilution of 13C nuclei to approximately 1%. Even a moderate MAS speed will remove this effect completely. Depending on the degree of crystallinity and the residual dynamics of the compound, the 13C solid-state NMR peaks can be very narrow. Compounds with high internal mobility, such as adamantane, can have line widths as narrow as 0.01 ppm. This is why in solid-state NMR, adamantane is used not only as a chemical shift standard, but also for optimizing experimental variables and for shimming probes. A typical line width encountered in crystalline organic compounds is of the order of 0.5 ppm, but the exact value will be strongly dependent on the mobility of the compound. It is not unusual to resolve the majority of carbon resonances in compounds containing 40 or more carbons. Before the development of the cross-polarization technique, the two serious limitations that precluded extensive use of carbon solid-state spectroscopy were low 13C sensitivity and its long T1 relaxation times. Both of these limitations are largely removed by cross-polarization of the proton intensities to carbons. CP relies on a strong pool of proton magnetization that is transferred during the cross-polarization step by spin locking pulses to carbons.87–88 The simultaneous spin locking pulses on proton and carbon channels must be of the speciﬁc rf amplitudes required by the Hartman–Hahn matching condition.87 Since the magnetization in the CP experiment starts at protons, it is the proton T1 relaxation that determines the recycle delay between successive scans. Because of dipole–dipole coupling, the proton relaxation is more efﬁcient than carbon relaxation, leading to shorter 1H T1 times. Therefore, the CP experiment improves carbon sensitivity and, at the same time, shortens the necessary recycle delays. 3. Assignment of Spectral Features Knowledge of carbon-speciﬁc assignments of 13C-NMR resonances is not absolutely essential to differentiate polymorphic substances. However, if the assignments are available, the chemical shift differences between polymorphs can be related to conformational changes at particular molecular sites (Figure 6). Recently it has been demonstrated that assignments can be made

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13 C assignments of vitamin B12 , recrystallized from ethyl alcohol (‘‘wet’’ form) 154. In the top right scheme of vitamin B12 (depicted in bold) are those carbons that exhibit shifts between the ‘‘wet’’ and ‘‘dried’’ forms. All 75.8 MHz spectra have been acquired at ambient conditions using (a) the conventional CP-MAS sequence, (b) cross-polarization followed by a period of dipolar dephasing (methyl and nonprotonated carbons observed only), (c) INEPT based spectral editing (methyl, methine and nonprotonated carbons observed only) reference 108, and (d) cross-polarization with short contact time (mainly methine and methylene carbons observed). The solid-state assignments are based on combining known solution assignments, reference 171, with spectral editing techniques.

FIGURE 6

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by relying solely on solid-state NMR data. For instance, a tripeptide sample has been completely assigned in the solid state.93 However, for small molecules with a limited number of resonances and good spectral dispersion, most of the assignments can be deduced by direct comparison of the liquids and solid-state spectra. In typical applications of pharmaceutical interest, solution NMR assignments are either already available or can be completed before proceeding with the solid-state analysis. Solid-state NMR resonances usually fall within several parts per million (ppm) from solution shifts. The origins of differences between the solution-phase and solid-phase spectra are primarily due to conformational changes and crystal-packing effects. A larger contribution, typically not exceeding 12 ppm, comes from the differences between the average molecular conformation or conﬁguration in solution and the solid state. A smaller contribution, typically not exceeding 3 ppm, comes from the packing of molecules in the crystal lattice.94 The latter effect is nonexistent in solution because of the fast tumbling of the molecules. Most of the assignments can be inferred by comparing the solid-state spectra with their assigned solution counterparts. The remaining resonances can be assigned from the application of simple editing techniques. The two simplest editing experiments, affording complementary information, are short CP and dipolar dephasing techniques.95–97 The short CP experiment limits the duration of the cross-polarization period to a short interval of approximately 100 ms, whereas conventional CP times are in the range of 1–10 ms. Only protonated carbons show signiﬁcant intensities due to their strong dipolar couplings (Figure 6d). Having no close proton neighbors and relying only on relatively distant proton–carbon dipolar couplings, quaternary carbons do not have enough time to build up sufﬁcient polarization. On the other hand, the dipolar dephasing experiment uses strong heteronuclear coupling to dephase the protonated carbons. Immediately following the cross-polarization step, a short period of typically 50 ms is applied during which the proton decoupling is turned off. The signals from strongly dipolar coupled carbons are completely dephased during this interrupted decoupling period, whereas the weakly coupled quaternary and methyl carbons are largely unaffected (Figure 6b). Despite their short proton–carbon distances, methyl carbons are only weakly coupled due to the self-decoupling effect of fast steering wheellike rotations around the C–C bond, taking place even in the solid state. Assignment ambiguities may arise when methylene carbons resonate close to methines. These groups have similar dipolar couplings and are not differentiated by either dipolar dephasing or short CP editing. More complex dipolar coupling-based editing techniques can be applied in these instances.98–110 Editing techniques, relying on spin-spin J-coupling rather a dipolar coupling, have also been introduced.111–112 These are essentially solid-state versions of the solution attached proton test (ATP) experiment, in which signals from quaternary carbons and methylene groups are of opposite phase from those of methine and methyl groups. For this experiment to work, J-coupled multiplets must be resolved in the solid state. Further help with the assignments can be gained from two-dimensional techniques, and there are many variants of multidimensional correlation

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experiments. Information-rich homonuclear correlations between carbons, which are based on the solids analog of the solution INADEQUATE experiment, require isotope labeling and as such are only of limited use for pharmaceutical applications.111,113–114 Heteronuclear correlations between protons and carbons or nitrogens can be achieved by through-space dipolar couplings 109,115–117 or through-bond J-couplings.93,113,118–121 The dipolar coupling based solid-state version of the solution HETCOR (Heteronuclear Correlation) experiment includes not only correlations between directly bonded carbons and protons but also correlations between protons and carbons close in space. Therefore, quaternary carbons show correlations in the dipolar version but not in the J-coupled version. In both cases, the resolution in the proton domain of these spectra suffers from the same limitations as for the CRAMPS experiment, restricting the limits of these correlation experiments for assignment purposes. Another group of experiments correlates carbon isotropic shifts with anisotropic contributions from the same nuclei.122–125 The assignment power of these experiments stems from the fact that different types of carbons can have similar isotropic shielding but different anisotropic chemical shifts. Therefore, based on the magnitude of the chemical shift anisotropy, the carbons in question can be assigned. As an added advantage, the chemical shift anisotropy could, in many cases, be more sensitive than isotropic chemical shifts to structural variations between polymorphs. Empirical as well as ab-initio quantum mechanical calculations of chemical shifts are likely to become an important tool for resonance assignments. Speciﬁcally, the ab-initio–type calculations are likely to prove useful in predicting conformations of polymorphs by comparing the, derived from guessed 3-D molecular structure inputs, with experimental chemical shifts.

B. SS-NMR Studies of Polymorphism Polymorphs are typically differentiated by SS-NMR based on their chemical shift differences, but other NMR properties (such as relaxation) can be used as well. Chemical shifts are very sensitive to molecular conformations and crystal packing. In order for the polymorphs to be distinguished by SS-NMR, they must show differences in either conformations or crystal packing. Even though many pharmaceutical applications of SS-NMR can be found in the literature,122–123,126–154 the full potential of this technique has not yet been realized. Several review articles have been published offering a comprehensive review of the literature in this ﬁeld.55,155–160 A powerful technique for the study of polymorphism is single crystal X-ray diffraction, since it provides a detailed description of the crystal structure, including molecular conformation and crystal packing. However, the technique requires good-quality single crystals, which may not be available in many instances. The need for single crystals also precludes application of this technique to the routine analysis of polymorph mixtures. Both of these limitations are avoided through the use of X-ray

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powder diffraction (XRPD), which has established itself as a standard in polymorph determination. Solid-state SS-NMR and XRPD take advantage of different phenomena to evaluate polymorphism. XRPD is sensitive to differences in unit cell dimensions, whereas SS-NMR is sensitive to conformational changes and differences in magnetic environments.94 One can think of two hypothetical situations. If two polymorphic forms differ exclusively in their unit cell dimensions but the conformation of the molecule is preserved, then the XRPD patterns are likely to be substantially different, whereas SS-NMR may not register any changes. On the other hand, if the unit cell dimensions are preserved while the molecular conformation changes substantially, SS-NMR is likely to pick up the differences and the XRPD patterns might be the same. In this sense, both techniques provide information that is complementary to the other.94 1. Advantages of the SS-NMR Approach Solid-state NMR has several important advantages over other spectroscopic techniques that are commonly used to characterize polymorphism. Among these advantages are its sensitivity and high degree of resolution. Even minor changes of conformation or crystal packing among polymorphs can produce different local environments and consequently different chemical shifts (Figure 7). SS-NMR can often be more sensitive for polymorph detection than other techniques, including XRPD, IR, NIR, or Raman spectroscopies. For most of the solvates shown in Figure 7, XRPD shows virtually identical traces. Solid-state NMR can clearly differentiate these forms based on their 13C shifts. In contrast to more established spectroscopic techniques, SS-NMR spectra are virtually independent of the physical properties of the sample, such as particle size, homogeneity, or residual water content. Therefore, pharmaceutical solids can be studied by NMR without a need for special sample preparations. Samples viable for SS-NMR analysis include a whole range of pharmaceutical formulations such as tablets, lyophilized powders, capsules, suspensions, and ointments. SS-NMR does not suffer from a preferred orientation restriction, which often leads to an incorrect identiﬁcation of polymorphs when using XRPD. In addition, SS-NMR is a nondestructive technique that allows other analyses to be performed on the same sample after the NMR spectrum is acquired. Another advantage of SS-NMR is that the observed chemical shift differences between polymorphs can be related to particular molecular sites based on known assignments (see Figure 6). Site-speciﬁc mobility can also be determined by probing the relaxation properties of the compound. This is an important application, since mobility is usually related to polymorphic interconversions and solid-state reactions.94 Solid-state reactions between active pharmaceutical ingredients and excipients can also be followed by SSNMR. The study of polymorphic transitions can be performed by variable temperature experiments. Probably the most important advantage of SS-NMR for pharmaceutical applications is its suitability for the analysis of complex formulations.

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FIGURE 7 125 MHz 13C spectra of different solvates of the same compound. Solvents used to prepare the different solvatomorphs were (A) water, (B) water and cyclohexane, (C) water and MTBE, (D) water (different stoichiometry from (A)), (E) water and i-propanol, (F) water and n-propanol, (G) water and ethanol, (H) water and propylene glycol. Spectrum (I) represents an amorphous form. Unlike SS-NMR spectra, the XRPD patterns of forms D through H are virtually identical.

Typically, excipients found in drug formulations resonate in limited spectral regions. Most excipient peaks can be found between 60 and 100 ppm, and as such do not completely overlap the active ingredient resonances. Spectral regions containing only drug signals can often be found even in complex formulation. 2. Limit of Detection A particular form or a mixture of polymorphs is identiﬁed based on comparison of the sample NMR spectrum with standard spectra of

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each component in its pure form. If one or more fully resolved peaks can be found in the spectrum, the limit of detection is dictated solely by the overall signal-to-noise ratio. The well known drawback of NMR is its lack of sensitivity (deﬁned as signal-to-noise ratio, as opposed to sensitivity to changes of electronic environment discussed in the previous paragraph). Assuming sufﬁciently short acquisition times on the order of one hour, the typical limit of detection of most natural abundance 13C pharmaceuticals is approximately 1% (see Figure 8). In favorable cases, when internal mobility is present and sharp lines are observed, the limit of detection may be even lower. The sensitivity of SS-NMR can also be increased by partial or uniform spin labeling. This, however, would limit the application of SS-NMR only to investigative samples, excluding the analysis of production lots. Nuclei other than carbon can be pursued. Although many pharmaceuticals contain nitrogen, the 15N isotope has low natural abundance and poor relative sensitivity. 15N is, unlike the more abundant quadrupolar 14N, a well-behaved spin 1⁄2 nucleus. Unless isotope enrichment can be applied, its low natural abundance severely limits 15N SS-NMR applications of pharmaceutical utility.

FIGURE 8 125 MHz 13C spectrum of 1% solvate (A) in otherwise pure solvate (D) of the compound shown in Figure 7. Pure solvate (A) is shown as an overlay for comparison. The experimental time was approximately one hour, and 1024 scans were acquired.

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FIGURE 9 470 MHz 19F spectra of two different polymorphs of the same compound. The compound contains one fluorine atom per molecule, which is directly bonded to an aromatic part of the molecule. 35 kHz spinning speed with no proton decoupling was used in these examples (2.5 mm spinners containing approximately 10 mg of sample). Because of relatively unfavorableT1 relaxation properties, 32 scans were acquired in approximately one hour total time.

However, many active pharmaceutical ingredients contain ﬂuorine. 19F is almost as sensitive a nucleus as 1H, but does not face the same empirical limitations as protons. Since the number of ﬂuorine atoms per molecule is usually small, homonuclear 19F–19F couplings are relatively weak and can be removed by conventional magic angle spinning. However, proton decoupling and/or very fast magic angle spinning have to be applied to cancel the strong 1H–19F dipolar interaction, sometimes necessitating the use of special NMR hardware153 (see Figure 9). The presence of ﬂuorine also enables measurements of ﬂuorine–carbon distances by the rotational echo double resonance161–165 (REDOR) experiment without the need for isotope enrichment. This approach provides information similar to single crystal X-ray data for polymorph conformational analysis. Other sensitive but less frequently occurring pharmaceutical nuclei such as phosphorus, bromine, and sodium can be used. The latter two are characterized by quadrupolar nuclear spin. Advanced experiments, such as multiple quantum magic angle spinning (MQMAS) may be required to obtain narrow signals in these instances.166,167 3. Polymorphs Versus Multiple Sites per Unit Cell A problem often arises when multiple lines are encountered for some or all carbons in the sample. The multiple lines can be interpreted as either

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evidence for a mixture of several polymorphs, or conversely, they could indicate the presence of multiple nonequivalent molecules in the unit cell. To differentiate between these two instances, relative peak intensities can be used as a ﬁrst approximation when pure form standards are absent. If the peak ratios of all the carbons appearing as multiplets are approximately equal to one, then multiple sites are likely to be present. This is, however, not a proof of multiple sites since, by coincidence, one could have an equal mixture of different polymorphs. In some cases, the buildup dynamics of CP, which is a dependence of CP-MAS signal intensity on cross-polarization time, can be used to differentiate polymorphs from multiple sites.156 However, it is not straightforward to analyze the CP buildup intensity differences between carbons from the same molecular sites in terms of polymorphs versus multiple sites. In carbon detected proton relaxation experiments, observed differences in proton relaxation typically indicate presence of a polymorphic mixture. In contrast, crystals containing multiple sites show the same relaxation for all protons, as the short distance between the multiple sites makes the proton spin diffusion possible. Positive proof for the existence of polymorphs can be gained by varying the relative concentration of the two forms and observing the varying intensity ratios of corresponding carbons. This in turn requires sample manipulations, such as varying the temperature, humidity, or other conditions to change the ratio of polymorphs.

C. Quantitative Analysis of Polymorphs In contrast to solution-phase or direct polarization solid-state NMR experiments, CP-MAS is not a truly quantitative technique. As a result of the cross-polarization step, the integral intensities of peaks in CP-MAS spectra do not directly reﬂect the stoichiometry of nuclei in the molecule. CP-MAS signals originate from protons, and its transfer efﬁciency varies from carbon to carbon, based on proximity of protons and local mobility of the molecule. In most pharmaceutical applications, only relative concentrations of mixtures of two or more polymorphs need to be determined even in complex dosage forms. The absolute concentration of the drug in formulation is usually known or can be determined by other techniques. Several approaches can be followed when quantifying polymorphic composition. The most reliable, though laborious, method is quantiﬁcation by means of a calibration curve. When pure forms are available, solid-state NMR spectra of mixtures with different known concentrations of polymorphs are acquired, and a calibration curve is constructed. To avoid any dependence on the precise setting of all the experimental parameters, the calibration curve can be plotted as relative signal ratio of the two forms rather than as absolute intensities. Since this method does not rely on absolute signal intensities, the calibration graph constructed on pure drug polymorph mixtures can be applied to formulations, where the active ingredient is diluted by excipients. Logarithmic or other transformation of the resulting nonlinear calibration curves is desirable to optimize the ﬁt.

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When no pure forms are available or when a quick, approximate estimate of the polymorph content is sufﬁcient, the intensity ratio of similar carbon peaks, each belonging to a different form, can be used for quantiﬁcation. In general, carbons with very close chemical shifts are likely to have similar chemical environments. This can be explicitly conﬁrmed if chemical shift assignments are known. Therefore, assuming the same number of protons in close vicinity of these carbons, the CP intensities are likely to be similar, reﬂecting the approximate quantitative ratio of the two forms. More precise results can be achieved by ﬁtting the cross-polarization buildup curve168 in terms of T1 (rotating frame relaxation time during the spin-locking pulse, which characterizes decrease of the signal during CP period due to the spin relaxation) and TCH (cross-polarization time constant, which characterizes the CP signal buildup speed). After the two CP constants T1 and TCH are calibrated on pure standards of all polymorphs present in the mixture, the known CP characteristics can be used to quantify the unknown mixtures. If the total experimental time is not critical, direct observation of carbon signal without the CP step can be used. Although the direct polarization carbon signal is fully quantitative, its signal-to-noise ratio will be inferior to that for CP-MAS because of the reasons discussed above. Not only is the signal per scan weaker, but also the relaxation delay must be set much longer. Alternatively, when the carbon relaxation properties of both polymorphs are known, the intensities can be corrected for relaxation without waiting for full relaxation between successive scans. The easiest and usually most precise quantiﬁcation of polymorphic composition is achieved when a sensitive heteronucleus (such as ﬂuorine or phosphorus) is present in the molecule, and its spectrum shows chemical shift differences between the polymorphs. Direct polarization experiments of these nuclei are sensitive, and therefore the polymorphs can be quantiﬁed quickly directly from the peak ratios. The limit of quantiﬁcation can be well below 1% of one form in the presence of other forms. As an added advantage, the spectra of these nuclei are usually very simple to analyze, since only a very limited number of resonances are typically present in the spectrum. Figure 9 shows the 19F spectra of two polymorphs of the same compound. If these were both present in a mixture, levels of less than 1% could be reliably quantiﬁed with acquisition times of approximately one hour, despite the relatively unfavorable T1 relaxation properties encountered for this compound. Many pharmaceutical applications involve quantiﬁcation of an amorphous phase in the presence of a crystalline phase of the same compound. Even though amorphous spectra are often substantially different from their crystalline counterparts, it is difﬁcult to precisely quantify small amounts of amorphous phase. The amorphous peaks present in low concentration can be lost in the baseline because of their broad line character. Quantiﬁcation is generally achieved by modeling the resulting spectrum as a sum of pure form standard spectra. Alternatively, if pure forms are not available, one can take advantage of the different properties of amorphous and crystalline phases

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such as different relaxation rates169,170 to selectively ﬁlter one component out of the spectrum. The glass transition temperature (Tg) of amorphous compounds is likely to be lower than the melting temperature of the crystalline phase. Heating the sample above Tg therefore results in observation of only the crystalline phase. The signal from the amorphous phase is lost, since this phase reaches a liquidlike mobility, and therefore the dipolar coupling that the CP-MAS experiment relies on is averaged out to zero.

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4 IMPURITIES IN DRUG PRODUCTS KENNETH C.WATERMAN, ROGER C. ADAMI, AND JINYANG HONG Pfizer, Inc.,Groton,CT 06340

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.

INTRODUCTION WATER PEROXIDES ALDEHYDES METAL IMPURITIES SMALL MOLECULE CARBOXYLIC ACIDS LEACHABLES/EXTRACTABLES ALCOHOLS AS IMPURITIES BIOLOGICAL IMPURITIES ADDITIVES IN EXCIPIENTS FINAL OBSERVATIONS SUMMARY REFERENCES

I. INTRODUCTION Impurities in drug products can come from the drug or from excipients or can be brought into the system through a process step or by contact with packaging. Though numerous impurities could be detected in any drug product sample, there are relatively few impurities that have an inﬂuence on the stability or performance of the dosage form. In general, most of the detrimental impurities are small molecules. This is especially true in solid dosage forms where the limited mobility restricts the reactivity of larger molecules. For most drugs, the reactive species consist of water (which can hydrolyze some drugs or affect the dosage form performance), small electrophiles (e.g., aldehydes and carboxylic acid derivatives), peroxides (which can oxidize some drugs), and metals (which can catalyze oxidation and other drug degradation pathways). These impurities are discussed below, with a focus on how these impurities are likely to affect dosage forms via chemical reactivity and physical changes to the systems.

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Additionally, some impurities cause toxicological problems. These impurities may not directly affect the performance or stability of a dosage form, but must be controlled to make a safe drug product.

II. WATER Water is the most commonly found impurity in drug products. Water is prevalent in excipients and drug substances and is used in dosage form preparation. In addition, water can be brought into a system from the atmosphere. Not only is water omnipresent in drug products, but it is often detrimental to chemical stability or dosage form performance. Chemical stability issues with water are generally associated with hydrolysis, particularly of carboxylic acid derivatives (esters, amides), acetals, and other susceptible functionalities.1 Even in nonaqueous liquid formulations, water can be present in amounts sufﬁcient to lead to drug degradation. For some drugs, the moisture level will determine if a hydrate can form. In some cases, hydrate formation or generation of an anhydrous form (by having a sufﬁciently low humidity level) can lead to a loss in crystallinity, which in turn can lead to greater chemical instability of the drug. In addition, changes in crystal morphology may lead to changes in drug dissolution rates. Water is present in a number of excipients as supplied by the vendor. This moisture content can be important when a dosage form is packaged such that equilibration with the environment does not occur. Under these conditions, the moisture brought into the system through the excipients can be sufﬁcient to bring about hydrolysis. Typical moisture contents for a number of solid excipients are listed in Table 1. What is not known is how readily each excipient will give up its moisture; that is, how tightly the water is bound. Water that is tightly bound as a crystalline hydrate is generally less likely to inﬂuence hydrolysis than water that is only loosely associated with an excipient. Ultimately, the water activity at the drug surface will determine the hydrolysis rate. This water can also induce hydrolysis of excipients, which in turn can adversely affect the performance of a dosage form.1 When excipients and dosage forms are exposed to various humidity conditions, water vapor sorption will occur. The relative water vapor sorption levels for a number of excipients have been studied.2 It was found that the total water vapor sorption in solid dosage forms can be predicted from the individual contributions of the excipients as powders. The moisture sorption tendency of an excipient will not only depend on the chemical nature of the excipient, but also on the particle size.3 Because of these effects, without packaging protection, water sorption can be signiﬁcant for a number of dosage forms. Water is often used in the process of preparing dosage forms. For example, lyophilization involves the dissolution or dispersion of a drug and excipients in water, freezing of the mixture and removal of the water by sublimation. The residual moisture level in lyophiles is typically 1–5% by weight, an amount adequate to induce chemical instability with a number

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TABLE 1 Typical Moisture Content of CommonTableting Excipients1 Excipient

Moisture content (wt%)

Comments

Calcium phosphate, dibasic anhydrous

0.1–0.187

Moisture surface adsorbed; does not form hydrate

Calcium phosphate, dibasic dihydrate

20

H2O lost < 100 C

Microcrystalline cellulose (MCC)

5

Moisture content varies with grade; hygroscopic

Colloidal silicon dioxide

0–15 (< 80% RH); 80 (> 80% RH)

Absorbs H2O without liquification; hydrophobic surface treatments decrease hygroscopicity

Crospovidone

60%

Hygroscopic

Hydroxypropylmethylcellulose

0–10 (< 80% RH); 35 (> 80% RH)

Moisture content depends greatly on previous storage conditions

Lactose, anhydrous

1

Varies with manufacturer and grade

Lactose, monohydrate

4.5–5.5

Varies with manufacturer and grade

Magnesium stearate

5–15 (< 75% RH); 35 (> 75% RH)

Varies with manufacturer and grade

Mannitol

0–1 (< 75% RH); 10 (> 75%RH)

Should be stored tightly closed under low RH

Methylcellulose

5

Slightly hygroscopic; store in cool, dry area; tightly closed

Sodium lauryl sulfate (SLS)

5

Nonhygroscopic

Sodium starch glycolate (Explotab)

5–20 (< 75% RH); 60 (> 75% RH)

Cakes if exposed to high humidity

Stearic acid

< 0.1

Nonhygroscopic

of drugs. The effects of moisture on the lyophile stability include direct hydrolysis reactions, affecting the microenvironmental pH and polarity and affecting the molecular mobility in the solid.1 These effects in lyophiles can lead to hydrolysis rates with more than linear dependency on water concentration.4 Other examples of water used for processing include wet granulations and ﬁlm coatings. In both cases, process water is later largely removed by a drying step. In some cases, the residual moisture can be an important factor in stability or dosage form performance. The moisture content within dosage forms can inﬂuence not only chemical stability but also the dosage form performance. For example, tablet hardness, tensile strength, and porosity vary with moisture content.5–7 The resulting dosage forms can show different properties, especially for controlled-release dosage applications. For example, theophylline release from amylodextrin tablets shows dramatic effects of moisture content.8 These moisture effects are explained as resulting from different degrees of porosity introduced from decompression during tableting, because of different degrees of elastic relaxation. Since water plasticizes amylodextrin,

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the lower Tg as a function of water content allows greater relaxation and formation of denser tablets for a given compression force. In general, higher moisture resulted in lower tablet porosity and less tablet disintegration during dissolution. The moisture content of the component excipients can affect important properties in making the dosage form itself. For example, the moisture content of excipients can affect the ﬂow of powders9 and the compactibility of tablet blends.3,9,10 Many of these effects are explained as resulting from changes to the interparticle and intermolecular forces that are due to moisture sorption and distribution.3,11 For lyophilized dosage forms, there is sometimes an optimum moisture content for stability, especially for proteins. The choice of excipient can determine the moisture content of the lyophilized cakes based on the moisture sorption levels.12 Controlling moisture levels to this optimum also requires that the dosage form be protected from environmental moisture, usually by packaging in glass.

III. PEROXIDES Peroxides are reactive materials present in a number of excipients.13 Peroxides can be found either in the form of hydrogen peroxide (HOOH) or as organic peroxides (ROOH). These species can act to oxidize susceptible drugs via direct reaction with the drugs.14 In addition, peroxides can thermally generate radicals (alone or with metals), which can act to initiate radical chain processes or themselves react with drug. This latter problem is particularly signiﬁcant with organic peroxides. Even when acting noncatalytically, peroxides can induce signiﬁcant drug degradation, especially in situations with high excipient-to-drug ratios (low dose). With some excipients, oxidative degradation of the excipient generates peroxide products, which can in turn react with the drug. Peroxides can be found in excipients either as a result of the excipient manufacturing process or due to oxidative instability of the excipient. In both cases, the issue is most prevalent with polymeric excipients. Peroxides are sometimes present as initiators in polymerization processes, though which excipients have this source of peroxides is difﬁcult to determine because of the proprietary nature of the processes used to prepare a number of excipients. In some cases, peroxides can form in the process of transforming natural or synthetic polymers into the desired ﬁnal products. Two classes of excipients are most associated with peroxide impurities, although peroxides may be found in other excipient classes, generally at lower levels. The ﬁrst group consists of polymeric ethers. These ethers include polyethylene glycols (PEGs),15,16 polyethylene oxides (PEOs), polysorbates, polyoxyethylene alkyl ethers, polyoxyethylene stearates, and other ethylene oxide-based materials.13,17 Such materials generally have some level of peroxides present as supplied by the vendor. Polymeric ethers are also subject to autoxidation, which results in the formation of more peroxides.14 To minimize this degradation pathway, the above materials are generally

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supplied with an antioxidant, typically butylated hydroxytoluene (BHT). BHT itself can be a concern for some pharmaceutical formulations due to the formation of strongly colored by-products. Under some storage or packaging conditions, the BHT will volatilize, resulting in reduced levels in the dosage form and correspondingly greater instability.18 The second group of excipients commonly associated with peroxide impurities consist of polyvinyl pyrrolidone (PVP)–based excipients. These include Povidone and Crospovidone.19,20 Both excipients commonly contain 100–200 ppm of peroxide present as process impurities. Peroxides can form by autoxidation of PVP, and greater amounts can form under such shear conditions as found during granulation and tableting. Peroxide formation is relatively slow for solid dosage forms under standard aging conditions; however, the age and storage conditions of the excipient can lead to variable peroxide levels.20 Some organic solvents and oils have also been reported to contain peroxides.21 The level of peroxides in susceptible solvents can increase with time, depending on the storage conditions.

IV. ALDEHYDES Aldehydes, even in trace amounts, are often found to adversely affect the stability and efﬁcacy of drugs via direct reaction with the active pharmaceutical ingredient (API). Formaldehyde, acetaldehyde, furfural, and glyoxal are the aldehydes most often responsible for stability issues in drug products. In solid dosage forms, these aldehydes are sufﬁciently small to be mobile enough to react effectively with APIs. Many commonly used pharmaceutical excipients contain trace low molecular weight aldehydes. Aldehyde impurities in excipients are often due to excipient degradation. With organic excipients, degradation to aldehydes is generally associated with oxidation. For example, benzyl alcohol oxidation generates benzaldehyde,22 and autoxidation of unsaturated fats, polyethylene glycol, and polysorbates generates small molecule aldehydes.13 In some cases, aldehydes are produced via hydrolytic reactions. This is seen in the formation of furfural and its adducts in the acid-catalyzed degradation of hemicellulose and other sugar-based excipients.23 For example, 5-hydroxymethylfurfural is found in spray-dried lactose because of thermal decomposition occurring during the spray-drying process.24 A second source of aldehydes involves functional additives present in the excipients, either as aldehydes themselves or as materials that oxidize or hydrolyze to give aldehydes. Examples include preservatives, cross-linking agents, and ﬂavoring agents. Corn starch often contains hexamethylenetetramine as a preservative, which hydrolyzes to give ammonia and formaldehyde.25 Glyoxal can be found in hydroxyethylcellulose as a crosslinking reagent22 and is found as an impurity in hydroxypropylmethyl celluloses.13 Many dye and ﬂavoring reagents (for example, vanillin) contain aldehyde impurities.17 A third source of aldehydes involves materials leached from elastomeric stoppers26,27 (see Section VII).

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A number of excipients contain reducing sugar impurities (aldehydes) such as glucose, maltose, and lactose,28,29 which can react with the API or other excipients.30 The mechanism of the deleterious effects of aldehydes on drug products is generally well understood. For drug substances with primary and secondary amines, nucleophilic attack on the aldehyde produces an -hydroxyl amine, which can dehydrate to give an imine (Schiff’s base) as shown below:

With nucleophiles such as alcohols (enolates) and thiols, reaction with aldehydes yields the corresponding addition products.31

Aldehydes can react with many excipients in drug products, which in turn can affect the performance of the drug by altering drug release and delivery. An example of this is seen with gelatin,25,32 where formaldehyde reacts with the amino groups on lysine residues causing protein cross-linking, which in turn changes the dissolution characteristics of gelatin capsules. A similar situation is seen when the aldehyde decomposition product of the antioxidant BHT, 2,6-di-t-butyl-4-hydroxybenzaldehyde, cross-links gelatin capsules, affecting the overall dissolution rate.33

V. METAL IMPURITIES Metals are found in almost all excipients. The types of metals and levels present in excipients can vary signiﬁcantly depending on the excipient type, source, and production process used to extract or produce the excipient.

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Metals are deleterious to drug products for the following reasons: inherent toxicity of the metal, formation of insoluble metal complexes, and oxidative14 and hydrolytic1 catalysis. Common metals found in excipients include arsenic, bismuth, cadmium, chromium, copper, iron, lead, mercury, nickel and sodium.22,34,35 Metals can also be brought into drug products during processing or can be leached from packaging (see Section VII). Certain excipients inherently contain high levels of metals, such as minerals (talc or kaolin) or inorganic compounds derived from minerals such as buffers and oxides (e.g., phosphates, silicates, titanium dioxide, etc). Additionally, excipients produced using metal catalysts, for example, hydrogenated oils and fats, may contain high concentration of metals such as platinum or palladium. The process equipment and storage container may lead to contamination by leached metals22 (see also Section VII). Levels are usually reported in parts per million (ppm) concentration. Since some of the issues associated with metals involve catalysis, even relatively low levels of metals may impact dosage form performance. Numerous processes for producing dosage forms involve contact of materials with metal surfaces. The degree of metal incorporation into dosage forms will depend on a number of factors. In liquid dosage forms, one is often concerned with direct dissolution of metal ions by contact of a liquid with a metal surface. This will tend to be a greater issue for lower pH solutions. With solid dosage forms, not only is direct dissolution an issue, but also abrasion of metals can enable incorporation of metal particles into the dosage forms. Toxicity that is due to heavy metals is the primary safety concern stemming from metal impurities in excipients. Toxicology studies have demonstrated that the most toxic metals are cadmium, mercury, chromium, lead, nickel, and arsenic. Depending on the level and route of ingestion, these metals can cause death, cancer, damage brain and nerve function, and speciﬁcally poison the liver and kidneys.36 Because of their toxicity, these metals have pharmacopeial speciﬁcations to limit the amount present in solutions.22 There is, however, variability in pharmacopeial speciﬁcations, even with a single country’s monographs. For example, lead is restricted to 5 ppm in USP/NF monographs, yet is limited to 3 ppm as an impurity in calcium carbonate.35 Harmonization of these limits has been attempted to facilitate drug development.34 Insoluble heavy metal salts may be an issue, depending on the solubility product (Ksp) of the drug being formulated in the presence of metals. Although precipitates in liquid dosage forms can sometimes be removed by ﬁltration, this approach is often unacceptable for either quality or cost concerns. Precipitates adversely impact the performance of injectable dosage forms and can cause in vivo reactions such as phlebitis.37 Different salts may precipitate from solution, depending on the buffers used and their concentrations. Calcium salts may be very insoluble and can lead to precipitate or particulate formation. Insoluble chlorides, hydroxides, and sulfates are commonly formed with calcium, mercury, iron, and magnesium because of low solubility products.38 These insoluble precipitates will lead to undesirable dosage form properties and can be quite harmful, depending on the route of administration. Trace metal impurities in excipients can lead to oxidative catalysis resulting in drug substance degradation.14 The metals most commonly

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associated with oxidation are iron and copper. Both of these metals act as catalysts by facilitating the reduction of molecular oxygen and thus rendering it more reactive. There are three distinct oxidative reactions that can occur with metals: (1) direct metal catalysis, whereby the metal acts as an electron exchange to reduce oxygen, (2) metal binding to drug and oxygen simultaneously, and (3) Fenton-type reactions where transition metal ions reduce peroxide to generate the highly reactive hydroxyl radical. Metals are also associated with catalysis of hydrolysis.1

VI. SMALL MOLECULE CARBOXYLIC ACIDS Small molecule carboxylic acids can be found in many polymeric excipients, sugars, and unsaturated fats. Generally, the most reactive carboxylic acids found as impurities in excipients include formic acid, acetic acid, and glyoxalic acid. In polymers made from carboxylic acids, unreacted monomeric carboxylic acids can be carried over from the polymerization process. For example, with poly(lactide), lactic acid is present at concentrations of 0.2–1.0%.39,40 The autoxidation of many polymers such as polyethylene glycols and non-ionic surfactants generates carboxylic acids, particularly formic acid.41,42 Under humid conditions, slow hydrolysis of polylactide-co-glycolide (PLGA) and polyanhydrides results in formation of carboxylic acids. Unsaturated fats also undergo autoxidation easily and generate carboxylic acid degradants in addition to peroxides.43 Oxidation of aldose sugar produces aldonic acid.44–46 Because of the higher oxidation propensity of aldehydes, carboxylic acid impurities are often found in systems that generate aldehydes (see Section IV). Small molecule carboxylic acids interact with drug products by three mechanisms: (1) they induce a change in acid content of a formulation, which can shift the formulation into a less stable pH region and initiate or accelerate the degradation of the API; (2) carboxylic acids can react with drug molecules or excipients with nucleophilic functional groups such as primary or secondary amines or with hydroxyls to form amides and esters, respectively;47 (3) salt formation between carboxylic acids and basic drugs can inﬂuence the solubility, dissolution, or chemical stability of the drug.48,49

VII. LEACHABLES/EXTRACTABLES Leachable /Extractable chemicals from packaging are a major source of contaminants in drug products, especially in liquid dosage forms.

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Leachable/Extractables can come from glass, rubber stoppers, and plastic packaging materials. Metal oxides such as Na2O, SiO2, CaO, MgO, and Al2O3 are the major components leached/extracted from glass.50 Manganese and iron can also be leached from amber glass vials. Because of the complexity of different synthetic materials, the leachable/extractable proﬁles from rubber and plastic are far more complicated than from glass. Generally, most synthetic materials contain leachable oligomers/monomers, vulcanizing agents, accelerators, plasticizers, and antioxidants.51 Some examples of leachables/extractables from synthetic materials include styrene from polystyrene,52 diethylhexylphthalate (DEHP, plasticizer in PVC),3 dioctyltin isooctylmercaptoacetate (stabilizer for PVC),53 zinc stearate (stabilizer in PVC and polypropylene),52,54 2-mercaptobenzothiazole (accelerator in rubber stopper),55 and furfural from rayon.27 Pharmaceutical and analytical scientists should obtain detailed composition information from packaging vendors, since such information is often proprietary and varies from vendor to vendor. The detrimental effects of leachables/extractables vary, depending on the nature of the extracted material. For example, glass leachables/extractables can decrease the formulation pH.50 Speciﬁc metals such as iron catalyze certain degradation reactions.14 Antioxidants, when combined with metal ions, can accelerate drug oxidation.56 Many accelerators are free radical initiators. Some leachables/extractables, such as 2-mercaptobenzothiazole, also have toxicological concerns.55

VIII. ALCOHOLS AS IMPURITIES Alcohols as impurities are generally not a problem in most drug products. The reactivity of alcohols is low under typical formulation conditions. Levels of alcohol impurities in solid dosage forms are usually not sufﬁciently high to cause signiﬁcant issues. Alcohols are often used in granulation steps and can therefore be expected to be found at some level in the ﬁnal products. In liquid dosage forms, alcohol impurities may be present in ethanol and long-chain poly-alcohol polymers.

IX. BIOLOGICAL IMPURITIES Biological impurities in excipients consist of bacteria, fungi, viruses, endotoxins, and prions. These impurities can enter excipients in a variety of ways and have different consequences, depending on the dosage form. Safety is the main concern of biological contamination. Infectious microorganisms can cause severe illness or death, depending on the quantity and type of organism. Chemically, certain organisms can degrade drug substances and excipients in the drug product via direct metabolism, although this is of less concern than the primary safety issue. Parenteral dosage forms require extensive testing of sterility and biological impurity contamination because of the high sensitivity of this administration route. These same sterility standards do not apply to solid and

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oral liquid dosage forms, yet it may sometimes be important to assess certain types of biological impurities (e.g., prions) in some excipients regardless of the ﬁnal dosage form. The term referring to the degree of biological contamination by microorganisms is bioburden and is quantiﬁed by measuring the number of colony forming units (CFU) present in an excipient. For example, in the EU the current limit is less than or equal to 100 CFU per 100 mL of sterile product. Microorganisms are introduced as contaminants in excipients at several stages of production including both manufacturing and processing. This can occur, for example, because of poor water quality control or handling during the manufacturing. Contamination can also occur by handling of the ﬁnal excipient when received for dosage form manufacturing. Non-aseptic storage of excipients may contaminate dry powder excipients that are opened for sampling via spores from the environment. Limits have been set for a variety of excipients. For example, the USP/NF microbial limits are not more than (NMT) 100 CFU/g for lactose monohydrate and NMT 1000 CFU/g for magnesium stearate and microcrystalline cellulose. Limits on yeasts and molds are usually about half the levels allowed for bacteria.34 Excipients derived from bacterial transformation or enzymatic processes, such as sugars (e.g., mannitol or dextrose), pose special concerns due to potential contamination by endotoxins. Endotoxins, or pyrogens, are lipopolysaccharides from gram-negative bacteria that can induce severe fever upon parenteral administration. Parenteral products have different limits for allowable endotoxin levels.57 Prions are found in animal-derived excipients. These infectious proteins induce transmissible spongiform encephalopathy (TSE) and cause degradation of bovine brain tissue. They may also lead to a human disease variant known as Creutzfeldt-Jacob disease.22 Excipients derived from animal sources, primarily bovine, sources are at the highest risk for prion contamination. Excipients such as gelatins or those derived from animal fats, such as magnesium stearate, are most likely to be contaminated by prions. Additionally, lanolin, lanolin derivatives, and milk-based excipients are at risk of TSE contamination. Limits on the risk of transmission were set in 1992 in the CPMP guidelines for minimizing the risk of transmission of agents causing spongiform encephalopathy via medicinal products.22 In addition to microorganism-based impurities, there are other biological impurities that may pose a concern for drug products. For example, allergens from peanuts can contaminate excipients by cross-contamination with processing equipment used to process peanuts and excipient raw materials. These contaminants can cause severe allergic reactions in sensitive individuals.58 Additionally, gluten present in excipients manufactured from wheat starch may cause celiac disease.22

X. ADDITIVES IN EXCIPIENTS A number of excipients contain additives that serve as stabilizers or to improve material properties. Stabilizers can themselves affect drug stability,

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either by direct interaction or by affecting excipient decomposition rates. As such, it is often important to control such additives. Examples of these stabilizers include antioxidants and antimicrobials. For example, PEOs typically contain 100–500 ppm of BHT;18 anhydrous lanolin and certain fat derivatives contain approximately 200 ppm of BHT; soybean lecithin and certain fats and fat derivatives contain 0.1–0.2% -tocopherol; and some aqueous dispersions used in tablet coating contain a few ppm of hydrogen peroxide (as an antimicrobial).22 Additives associated with material properties often involve materials designed to plasticize polymeric excipients. Latex dispersions often have additives to promote coalescence of particles upon drying. These often include surfactants and plasticizers. These additives can be of concern when they interact with the drug. For example, if they dissolve some of the drug, that material may become more reactive toward chemical decomposition.

XI. FINAL OBSERVATIONS In a number of cases, chemical stability issues associated with an excipient are due to impurities in the excipient rather than the excipient itself. The most reactive impurities tend to be small molecules. This is especially true in solid dosage forms, where the mobility of large molecules is severely restricted. In addition to chemical stability, impurities in drug products can cause issues associated with toxicity or dosage form performance. It is often valuable to quantitatively determine the level of important impurities in drug products, and to trace the origin of those impurities to their source. If the source is from an excipient, variability from lot to lot may make a marginal product unacceptable for reliability. If the source is related to the dosage form preparation process, it may be worthwhile considering technologies and processes that provide less of the undesirable impurity.

XII. SUMMARY This overview has focused on those impurities brought into the drug product system by excipients or dosage form processing likely to cause stability or performance issues in the dosage form. The sources of the impurities are discussed as well as how they affect dosage forms or represent product issues. In particular, those impurities that lead to drug stability issues are reviewed.

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3. Malamataris, S., Tsiri, K. and Goidas, P. Sorption of moisture of different size fractions of some direct compression excipients and tensile strength of the corresponding tablets. Congr. Int. Technol. Pharm. 6th, 5:195–204, 1992. 4. Bell, L. N. and Labuza, T. P. Aspartame degradation as a function of ‘‘water activity.’’ In Water Relationships in Food. (H. Levine and L. Slade, Eds.) pp. 337–349, Plenum Press, NY, 1991. 5. Schepky, G. and Fischer, M. Effect of production-related variations in hardness and moisture content on the sorption characteristics of tablets. Eur. J. Pharm. Biopharm. 39:53– 60, 1993. 6. Matsunaga, Y., Ohta, R., Bando, N., Yamada, H., Yuasa, H. and Kanaya, Y. Studies on internal structure of tablets IV: Effects of water content on physical and chemical stability of tablets containing an anticancer drug TAT-59. Chem. Pharm. Bull. 41:720–724, 1993. 7. Nyqvist, H., Nicklasson, M. and Lundgren, P. Studies on the physical properties of tablets and tablet excipients III: Water sorption and its effect on hardness and disintegration. Acta Pharm. Suec. 18:305–314, 1981. 8. Steendam, R., Eissens, A. C. E., Frijlink, H. W. and Lerk, C. F. Plasticization of amylodextrin by moisture. Consequences for drug release from tablets. Int. J. Pharm. 204:23–33, 2000. 9. Hoon, T. S. and Pathak, Y. V. Evaluation of maltodextrins as tablet excipients 3 Moisture absorption studies. Congr. Int. Technol. Pharm. 6th, 5:185–194, 1992. 10. Kawashima, Y., Takeuchi, H., Hino, T., Niwa, T., Lin, T. L., Sekigawa, F. and Ohya, M. The effects of particle size, degree of hydroxypropyl substitution and moisture content of low-substituted hydroxypropyl cellulose on the compactibility of acetaminophen and the drug release of the resultant tablets. S. T. P Pharma Sci. 3:170–177, 1993. 11. Malamataris, S., Goidas, P. and Dimitriou, A. Moisture sorption and tensile strength of some tableted direct compression excipients. Int. J. Pharm. 68:51–60, 1991. 12. Fakes, M. G., Dali, M. V., Haby, T. A., Morris, K. R., Varia, S. A. and Serajuddin, A. T. M. Moisture sorption behavior of selected bulking agents used in lyophilized products. PDA J. Pharm. Sci. Technol. 54:144–149, 2000. 13. Crowley, P. and Martini, L. Drug-excipient interactions. Pharm. Technol. Europe, 15:26–34, 2001. 14. Waterman, K. C., Adami, R. C., Alsante, K. M., Hong, J., Landis, M. S., Lombardo, F. and Roberts, C. J. Stabilization of pharmaceuticals to oxidative degradation. Pharm. Dev. Technol. 7:1–31, 2002. 15. McGinity, J. W., Hill, J. A. and LaVia, A. L. Influence of peroxide impurities in polyethylene glycols on drug stability. J. Pharm. Sci. 64:356–357, 1975. 16. Johnson, D. M. and Taylor, W. F. Degradation of fenprostalene in polyethylene glycol 400 solution. J. Pharm. Sci. 73:1414–1417, 1984. 17. Kibbe, A. H. (Ed.) Handbook of pharmaceutical excipients, 370–371, 407–411, 576–577. American Pharmaceutical Association: Washington, D. C. 2000. 18. Union Carbide Corporation Sentry2 Polyox2 water soluble resin storage stability. Personal Communication, 1996. 19. Laicher, A., Junger, H. and Klemm, F. H. Stability behaviour of molsidomine-containing pellet formulations. J. Pharm. Pharmacol. 49:131–134, 1997. 20. Hartauer, K. J., Arbuthnot, G. N., Baertschi, S. W., Johnson, R. A., Luke, W. D., Pearson, N. G., Rickard, E. C., Tingle, C. A., Tsang, P. K. S. and Wiens, R. E. Influence of peroxide impurities in povidone and crospovidone on the stability of raloxifene hydrochloride in tablets: identification and control of an oxidative degradation product. Pharm. Dev. Technol. 5:303–310, 2000. 21. Shertzer, H. G. and Tabor, M. W. Peroxide removal from organic solvents and vegetable oils. J. Environ. Sci. Health, A20:845–855, 1985. 22. Ja¨kel, D. and Keck, M. Purity of Excipients. Excipient Toxicity & Safety, 103:21–58, 2000. 23. Janicki, C.A. and Almond, H.R. Jr., Reaction of haloperidol with 5-(hydroxymethyl)-2furfuraldehyde, an impurity in anhydrous lactose. J. Pharm. Sci. 63:41–43, 1974. 24. Brownley, C. A., Jr. and Lachman, L. Browning of spray-processed lactose. J. Pharm. Sci. 53: 452–454, 1964.

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25. Digenis, G. A., Thomas, B. and Shah, V. P. Cross-linking of gelatin capsules and its relevance to their in vitro–in vivo performance. J. Pharm. Sci. 83:915–921, 1994. 26. Battersby, J. E., Hancock, W. S. and Lawlis, V. B., Jr. Container system for storage stable pharmaceuticals. PCT Int. Appl. WO 9210747, 1992. 27. Hartauer, K. J., Mayer, R. F., Schwier, J. R., Bucko, J. H., Cooke, G. G. and Sullivan, G. R. The effect of rayon coiler on the dissolution stability of hard-shell gelatin capsules. Pharm. Technol. 17, 76, 78, 80:82–83, 1993. 28. Kitson, G. E., Hudson, H. E., and Dickinson, N. A. The effect of various sugars on the stability of 2-amino-6-methyl-5-oxo-4-n-propyl-4,5-dihydro-s-triazolo [1,5-a] pyrimidine. Pharm. Acta. Helv. 51:181–185, 1976. 29. Kitson, G. E., Hudson, H. E. and Dickinson, N. A. The effect of various sugars on the stability of 2-amino-6-methyl-5-oxo-4-n-propyl-4,5-dihydro-s-triazolo [1,5-a] pyrimidine Part II: The use of aldehydic and ketonic compounds to investigate the functional groups involved. Pharm. Acta. Helv. 54:359–362 1979. 30. Wirth, D. D., Baertschi, S. W., Johnson, R. A., Maple, S. R., Miller, M. S., Hallenbeck, D. K., and Gregg, S. M. Maillard reaction of lactose and fluoxetine hydrochloride, a secondary amine. J. Pharm. Sci. 87:31–39, 1998. 31. March, J. Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 4th ed., McGraw-Hill Book Co., New York. 1992. 32. Doelker, E. and Vial-Bernasconi, A. C. Interactions contenant-contenu au sein des capsules gelatineuses et evaluation critique de leurs effets sur la disponibilite des principes actifs. STP. Pharma, 4:296–306, 1988. 33. Desai, D. S., Ranadive, S. A., Lozano, R., and Varia, S. A. Dissolution instability of encapsulated marketed tablets. Int. J. Pharm. 144:153–158, 1996. 34. Chowhan, Z. T. Harmonization of Excipient Standards, Chapter 14. In Excipient Toxicity and Safety, M.L. Weiner and L.A. Kotkoskie (Eds.) Marcel Dekker, Inc., NY, pp. 321–354. 1999. 35. Chowhan, Z. T. A rational approach to setting limit tests and standards on impurities in excipients. Pharm. Tech. 19:43–48, 1995. 36. Duffus, J. and Worth, H. 2001. The Science of Chemical Safety Essential Toxicology, An Educational Resource. IUPAC. 37. Yalkowsky, S. H., Krzyzaniak, J. F. and Ward, G. H. Formulation-related problems associated with intravenous drug delivery. J. Pharm. Sci. 87:787–796, 1998. 38. Ebbing, D. D. General Chemistry, 5th ed. Houghton Mifflin Company, Boston, MA. 1996. 39. Cam, D., Bracci, B. and Marucci, M. Influence of residual monomers and metals on poly D,L-(lactide) thermal stability. Front. Biomed. Polym. Appl. 2:189–193, 1999. 40. Hyon, S.-H., Jamshidi, K. and Ikada, Y. Effects of residual monomer on the degradation of D,L-lactide polymer. Polym. Int. 46:196–202, 1998. 41. Hamburger, R., Azaz, E. and Donbrow, M. Autoxidation of polyoxyethylenic non-ionic surfactants and of polyethylene glycols. Pharm. Acta Helv. 50:10–17, 1975. 42. Sakharov, A. M., Mazaletskaya, L. I. and Skibida, I. P. Catalytic oxidative deformylation of polyethylene glycols with the participation of molecular oxygen. Kinetics and Catalysis, 42:662–668, 2001. 43. Gardner, H. W. Oxygen radical chemistry of polyunsaturated fatty acids. Free Radical Biol. Med. 7:65–86, 1989. 44. Volt, D. and Volt, J. G. Biochemistry, p. 250. John Wiley & Sons, New York, 1990. 45. Krebs, A. The role of heavy metals in the autoxidation of sugar solutions. Biochem. Z. 180: 377–394, 1927. 46. Wehmeier K. R. and Mooradian, A. D. Autoxidation and antioxidation potential of simple carbohydrates. Free Radical Biol. Med. 17:83–86, 1994. 47. Johnson, D. M. and Taylor, W. F. Degradation of fenprostalene in polyethylene glycol 400 solution. J. Pharm. Sci. 73:1414–1417, 1984. 48. Bastin, R. J., Bowker, M. J. and Slater, B. J. Salt selection and optimization procedures for pharmaceutical new chemical entities. Org. Process Res. Dev. 4:427–435, 2000. 49. Gould, P. L. Salt selection for basic drugs. Int, J. Pharm. 33:201–217, 1986. 50. Sanga, S. V. Review of glass types available for packaging parenteral solutions. J. Parenteral Drug Assn. 33:61–64, 1979.

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51. Paskiet, D. M. Strategy for determining extractables from rubber packaging materials in drug products. PDA J. Pharm. Sci. 51:248–251, 1997. 52. Hamon, M. Plastic materials in pharmaceutical packaging I: Behavior in relation to various parameters. Sci. Tech. Pharm. 10:277–281, 1981. 53. Aignasse, M. F., Prognon, P., Stachowicz, M., Gheyouche, R. and Pradeau, D. A new and rapid HPLC method for determination of DEHP in PVC packaging and releasing studies. Int. J. Pharm. 113:241–246, 1995. 54. Fras, I., Cassagnau, P. and Michel, A. Influence of processing conditions on the leaching of thermal stabilizers from plasticized poly(vinyl chloride) in the presence of water. J. Appl. Polym. Sci. 70:2391–2400, 1998. 55. Reepmeyer, J. C. and Juhl, Y. H. Contamination of injectable solutions with 2-mercaptobenzothiazole leached from rubber closures. J. Pharm. Sci. 72:1302–1305, 1983. 56. Hovorka, S. W. and Scho¨neich, C. Oxidation degradation of pharmaceuticals: theory, mechanism and inhibition. J. Pharm. Sci. 90:253–269, 2001. 57. Center for Drug Evaluation and Research. Guidance for Industry: Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices, p. 10. CDER FDA. Rockville, MD, 1997. 58. Schappi, G. F., Konrad, V., Imhof, D., Etter, R. and Wuthrich, B. Hidden peanut allergens detected in various foods: Findings and legal measures. Allergy, 56:1216–1220, 2001.

5 STRATEGIES FOR INVESTIGATION AND CONTROL OF PROCESS- AND DEGRADATION-RELATED IMPURITIES BERNARD A.OLSENa AND STEVEN W. BAERTSCHIb a

Eli Lilly and Company, Lafayette, IN 47909 Eli Lilly and Company, Indianapolis, IN 46285

b

I. INTRODUCTION II. GOALS AND STRATEGIES A. Goals B. Chemistry-Guided VersusTechnique-Oriented Strategy C. Screening Methods D. Targeted Methods E. Focused Methods for Control Use F. Pharmacopeial Methods III. PROCESS-RELATED IMPURITIES IV. DEGRADATION-RELATED IMPURITIES A. Definitions B. Strategy C. Thermolytic Degradation D. Hydrolytic Degradation E. Photolytic Degradation F. Oxidative Degradation G. Common Problems Encountered During StressTesting with HPLC-UV H. Detectors I. Drug Product StressTesting V. SUMMARYAND CONCLUSIONS VI. ACKNOWLEDGMENTS REFERENCES

I. INTRODUCTION The investigation of impurities is a critical part of drug development. The overall goal of these investigations is to provide information that will contribute to reproducible production of a high quality product that has been demonstrated to be safe through toxicological and clinical trials.

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Understanding the origin of impurities and the mechanisms for their minimization or removal are necessary to develop drug substance and drug product manufacturing processes and storage conditions that will assure quality at release and throughout the allowed storage period. The analytical evaluation of impurities is used to guide development and is required for drug registration. As mentioned in Chapters 1 and 2, the International Conference on Harmonisation (ICH) guidelines on impurities in drug substances1,2 and drug products3,4 classify impurities as organic or inorganic. Speciﬁc thresholds for identiﬁcation, safety qualiﬁcation, and reporting of organic impurities have been set in these guidelines (Table 1). These thresholds deﬁne the level of signiﬁcance of an impurity that can be used to guide development efforts. The investigation of impurities covers topics such as method development and impurity identiﬁcation techniques, both of which have been reviewed in the literature.5–10 These reviews have often focused on speciﬁc examples or particular analytical techniques. This chapter describes strategies for the investigation of process-related and degradation-related impurities in drug substances and drug products. Particular emphasis will be given to a chemistry-guided approach and the iterative nature of investigations as knowledge is gained. The delineation of impurity type and source that will be used for this discussion is given in Table 2. Although the scope of the discussion will be limited to drugs prepared by chemical synthesis, the strategies described may also apply to biosynthetic or semisynthetic molecules. Considerations for investigating and controlling crystal forms,11 residual solvents12 and microbiological impurities11 have been addressed in other ICH guidelines and will not be discussed here.

TABLE 1 ICH Reporting, Identification, and QualificationThresholds Thresholds* Maximum daily dose

Reporting

Identification

Qualification

Drug substance 2g

0.05%

0.10% or 1.0 mg/day intake

0.15% or 1.0 mg/day intake

>2g

0.03%

0.05%

0.05%

Drug product 1g

0.1%

>1g

0.05%

< 1 mg

1% or 5 mg/day intake

1% or 50 mg/day intake

1–10 mg

0.5% or 20 mg/day intake

1% or 50 mg/day intake

10–100 mg

0.2% or 2 mg/day intake

0.5% or 200 mg/day intake

100 mg–2 g

0.2% or 2 mg/day intake

0.2% or 2 mg/day intake

>2g

0.1%

0.1%

*When a percentage and amount/day intake are both given, use whichever is lower.

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TABLE 2 Impurity Descriptions Impurity type Process-related drug substance

Process-related drug substance

Degradation drug substance or drug product Degradation drug product

Impurity source 8 Organic > > > > < Starting material Intermediate > > > By-product > : Impurity in starting material

Organic or Inorganic Reagents; catalysts; etc: Organic Degradation products Organic Excipient interaction products

Analytical method development is at the heart of impurity investigations. Since appropriate methods must be used to detect and quantify impurities, a key question for investigating impurities is How much investigation is enough? The answer determines how many techniques and conditions will be used to provide conﬁdence that all impurities of signiﬁcance have been detected. With a threshold of 0.10% for the drug substance, one can never say with 100% conﬁdence that all impurities have been detected in a drug. However, with an appropriate strategy, a high level of assurance can be provided that all signiﬁcant impurities have been detected.

II. GOALS AND STRATEGIES A. Goals The goals for the investigation of impurities are outlined in Table 3. A common goal for investigation of both process- and degradation-related impurities is to determine which of the many potential impurities are, in fact, produced in the manufacturing process and which occur under a given set of storage conditions. During new drug development this knowledge can be used to modify the process in order to eliminate or minimize levels of impurities. Knowledge of stability characteristics can be used to store the drug substance and drug product in appropriate packaging and under appropriate environmental conditions to minimize or eliminate unacceptable degradation. Knowledge of the degradation behavior of the drug and interactions with excipients is also useful in developing the formulation. Information from impurity investigations is essential in establishing speciﬁcation tests and acceptance limits at various control points in the manufacturing process.

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TABLE 3 Goals of Impurity Investigations Process-related impurities

Degradation-related impurities

Identify significant impurities and demonstrate the absence of potential or postulated impurities

Identify potential degradation products through stress testing and actual degradation products through stability studies

Determine origin of impurities and methods for elimination or reduction

Understand degradation pathways and methods to minimize degradation

Establish a control system for impurities involving:

Establish a control system for impurities involving:

1. Processing/manufacturing conditions 2. Suitable analytical methods/ specifications

1. Processing/manufacturing conditions 2. Suitable analytical methods/ specifications 3. Long-term storage conditions, including packaging 4. Formulation

B. Chemistry-Guided Versus Technique-Oriented Strategy One strategy for impurity investigation might be termed a ‘‘techniqueoriented approach.’’13,14 The main feature of this approach is to use as many techniques as possible to search for impurities. This would include various types of chromatographic techniques with multiple detection schemes. Of course the characteristics of the drug substance, process intermediates, and any obvious by-products or degradation products would strongly inﬂuence the primary method(s) employed. Quite often the primary technique is reversed-phase HPLC (RP-HPLC) with UV detection. The search for unknown impurities might continue with orthogonal separation techniques such as normal phase HPLC, TLC, CZE, GC, and SFC and multiple detection modes such as PDA, MS, ELSD, CLND, RI, FID, TC, and multiple visualization reagents for TLC. This approach is sometimes focused on analysis of the drug substance and may not include investigation of impurities in synthetic intermediates and reaction mixtures. Determining when enough investigation has been performed and when the point of diminishing returns is reached are the main issues with this approach. We advocate a ‘‘chemistry-guided’’ approach for the investigation of impurities. For process-related impurities, the chemistry of the process is carefully examined to postulate potential by-products or other impurities at each step. These, in addition to the synthetic intermediates, can form a basis for method development and further impurity investigation. For degradation products, well-designed stress degradation and excipient interaction studies serve to direct development efforts. Some degradation products can also be predicted based on the structure of the molecule. Evaluation of stress-degraded samples using a broad screening method can reveal potential degradation products, and an evaluation of the degradation

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pathways can help assess whether degradation products have been investigated adequately.

C. Screening Methods A method capable of screening various samples for a broad range of impurities is usually desired. Reversed-phase HPLC with UV detection and gradient elution is recommended to cover a broad polarity range and is often employed for impurity screening. Screening laboratory samples with gradient elution over a wide polarity range is useful in establishing the presence or absence of both polar and nonpolar impurities. The gradient range can then be adjusted accordingly to provide elution of the most nonpolar (i.e., the mostretained) impurities. When gradient baseline artifacts are problematic, it can be useful to employ dual isocratic screening methods. Low solvent strength systems may be needed to separate relatively polar compounds while a stronger solvent strength system can be used to determine nonpolar impurities. Compatibility of the mobile phase with mass spectrometric detection is highly desirable for providing information to help identify unknown components that may appear in the chromatogram. Other separation or detection modes may be necessary, depending on the nature of the drug substance, but the key concept here is to develop and use methodology that will provide the greatest overall separation and most universal detection.

D. Targeted Methods It is ideal when a screening method is capable of detecting all potential impurities, but this is rarely the case. An obvious example is encountered with single-enantiomer drug substances where a chiral method must be used to determine low levels of the undesired isomer. Such methods can be considered ‘‘targeted’’ toward a speciﬁc impurity rather than used for general screening purposes. There are abundant examples of chiral methods in the literature so they will not be discussed further here. Determination of other impurities that are not related in structure to the drug substance, such as metal catalysts or inorganic salts used in the synthetic process, usually requires targeted methods since they are not likely to be detected with the screening method. Targeted methods are often used during development to help deﬁne the overall control strategy even if the methods are not ultimately needed for routine use. Impurities that are known or suspected to be toxic present a special concern during development. The goal for investigation of such impurities is to demonstrate that they are undetectable or well below an appropriate level of concern. A targeted method is usually required for investigation of these impurities.

E. Focused Methods for Control Use One of the main goals of impurity investigations is to determine which impurities need to have speciﬁcation limits for routine monitoring.

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Knowledge gained with the screening and targeted methods described above can be used to establish the points in a process at which signiﬁcant impurities need to be controlled. When the synthetic process and product formulation are ﬁnalized and impurity investigations are completed, the analytical methods needed to monitor impurities can be optimized. For example, a broad polarity range gradient might be used as a screening method in the search for impurities. If there are no signiﬁcant late-eluting impurities, the gradient range and time can be optimized to reduce run time and provide ruggedness across different laboratories. An isocratic method might even be possible if it can determine all of the important impurities. These focused methods can then be registered in marketing applications and used in quality control laboratories for product release and stability. Although the conditions of targeted methods used during development may not require focusing, the number of targeted methods to carry forward for routine control laboratory use needs to be determined. This decision is based on the results obtained during development. Targeted methods are usually not required if they were used to demonstrate the absence of a postulated impurity in several development batches, including validation batches. Good manufacturing practices and maintenance of a validated process provide assurance that such postulated impurities will not suddenly appear. Determination of some impurities using targeted methods may need to be continued routinely because of regulatory expectations. The set of broad screening and targeted methods can be used as necessary to investigate impurities in connection with process changes or deviations. As recommended above for initial impurity investigations, chemical considerations should ﬁrst be used to guide the investigation by postulation of new impurities that may require modiﬁed or new methods. If new impurities are highly unlikely, the existing methods can be used to establish comparability of material before and after a change or a deviation from the established process. These results can also demonstrate that focused methods for routine use are still valid, i.e., the important impurities are detected.

F. Pharmacopeial Methods Speciﬁcations (tests, methods, and acceptance limits) for impurities given in pharmacopeial monographs provide public standards for generic suppliers of drug substances and drug products. Since drug substances can usually be prepared using many different synthetic routes with varying degrees of puriﬁcation, the suitability of monograph methods to monitor the purity of a drug substance from a particular supplier is an important issue. The monograph method for related substances or compounds is often focused on the impurities found to be important in material from the monograph sponsor, usually the innovator company. For example, ﬂuoxetine hydrochloride may be synthesized using routes that can produce different impurity proﬁles.15 Some of these differences can be discerned by the isocratic monograph impurity method while others require the use of a nonofﬁcial gradient method.16 These differences highlight the need to conduct an

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impurity investigation as described above for drug substances that may be of different origin than that used to establish the monograph methods. The investigation may indeed show that the methods are suitable or it may show the need for different or additional methods. This approach is taken in the European Pharmacopoeia process for certiﬁcation of suitability of drug substances from different sources. There are certainly areas of overlap of the technique-oriented and the chemistry-guided approaches. For example, chemistry-guided approaches still require a multitechnique or multidimensional approach, although the selection of which additional techniques are used is driven by scientiﬁc evaluation rather than a predetermined scheme. It should be noted that neither approach can guarantee that all impurities have been accounted for. More detailed descriptions of the chemistry-guided approach are given below in separate sections on process impurities and degradation products. Examples illustrate how this approach can provide assurance that impurities have been investigated in a thorough and efﬁcient manner.

III. PROCESS-RELATED IMPURITIES A generalized scheme for the synthesis of a drug substance is shown in Figure 1. Every reactant, reagent, solvent, intermediate, and by-product can be considered a potential impurity and should be addressed. Not all byproducts are known when development begins, so efforts to discover and identify them must be included in the investigation of impurities. As described above, one of the main goals of process-impurity investigations is to determine which of the many potential impurities are important and how these should be controlled during the synthetic process to produce a high-quality drug substance. A simpliﬁed way of looking at this goal is shown in Figure 2. Knowledge of impurities introduced through starting materials and reagents, those formed during processing, and those that carry through to the drug substance enables development of reaction conditions, criteria for forward processing and ﬁnal speciﬁcations. Stability

FIGURE 1 Generalized synthetic scheme for a drug substance.

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FIGURE 2 Development of an impurity control strategy for drug substances.

characteristics will dictate appropriate storage conditions needed to maintain the quality of the drug substance. As with the technique-oriented approach, a screening method that detects the compounds involved in the synthesis is typically the starting point for the chemistry-guided approach to impurity investigations. In addition to the drug substance, each isolated intermediate is usually examined to determine impurities present at that point in the synthesis. The nature of the intermediates will determine whether a similar analytical screening method may be used at each step or whether quite different methods/conditions are required. Unknowns are often discovered using the screening method. The conditions of the screening methods can be modiﬁed if needed for different steps of the synthesis. More speciﬁcally, tailored methods can be developed to focus on known or postulated impurities not detected using the screening conditions. Process-related impurities resulting from impurities in reagents, solvents, and starting materials are also possible. The development of selective methods to detect these impurities serves to broaden the scope of the impurity investigation beyond just the known intermediates and by-products and increases the likelihood of discovering other unknowns. A key aspect of the chemistry-guided approach is the determination of impurity origin and fate at each synthetic step. Analysis of reaction mixtures, ﬁltrates, and isolated intermediates can provide valuable information concerning these questions. Determination of the identity and mechanism of formation of an impurity can lead to modiﬁcation of reaction conditions that can minimize or eliminate the impurity. The effectiveness of puriﬁcation procedures such as crystallization and washing can be determined by analysis of ﬁltrates and wash solutions. Showing effective control of an impurity at an intermediate step can make it possible to rule out the impurity as a possibility in the ﬁnal drug substance. For example, referring to Figure 1, a negligible amount of starting material B present in intermediate C eliminates the need to monitor B in the drug substance. Conversely, if an impurity is difﬁcult to remove, the investigation will determine what acceptance limits must be instituted at intermediate steps to assure that acceptable levels will be present in the drug substance. As impurity investigations proceed, laboratory and scale-up samples from throughout the process should be analyzed for impurities. Signiﬁcant unknowns that are discovered are identiﬁed. As knowledge about impurities

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increases, the synthetic process may be modiﬁed to reduce impurity levels and improve reproducibility. These efforts will, in turn, generate further samples for evaluation. Depending on the nature of the process change, modiﬁcations of the screening method or new methods may need to be considered. This iterative cycle continues as the process and methods approach their ﬁnal forms for registration and commercial production. The following example illustrates this strategy. The synthetic scheme for LY297802 tartrate is shown in Figure 3. The intermediates and drug substance were used to develop initial separation conditions compatible with mass spectrometric detection (Figure 4). Samples

FIGURE 3 Partial synthetic scheme for LY297802 tartrate.

FIGURE 4 Separation of LY297802 and intermediates, using LC-MS compatible conditions. Conditions:YMC basic column, 25 cm 3 4.6 mm id, 5 m; A 5 0.05% trifluoroacetic acid in water, B 5 0.05% trifluoroacetic acid in acetonitrile, gradient elution from 25^60% B in 15 min, hold 10 min; 1.0 mL/min, 280 nm.Unlabeled peaks were unknowns.

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of isolated intermediates, reaction solutions, and ﬁltrates were also amenable to analysis using similar LC-MS conditions. Analysis of laboratory samples revealed several unknowns. Molecular weight information obtained via mass spectrometry was used to tentatively identify unknown impurities. Ultraviolet spectra obtained from photodiode array detection were also used to support the identiﬁcation in some cases. Some of the impurities detected had been predicted by examination of the process chemistry and the identities of others were postulated from the mass spectral data. The suspected impurities were then synthesized for comparison with the components observed in samples. Identical retention times, UV spectra, and mass spectra provided strong evidence for positive impurity identiﬁcation. A summary of related substance impurities investigated for LY297802 is given in Figure 5. Two impurities were identiﬁed in the reaction of I1 to form I2 (Figure 6). The hydroxylated and elimination by-products were able to undergo the next reaction in the synthesis in the same way as the desired monochloro intermediate. Therefore, the impurities at this step would lead to the analogous impurities in the drug substance unless the initial impurities were less reactive or puriﬁcation was effected at some point in the process. To determine the signiﬁcance of the hydroxy and elimination by-products at the intermediate step, it was necessary to determine at what levels the downstream impurities, BP1 and BP2, were present in the ﬁnal drug

FIGURE 5 LY297802-related substance impurities.

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FIGURE 6 Impurities formed during preparation of I2. BP1 and BP2 are the corresponding impurities in the drug substance.

substance. Impurities BP1 and BP2 were synthesized and their chromatographic retention properties examined using the LC-MS screening conditions. The hydroxy by-product, BP1, was separated from the drug substance, but the elimination by-product, BP2, coeluted with the drug substance. Therefore, the low pH LC-MS screening conditions could not be used to determine levels of BP2 in the drug substance. Determination of BP2 coeluted with the drug substance peak might have been possible with mass spectrometric detection, but a simpler method with separation of the impurity was desired. The effect of pH was investigated to check the potential to improve separation of the drug substance and BP2. Mobile phase pH can be a powerful tool for changing the separation selectivity of acidic or basic compounds. This was indicated in a preliminary evaluation of retention versus pH for I1, I2, and LY297802. Based on the potential for a change in selectivity, a pH 5.0 mobile phase was used for the separation of BP2 and other potential impurities. Figure 7 shows that BP2 as well as almost all other impurities were separated using these conditions. The pH 5 conditions revealed that signiﬁcant levels of BP2 appeared in laboratory samples of drug substance. Additional process optimization was performed to reduce the hydroxy and elimination by-products formed in the intermediate reaction to insigniﬁcant levels. Further control could be provided with limits on the impurities in isolated I2. Response factors and detectability of potential impurities must also be considered during impurity investigations. A potential impurity in the thiolformation reaction was the hydrido compound, BP5. Although well separated using the pH 5.0 screening conditions, this impurity had a very low response at 280 nm. The response at 250 nm was much higher and provided a better means of tracking the impurity. Figure 8 shows chromatograms recorded at 250 and 280 nm for crude and recrystallized drug substance. While the hydrido impurity was detectable at 280 nm in crude material, detection at

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FIGURE 7 Screening for LY297802 impurities using pH 5 mobile phase. Conditions: YMC basic column, 25 cm 3 4.6 mm id, 5 mm; A 5water/acetonitrile/acetic acid/triethylamine 950/ 50/3/5, B 5 water/acetonitrile/acetic acid/triethylamine 100/900/3/5, gradient elution from 0 to 100% B in 30 min; 1.0 mL/min, 280 nm.

FIGURE 8 Impurity profiles demonstrating superior detectability of BP5 at 250 nm. Conditions as in Figure 7 with extended hold time at final conditions.

250 nm afforded a more sensitive means of establishing the effectiveness of the recrystallization in removing this impurity. The need for a targeted method arose because of the n-butyl chloride reactant used in the synthesis of LY297802. This starting material contains as impurities the n-propyl and n-pentyl chloride homologs as well as the isobutyl isomer. Since these impurities will react to form the corresponding impurities in the drug substance (SM1–3), the quality of the n-butyl chloride

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FIGURE 9 Targeted method for determination of the isobutyl isomer of LY297802. Conditions:YMC ODS column, 25 cm 3 4.6 mm id, 5 mm; 77% trifluoroacetic acid (0.05%) in water, 23% tetrahydrofuran,1.0 mL/min.

must be controlled. It is important in terms of speciﬁcations and cost to determine what levels of reactive impurities can be tolerated in the n-butyl chloride to provide acceptable drug substance. This is usually done by spiking the starting material impurities at various levels into reactions and checking the extent of formation and carry-through of the corresponding reaction product impurities. In the case of n-butyl chloride, the homologous reaction products (SM1 and SM3) were separated from LY297802 in the pH 5 screening method, but the isobutyl isomer impurity (SM2) was not. The spiking study required a method capable of separating the isobutyl analog of LY297802. The necessary selectivity was obtained using tetrahydrofuran as a mobile phase modiﬁer rather than acetonitrile (Figure 9). These conditions could be used to determine the relationship between the amount of isobutyl chloride in the starting material and the amount of SM2 in the drug substance, thereby establishing a sound basis for a starting material impurity speciﬁcation limit. Appropriate control of isobutyl chloride in the starting material eliminates the need to routinely monitor SM2 in the drug substance. For impurities that are suspected to be toxic, it is desirable to demonstrate their absence in an intermediate compound early in the process, if possible. A speciﬁcation for the impurity in the drug substance may be necessary if the impurity is introduced late in the process or can carry through to a late intermediate or ﬁnal step. Methods for toxic impurities are usually targeted to determine the speciﬁc impurity at trace levels. An appropriate detection limit must be based on toxicological considerations of the impurity as well as the dose, formulation, and indication of the drug. For example, a toxic impurity in a drug for chronic use may be of concern at lower levels than in a drug for an acute indication. An example of use of a targeted method is the determination of a trace impurity in paroxetine HCl (Figure 10).17 The impurity is very similar in

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FIGURE 10 Paroxetine and trace level impurity.

structure to a neurotoxin known to cause Parkinsonism.18 A method utilizing HPLC (70-minute gradient elution proﬁle) with dual-stage mass spectrometric detection was described. The daughter ion with mass-to-charge ratio 44 produced from the parent ion with mass-to-charge ratio 192 was monitored. The limit proposed for the impurity was 5 ppm. Another example is the determination of mesylate esters as impurities in drug substances isolated as mesylate salts. Figure 11 shows GC-MS chromatograms obtained using single ion monitoring for methyl and isopropyl mesylate in a drug substance. Standards at concentrations corresponding to conservative safety limits gave readily detectable signals. No responses for the mesylate esters were observed in several samples, helping to establish the absence of the impurities in the process.

IV. DEGRADATION-RELATED IMPURITIES As outlined in Table 3, the goals of degradation-related impurity investigations are parallel to the goals of process-related impurity investigations. ‘‘Potential’’ degradation products, i.e., those formed upon stressing or forcing conditions, are analogous to ‘‘potential’’ or postulated process-related impurities resulting from the synthetic reactions utilized to create the drug substance compound. Understanding how the ‘‘potential’’ degradation products form (via speciﬁc degradation pathways or mechanisms) provides a basis for minimizing or eliminating degradation (e.g., through processing conditions, formulation, storage conditions, or packaging). Degradation studies also provide for the development of analytical methods that are ‘‘stability indicating.’’ Thus, degradation-related impurity investigations need to start early in development to enable a clear understanding of stability issues and to provide for a safe and efﬁcacious product during clinical trials, eventually leading to a marketed product.

A. Definitions It is important to have a clear deﬁnition of terms to facilitate the discussion. The ICH guideline on stability19 differentiates the terms ‘‘stress testing’’ and ‘‘accelerated testing,’’ which have historically been used interchangeably. The guideline deﬁnes accelerated testing as ‘‘Studies designed to

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FIGURE 11 GC-MS determination of mesylate esters in a drug substance. A 5 isopropyl

mesylate, B 5 methyl mesylate; chromatograms (from bottom to top): blank, standard solution, drug substance sample solutions. Conditions: DB-WAX, 30 m 3 0.32 mm 3 0.25 mm film; oven temperature held at 90 C for 3 min, increased to146 C at 8 C/min, increased to 180 C at 20 C/min, and then held isothermal for 2 min; injector temperature 5 250 C; flow rate 2 mL/min splitless with purge on at 0.2 min. Isopropyl mesylate was monitored at 79 and 123 m/z and methyl mesylate was monitored at 80, 95, and 110 m/z.

increase the rate of chemical degradation or physical change of an active drug substance or drug product using exaggerated storage conditions as part of the formal, deﬁnitive, storage program . . . .’’ For drugs to be stored at room temperature, i.e., 25 C, accelerated testing is deﬁned as 40 C / 75% relative humidity. For other storage conditions, accelerated testing is to be carried out at 15 C above the long-term storage temperature. Accelerated testing, along with long-term testing (formal stability studies under normal storage conditions) is part of the formal stability studies required for regulatory submission. In contrast, the guideline differentiates stress testing as an investigation of the stability of the drug molecule that provides the foundation for the formal, deﬁnitive stability studies. Stress testing is distinguished by both the severity of the conditions and the focus or intent of the results. Thus, the guideline indicates that stress testing is intended to ‘‘. . . determine the intrinsic stability of the molecule by establishing degradation pathways in order to identify the likely degradation products and to validate the stability-indicating power of the analytical procedures used.’’ A longer, more detailed deﬁnition is also given in the guideline. Determination of the ‘‘intrinsic stability’’ characteristics of the drug molecule

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includes developing an understanding of (1) conditions leading to degradation, (2) rates of degradation, (3) the chemical structures of the degradation products, and (4) the degradation pathways. Understanding the ‘‘intrinsic stability’’ characteristics of the drug molecule allows for prediction of stability-related concerns. This information can help direct both formulation and packaging development.

B. Strategy The overall strategy for investigation and control of degradation-related impurities can be illustrated as shown in Figure 12. Stress testing studies provide the foundation for the overall strategy. Various parts of the strategy are explored in more detail below. Stress testing studies involve exposure of the drug substance (and/or formulated drug product or drug-excipient mixtures) to the stress conditions of heat, humidity, photostress (UV and VIS), oxidative conditions, and aqueous conditions across a broad pH range. The intent is to induce 10–20% degradation of the parent drug. It is acceptable to conclude that a molecule is stable if no degradation occurs under reasonably stressing conditions. The inherent dilemma here is how to measure the degradation and detect degradation products prior to development of appropriate methods for the purpose. Without stress testing there is no way to assess whether or not the method will resolve and detect the degradation products. As discussed previously in the chapter, there are two major analytical approaches to the search for degradation-related impurities: chemistry-guided

FIGURE 12 Illustration of the overall strategy for investigating and controlling degradationrelated impurities.

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and technique-oriented. The technique-oriented approach involves the use of multiple analytical techniques to increase the chances of detecting unknown impurities. The chemistry-guided approach involves a scientiﬁc evaluation of the possible degradation pathways of the drug substance and choosing analytical techniques appropriate for the proposed degradation chemistry. The chemistry-guided approach should be iterative, i.e., it should involve multiple evaluation points as more information is gathered about the conditions leading to degradation and about the structures and spectroscopic characteristics of the resulting degradation products that are detected. As degradation products and pathways are elucidated, this information is used to reevaluate the relevance of the analytical methods. For example, if a drug degrades under a certain condition to yield a product whose structure indicates that a cleavage has occurred in the parent molecule resulting in (at least) two products but only one is detected, then the analytical conditions may need to be modiﬁed or a different analytical technique (or detector) may need to be employed as part of the investigation. Ideally, a method that resolves and quantitatively detects the parent drug and all the degradation products is desired. In reality, RP-HPLC with UV detection is by far the most common analytical technique currently used for detection of degradation and impurities. A good starting point for analyzing stressed samples is a broad screening RP-HPLC method with UV detection. Characteristics of such a method would include photodiode array UV detection, UV-transparent buffers for monitoring at low wavelengths (to increase universality of detection), and gradient elution to cover a wide polarity range. Such a method maximizes the chances of resolving, eluting, and detecting both polar and nonpolar degradation products. The broad screening method can be developed/optimized by analysis of partially degraded samples and the use of standard method development procedures and tools (e.g., optimization of the gradient, mobile phase solvent system, choice of column). The analysis of the stressed samples should reveal the degradation products formed under the various conditions. As information on the structures of the degradation products becomes available, degradation pathways can be proposed and evaluated. It should be remembered that the information about the stability and degradation of the drug substance from stress testing studies is predictive in nature (as opposed to deﬁnitive). That is, the degradation products observed during stress testing may or may not be relevant to actual storage conditions of the drug substance and/or to the degradation chemistry of the formulated product. As illustrated in Figure 13, the degradation products observed are ‘‘potential’’ degradation-related impurities. The ‘‘actual’’ or ‘‘signiﬁcant’’ degradation-related impurities that occur during long-term storage or shipping (as revealed by accelerated testing and long-term stability) are the impurities that must be controlled. If the stress-testing studies are thorough and well designed, the ‘‘signiﬁcant’’ degradation-related impurities should be a subset of the ‘‘potential’’ degradation products observed during stress testing. Focused methods can then be developed and optimized to monitor the ‘‘signiﬁcant’’ degradation products for regulatory registration in marketing applications and use in quality control laboratories for product release

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FIGURE 13 Cartoon illustration of hypothetical stress testing and accelerated or long-term storage stability HPLC chromatograms. Degradation products A^I are the degradation products formed during stress testing and are therefore classified as ‘‘potential’’ degradation products. Products B, C, D, E, and G are the products formed at significant levels during formal stability studies and are classified as the ‘‘actual’’or ‘‘significant’’degradation products.

and stability. The validity of this approach is apparent from an evaluation of the ICH deﬁnition of stress testing: . . .It is recognized that some degradation pathways can be complex and that, under forcing conditions, decomposition products may be observed that are unlikely to be formed under accelerated or long-term testing. This information may be useful in developing and validating suitable analytical methods, but it may not always be necessary to examine speciﬁcally for all degradation products if it has been demonstrated that in practice these are not formed.

The information gathered during stress testing of the drug substance is also used to guide the formulation of the drug product. As shown in Figure 12, drug-excipient compatibility studies should be performed to determine whether excipient blends or individual excipients have any adverse interactions with the parent drug. A broad screening method such as that developed for drug substance stress testing should be part of the analytical strategy to examine for excipient-catalyzed drug degradation. Once a suitable lead formulation has been developed, stress testing studies can be performed on the formulation leading to the detection/identiﬁcation of ‘‘potential’’ degradation products. In an analogous manner to the strategy for the drug

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TABLE 4 Recommended Conditions for Assessing Hydrolytic,Thermolytic, and Photolytic Degradation Pathways

Solid state

Thermolytic

Hydrolytic

Photolytic

55, 70, 85 C, Low humidity (e.g., < 30% RH or no humidity control); 4–6 weeks

55, 70, 85 C, High humidity (e.g., > 70% RH); 4–6 weeks

5–10 the ICH min. confirmatory exposure; (1) thin layer of powder; (2) thin film (optional)

Aqueous solutions or slurries, pH 1–13, RT-70 C, 2–4 weeks

Aqueous solutions or slurries, 5–10 the ICH minimum confirmatory exposure; at pH’s above and below relevant pK’s.

Solution

substance, the ‘‘actual’’ or ‘‘signiﬁcant’’ degradation products can be determined during accelerated and long-term stability studies. The key to the strategy outlined above is to have well-designed stress testing studies that form all potential degradation products. Thorough stress testing studies need to evaluate the four main degradation pathways of pharmaceuticals: (1) hydrolytic, (2) thermolytic, (3) photolytic, and (4) oxidative. Uncovering the potential hydrolytic, thermolytic, and photolytic degradations is relatively straightforward. The conditions outlined in Table 4 are suggested as practical stress conditions for assessing these pathways. The oxidative pathways are a bit trickier, and will be discussed in more detail later in this chapter.

C. Thermolytic Degradation To evaluate thermolytic pathways, elevated temperatures (e.g., 50–80 C) in the solid state and/or in solutions can be used. Above 80 C many compounds begin to degrade via different mechanisms, giving rise to degradation products that may not be observed under ordinary storage or shipping conditions. For solid-state stressing, the use of high- and lowhumidity atmosphere at the elevated temperatures is appropriate, and both thermolytic and hydrolytic pathways may be observed under such stressing conditions. Similarly, when aqueous solutions are stressed at elevated temperatures, both thermolytic and hydrolytic pathways may be observed.

D. Hydrolytic Degradation To evaluate hydrolytic pathways, aqueous solutions (using 100% aqueous conditions when solubility permits or adding an inert co-solvent such as acetonitrile) can be prepared at various pH conditions (e.g., pH 1–13)

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and samples can be stored at elevated temperatures if no (or slow) degradation is observed at room temperature.

E. Photolytic Degradation The evaluation of photolytic pathways should be performed in both the solid state and in solution. Samples should be photostressed by exposure to irradiation signiﬁcantly beyond the ICH minimum recommended conﬁrmatory levels,20 both in the ultraviolet and visible spectral regions. The photoexposure should mimic a worst-case scenario, and thus we recommend exposure to simulated outdoor sunlight (D65), as per Option 1 of the ICH Q1B photostability guideline. An excellent resource for conducting such studies can be found in a two-part review article by Thatcher et al.21,22

F. Oxidative Degradation As mentioned previously, oxidative degradation pathways can be complex, and it is useful to consider the chemistry of oxidative degradation. The oxidative degradation of pharmaceuticals has been discussed in the literature,23–27 and we assert that there are three major pathways, (1) autoxidation or radical-mediated oxidation, (2) peroxide-mediated, and (3) photochemically induced (see Table 5). Traditionally, dilute aqueous peroxide solutions have been used for oxidative stress testing of pharmaceuticals. Landmark papers by Boccardi23,24 in 1992 and 1994 identiﬁed autoxidation as the major oxidative degradation pathway for pharmaceuticals. Boccardi showed that the use of radical initiators such as

TABLE 5 Three Major Oxidative Degradation Pathways 1. Autoxidation

2. Peroxide-mediated oxidation

3. Photochemically induced oxidation

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azobisisobutyronitrile (AIBN) gave results more predictive of autoxidative degradation than dilute peroxide solutions. It is worth noting here that radical-initiated oxidative chemistry can also be induced by transition metals, and stressing of the drug substance with iron(III) and copper(II) (in separate stress experiments) is recommended. In addition to autoxidative processes, peroxide-mediated oxidative degradation can occur that may not be observed using radical initiators as described above. For example, tertiary nitrogens are often observed to oxidize to N-oxides during long-term storage, but may or may not be observed during stress testing using radical initiators in aqueous/acetonitrile solutions. Since most tertiary nitrogens would likely be in the cationic protonated form under neutral aqueous conditions, the protonation undoubtedly helps to protect the nitrogen from oxidation. The use of pressured oxygen (e.g., 150 psi) in conjunction with radical initiators appears to aid in the formation of N-oxides.28 The formation of N-oxides can occur via direct oxidation29 as a result of the presence of peroxides in the excipients (e.g., PEG,30,31 polysorbates,32 and povidone33). Therefore, the use of dilute aqueous hydrogen peroxide to oxidatively stress the drug substance is appropriate. Room temperature is sufﬁcient for such stressing, and elevated temperatures are not recommended because the oxidative degradation pathways catalyzed by peroxides at high temperature may not be predictive of real-world degradation. The third major oxidative pathway is photochemically induced oxidation (see Table 5). Such oxidation can occur from direct reaction of ground-state oxygen with the electronically excited state of the drug molecule or from photosensitization of triplet oxygen to singlet oxygen and direct reaction with the drug molecule. Photolytic stressing of the drug substance, as described above for assessing photolytic degradation pathways, is effective for the prediction of these oxidative pathways.

G. Common Problems Encountered During Stress Testing with HPLC-UV Although RP-HPLC with UV detection is by far the most common analytical technique used to search for and analyze degradation-related impurities, it presents signiﬁcant problems faced by virtually every analytical researcher. Questions such as (1) Is everything being eluted off the column?, (2) Are there nonchromophoric products?, (3) What are the responses of the unknown impurities?, and (4) Are all the impurities amenable to the separation technique (e.g., solubility, volatility, stability)?, need to be asked. These questions all revolve around the concept of ‘‘mass balance,’’ i.e., The process of adding together the assay value and levels of degradation products to see how closely these add up to 100% of the initial value, with due consideration of the margin of analytical precision.34 Since during impurity investigations (e.g., stress testing/degradation studies), the response factors and other characteristics of the impurities are usually unknown, the researcher will undoubtedly confront questions about mass balance. One practical example of a problem related to mass balance is shown in Figure 14, where aqueous solutions (at pH 7) of drug substance A were

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FIGURE 14 RP-HPLC-UV chromatogram (using an isocratic method) of an aqueous solution of drug substance A after stressing at 70 C for 14 days at pH 7.

stressed at 70 C for up to 14 days. RP-HPLC-UV analyses were performed using a previously developed isocratic method and the results indicated a loss of parent drug with only a minor increase in related substances (day 14 showed 4.9% degradation with only a 1.4% increase in impurities, calculated using the UV response factor of the parent drug). Close examination of the parent peak revealed a shoulder on the ‘‘tail’’ of the peak, indicating partial co-elution of a degradation product. To investigate this mass balance discrepancy, a broad-screening gradient HPLC method was developed. Analysis of the day-14 sample using this method (see Figure 15) revealed ﬁve additional degradation products not observed using the isocratic method, accounting for an increase in detected impurities of approximately 3%. This result is a dramatic example of the importance of using gradient HPLC methodology for impurity investigations. The use of highly resolving broadscreening gradient methods, with monitoring at low wavelengths using a PDA, can help to maximize the chances of resolving and detecting a wide range of impurities. Another example of a real-world problem encountered during degradation studies is illustrated in Figure 16. HPLC analysis of an aqueous solution of a drug substance (LY297802, Figure 18) after exposure to cool white ﬂuorescent light during a photostability study revealed a substantial loss of parent drug with no apparent degradation products. PDA-UV analysis of all wavelengths between 200 and 400 nm did not reveal any degradation-related peaks. Obviously there is a signiﬁcant ‘‘mass balance’’ issue! Closer examination of the photostressed solution revealed a hazy, insoluble ﬁlm adhering to the surface of the glass solution container. The ﬁlm was collected and analyzed by probe EI-MS, which quickly revealed that the ﬁlm was elemental sulfur (S8). Since LY297802 contains a sulfur atom in the thiadiazole ring, it was apparent that the chromophoric thiadiazole moiety

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FIGURE 15 RP-HPLC-UV chromatogram (using a gradient method) of an aqueous solution of drug substance A after stressing at 70 C for 14 days at pH 7.

FIGURE 16 HPLC-UV chromatograms of LY297802 after 3 days and 7 days in a photostability chamber (cool white fluorescent lamps, 17,000 lux). Conditions: Zorbax RX-C8 column, 25 cm 3 4.6 mm id, 5 mm; A 5 buffer/acetonitrile 95/5, B 5 buffer/acetonitrile, 25/ 75; buffer 5 25 mM potassium phosphate with pH adjusted to 6.5 with NaOH,UVdetection using photodiode array withWaters Maxplot2 200 ^ 400 nm; gradient elution from 0% Ato100% B in 30 min, 1mL/min flow rate.

was being photodegraded to nonchromophoric and/or volatile products. When LC-MS analysis failed to detect any new peaks, another separation technique that was amenable to volatile compounds (GC) was employed. Hexane extraction of the aqueous light-degraded solution, previously made basic to give the free base of LY297802, followed by GC-FID analysis revealed two major degradation products in the degraded samples (Figure 17). GC-EI-MS analysis quickly revealed the ‘‘missing’’ degradation products as n-butyl-thiocyanate and 1-aza-bicyclo[2.2.2]octane-3-carbonitrile (see Figure 18).

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FIGURE17 GC^FID analysis of a hexane extract from a photodegraded sample of LY297802.

Conditions: DB-1, 30 m 3 0.53 mm, 3 mm film thickness; oven temperature held at 40 C for 2 min, ramp to 70 C at 5 C/min, then ramp to 260 C at 10 C/min, hold isothermal for 2 min; detector FID at 300 C, helium carrier at 20 cc/min, helium makeup at 10 cc/min; 1 mL injection volume.

FIGURE 18 Structures of LY297802 and its photodegradation products.

The examples described here are illustrative of the ‘‘chemistry-guided approach.’’ An initial analysis is made based on good analytical chemistry, the results are evaluated carefully, and further steps are guided by scientiﬁc judgment. As the structures of degradation products are determined, the pathways of degradation can usually be proposed. The pathways help determine whether all the degradation products have been detected and what other possible products might be formed. These pathways and the conditions under which they occur form the basis for understanding the ‘‘intrinsic stability’’ characteristics of the compound, which once developed can be used to guide analytical and other development activities.

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H. Detectors The basic problem related to the determination of mass balance is the differential response of detectors to different compounds. As outlined in the ICH guidelines on impurities, it is acceptable to use the response factor of the drug substance to estimate levels of unknown impurities.1–4 In many cases, however, the response factor differences can be substantial and therefore, even with results that appear to show ‘‘mass balance,’’ uncertainty exists. The use of alternate detectors is one practical strategy for investigating issues related to mass balance. HPLC with MS detection is commonly employed as an alternate detector to UV. Mass spectrometry is generally regarded as a universal detector, but the response per unit weight depends greatly on the ionization type (e.g., positive or negative electrospray, atmospheric pressure chemical ionization, etc.) and on the ionization efﬁciency of the analyte under the given conditions. Three HPLC detectors that are viewed as giving a response that is proportional to mass are chemiluminescent nitrogen (CLN), evaporative light scattering (ELS), and refractive index (RI) detectors. RI detectors are useful for isocratic HPLC, but they suffer from variability in response, depending on the mobile phase composition, temperature, and dissolved gases, and are not very sensitive.35,36 ELS detectors should detect most nonvolatile substances, but the response will depend on the quantity and characteristics of the particles formed during solvent evaporation. For compounds of similar structures, one can expect response factors to be similar (þ/ approximately 10–20%). When compounds are substantially different, however, the responses can vary more than 10-fold.37 The CLN detector is based on combustion of the HPLC efﬂuent in an oxygen-rich furnace to produce nitric oxide from nitrogen-containing compounds. The nitric oxide is reacted with ozone to produce nitrogen dioxide in an excited state, and photons are emitted upon return to the ground state. This chemiluminescent response is proportional to the number of moles of nitric oxide, and correspondingly to the number of moles of nitrogen in the analyte (with the exception of N2 and N ¼ N bonds). Nitrogen-containing compounds will therefore produce a signal that is directly related to the number of moles of nitrogen present. As long as the molecular formula of the unknown is known, the mass of the unknown can be determined. Recent articles by Nussbaum et al.37 and Korner38 describe the capabilities and limitations of this detector in more detail. The use of alternate detectors is an important tool to consider when investigating degradation-related impurities. If mass balance can be achieved using a detector that provides a response that is proportional to the mass of the analyte, such as the CLN, further investigations using other analytical techniques can be avoided.

I. Drug Product Stress Testing Developing an understanding of the intrinsic stability of the drug substance does not eliminate the need for speciﬁc stress testing of the

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formulated product. Indeed, the literature is replete with examples of unexpected and unprecedented surprises from the formation of degradationrelated impurities speciﬁcally related to the excipients or packaging components, a few of which are cited here.39–41 Drug product stress testing begins with drug-excipient compatibility testing, although a discussion of this topic is beyond the scope of this chapter. An article by Serajuddin et al. provides an excellent discussion of drug-excipient compatibility testing.42 Stressing of the formulated product, both inside and outside of the packaging material is critical to avoiding surprises during formal stability studies that can affect the development time line. Stressing of the drug product should involve evaluation of elevated temperature, humidity, and photolytic stress as a minimum. It is also prudent to evaluate the effects of the solvents and pH conditions used during analytical sample preparation for assay and related substances testing. An interesting example of the identiﬁcation of a formulation-related impurity is described by Jansen et al. for the case of duloxetine hydrochloride.43 Duloxetine hydrochloride is a secondary amine that is a potent inhibitor of both serotonin and norepinephrine reuptake. Since the drug is unstable in acid, oral dosing requires encapsulation within an enteric coating that consists of hydroxypropylmethylcellulose (HPMC) and HPMC acetate succinate (HPMCAS). During the stress-testing studies, a signiﬁcant level of the succinamide impurity was formed, as shown in Figure 19. This formation occurred in spite of the fact that duloxetine was physically separated from the enteric coating by a barrier layer (see Figure 20) and did not react with succinic acid in stress studies. It was postulated that succinic anhydride was

FIGURE 19 Structures of duloxetine hydrochloride and a low-level impurity formed upon exposure of enteric-coated pellets to accelerated conditions of 40 C/75% relative humidity.

FIGURE 20 Cartoon representation of a cross-sectional view of the duloxetine hydrochloride enteric-coated pellets.

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FIGURE 21 Proposed pathway for formation of duloxetine succinamide.

cleaving from the HPMCAS backbone and that migration of either the drug substance or the anhydride facilitated the reaction (see Figure 21). The levels of this impurity that were forming under accelerated conditions threatened the viability of the formulation and the development time line, but the researchers were able to minimize the formation of the impurity by increasing the thickness of the barrier layer. It is clear that knowledge of the structure of this degradation product was critical to the development of an acceptable formulation for the marketed product.

V. SUMMARY AND CONCLUSIONS While the overall strategy for controlling process and degradation-related impurities should involve multiple analytical techniques, a chemistry-guided approach using the results in an iterative fashion can help determine which of the techniques will provide useful results in impurity-related investigations. For both process- and degradation-related impurities, the approaches described in this chapter can differentiate between those impurities that are ‘‘relevant’’ and those that are ‘‘potential.’’ A thorough knowledge of which potential process and degradation-related impurities are likely to be present after synthesis or upon storage (for both the drug substance and product) can be used to guide the development of efﬁcient and robust ‘‘focused’’ control methods. Such knowledge also guides the selection of appropriate packaging and long-term storage conditions. The ultimate goal of controlling impurities in drug products is to provide safe, pure, and effective drug products for the patient.

ACKNOWLEDGMENTS The authors gratefully acknowledge the technical contributions of Patrick Jansen, W. Kimmer Smith and Liu Yang.

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REFERENCES 1. International Conference on Harmonisation, Impurities in New Drug Substances, Q3A, January 1996. 2. International Conference on Harmonisation, Impurities in New Drug Substances, Draft, Q3A(R), February 2002. 3. International Conference on Harmonisation, Impurities in New Drug Products, Q3B, November 1996. 4. International Conference on Harmonisation, Impurities in New Drug Products, Draft, Q3B(R), October 1999. 5. Identification and Determination of Impurities in Drugs, Go´ro´g, S. (Ed.), Elsevier, Amsterdam, 2000. 6. Niessen, W. M. A. Chimia 53:478–483, 1999. 7. Hilhorst, M. J., Somsen, G. W. and de Jong, G. J. Electrophoresis 22:2542–2564, 2001. 8. Hilhorst, M. J., Derksen, A. F., Steringa, M., Somsen, G. W. and de Jong, G. J. Electrophoresis 22:1337–1344, 2001. 9. Ermer, J. and Vogel, M. Biomedical Chromatography 16:373–383, 2000. 10. Go´ro´g, S. Current Trends in Analytical Chemistry 1:11–18, 1998. 11. International Conference on Harmonisation, Specifications: Test Procedures and Acceptance Criterial for New Drug Substances and New Drug Products: Chemical Substances, Q6A, October 1999. 12. International Conference on Harmonisation, Impurities: Residual Solvents, Q3C, July 1997. 13. Go´ro´g, S., Babja´k, M., Balogh, G., Brlik, J., Csehi, A., Dravecz, F., Gazdag, M., Horva´th, P., Lauko´, A. and Varga, K. Talanta 44:1517–1526, 1997. 14. Giron, D. Boll. Chim. Farmaceutico 133:201–220, 1994. 15. Wirth, D. D., Miller, M. S., Boini, S. K. and Koenig, T.M. Org. Proc. Res. Dev. 4:513–519, 2000. 16. Wirth, D. D., Olsen, B. A., Hallenbeck, D. K., Lake, M. E., Gregg, S. M. and Perry, F.M. Chromatographia 46:511–523, 1997. 17. Pharmacopeial Forum, 26:193, 2000. 18. Markey, S. P. and Schmuff, N. R. Med. Res. Rev. 4:389–429, 1986. 19. International Conference on Harmonisation, Stability Testing of New Drug Substances and Products, Q1A, September 1994. 20. International Conference on Harmonisation, Stability Testing: Photostability Testing of New Drug Substances and Products, Q1B, November 1996. 21. Thatcher, S. R., Mansfield, R. K., Miller, R. B., Davis, C. W. and Baertschi, S. W. Pharmaceutical Technology, March 98–110, 2001. 22. Thatcher, S. R., Mansfield, R. K., Miller, R. B., Davis, C. W. and Baertschi, S. W. Pharmaceutical Technology, April 50–62, 2001. 23. Boccardi, G., Deleuze, C., Gachon, M., Palmisano, G., and Vergnaud, J. P. J. Pharm Sci. 81:2, 183, 1992. 24. Boccardi, G. Il Farmaco. 49:6, 431, 1994. 25. Hovorka, S. W. and Schoneich, C. J. Pharm. Sci. 90(3):253–269, 2001. 26. Johnson, D. M. and Gu, L. C. Autoxidation and antioxidants, In Encyclopedia of Pharmaceutical Technology (Swarbrick, J. and Boylan, J.C. Eds.) Vol. I, Marcel Dekker, Inc., New York, pp. 415–449, 1998. 27. Waterman, K. C., Adami, R. C., Alsante, K. M., Hong, J., Landis, M. S., Lombardo, F. and Roberts, C. J. Pharmaceutical Development and Technology 7:1, 1–32, 2002. 28. Unpublished results, Karen Alsante, Pfizer. 29. March, J. Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 3rd ed. John Wiley & Sons, New York, p. 1088, 1985. 30. Ginsburg, E. J., Stephens, D. A., West, P. R., Buko, A. M., Robinson, D. H., Li, L. C. and Bommiredi, A. R. J. Pharm. Sci. 89:6, 766–770, 2000. 31. McGinity, J. W., Hill, J. A. and LaVia, A. L. J. Pharm. Sci. 64:2, 356–357, 1975. 32. Chafetz, L., Hong, W.-H., Tsilifonis, D. C., Taylor, A. K. and Philip, J. J. Pharm. Sci. 73:8, 1186–1187, 1984.

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33. Hartauer, K., Arbuthnot, G., Baertschi, S. W., Johnson, R., Luke, W., Pearson, N., Rickard, E., Tsang, P. and Wiens, R. Pharmaceutical Development and Technology 5:3, 303–319, 2000. 34. International Conference on Harmonisation, Stability Testing of New Drug Substances and Products, Q1A, September 1994. 35. McNabb, T. J., Cremesti, A. E., Brown, P. R. and Fischl, A. A. Seminars in Food Analysis, 4:53–70, 1999. 36. Snyder, L. R., Kirkland, J. J. and Glajch, J. L. Practical HPLC Method Development, 2nd ed. John Wiley & Sons, NY, pp. 80–81, 1997. 37. Nussbaum, M.A., Baertschi, S.W. and Jansen, P.J. J. Pharm. Biomed. Anal., 27:983–993, 2002. 38. Korner, A., LC/GC, 20(4):364–373, 2002. 39. Qin, X.-Z, Ip, D. P., Chang, K. H.-C, Dradransky, P. M., Brooks, M. A. and Sakuma, T. J. Pharm. Biomed. Anal. 12(2): 2212–2233, 1994. 40. Wirth, D. D., Baertschi, S. W., Johnson, R. A., Maple, S. R., Miller, M. S., Hallenbeck, D. K. and Gregg, S. M. (1998). J. Pharm. Sci. 87(1): 31–39, 1998. 41. Schildcrout, S. A., Risley, D. S. and Kleeman, R. L. Drug Development and Industrial Pharmacy 19(10):1113–1130, 1993. 42. Serajuddin, A. T. M., Thakur, A. B., Ghoshal, R. N., Fakes, M. G., Ranadive, S. A., Morris, K. R. and Varia, S. A. J. Pharm. Sci. 88(7):696–704, 1999. 43. Jansen, P. J., Oren, P. L., Kemp, C. A., Maple, S. R. and Baertschi, S. W. J. Pharm. Sci. 87(1):81–85, 1998.

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6 REFERENCE STANDARDS PAUL A. CULBERT a AND BRUCE D. JOHNSONb a

Eli Lilly Canada Inc.,ON, M1N 2E8 Pfizer, Inc., Ann Arbor, MI 48105

b

I. INTRODUCTION II. DEFINITIONS III. LIFE CYCLE A. Discovery B. Lead Declaration C. Exploratory Development D. Full Development E. New Drug Application (NDA) Submission F. Product Launch G. Product Maturation IV. GOVERNANCE A. Control and Use Procedures B. Qualification Procedures V. QUALIFICATION PROCESS A. CharacterizationTechniques B. Form Selection C. Assignment of Purity Case1: DS Information: Case 2: DS Information: D. Bridging Standards VI. SUMMARY REFERENCES

I. INTRODUCTION Pharmaceutical reference standards are a critical aspect in all phases of drug research, development, and commercialization. Reference standards serve as the basis of evaluation for both process and product performance and are the benchmarks for the assessment of drug potency for patient consumption. Reference standards may be required not only for the active ingredients in dosage forms but also for impurities, degradation products, starting materials, preservatives, process intermediates, and excipients. At the same time, the availability of reference standards and the degree to which they are characterized and governed is often dependent on the stage of

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the drug development process. The FDA has not provided practical guidance on the topic of reference standards,1 but information does exist in the form of best practice documents from a variety of sources. Therefore, this chapter is intended to provide clarity to the overall role, qualiﬁcation, and governance of reference standards used in the pharmaceutical environment. The scope of this chapter will be limited to the discussion of nonbiological (i.e., synthetic small molecule) reference standards and will not include the discussion of compendial reference standards. Additionally, for reasons of clarity, the discussion will focus on reference standards of active pharmaceutical ingredients. However, the same considerations should generally be made for reference standards of synthetic impurities, degradation products, and the others mentioned above.

II. DEFINITIONS Terms used to describe reference standards differ among industries and also among companies in the pharmaceutical industry. The following deﬁnitions, consistent with literature references,2–13 will be used throughout this chapter. Drug substance: The drug substance (DS), also known as a new chemical entity (NCE) or the active pharmaceutical ingredient (API), in early phases of drug development consists of the following components: (a) active moiety, (b) counter-ion, and (c) water or solvent molecules that are known components of the DS crystal structure. If the single crystal structure of the DS is unavailable, the DS can be deﬁned as a hydrate or solvate based on the results of other analytical tests and the scientiﬁc judgment of the scientist. Active Moiety: The active moiety is the pharmacologically active species, expressed in terms of the neutral molecule (e.g., free base or free acid). For chiral substances, the active moiety is the enantiomer speciﬁed for clinical development. When racemates are speciﬁed for development, however, the active moiety refers to both enantiomers. For pro-drugs, the active moiety refers to the pro-drug and not the parent drug. Drug Product: The drug product (DP), also known as ﬁnished product or dosage form, is the DS formulated with inactive ingredients (excipients) for appropriate delivery to either human or veterinary patients. Reference Standard: A reference standard is broadly deﬁned as certiﬁed material or substance, supplied by a certifying body, which exhibits one or more properties that are sufﬁciently well established (and assigned) that it may be used for calibration of an apparatus, assessment of a measurement method, and assigning values to materials. Reference standards in pharmaceutical sciences generally fall within three categories: analytical reference standards, working standards, and authentic materials. Analytical Reference Standard: An analytical reference standard (ARS), also known as primary standard or gold standard, is deﬁned as a batch of drug substance, whose purity is independently established and accepted without reference to other standards. The ARS is the benchmark against

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which secondary standards are judged. The ARS is also used to determine the amount of active moiety present in the DS or DP. The material is of the highest purity (> 99.5%) that can be obtained by reasonable effort. Typically the material is obtained either by puriﬁcation of existing production material or, if necessary, speciﬁcally prepared by independent synthesis. The material is thoroughly characterized to assure its identity, strength, quality, and purity. The structural identity is performed via independent spectral interpretation, and all impurities should be determined on a weight basis if possible in order to establish full mass balance accountability. Additionally, there should be a complete understanding of the material’s inherent stability, appropriate storage containers and conditions, and other potential chemical transformations that may occur in stressed samples. Ultimately, the ARS must satisfy all identity, assay, and purity aspects of the API speciﬁcation and have a satisfactory physicochemical measurement proﬁle. Working Standard: The working standard (WS), also known as secondary standard, in-house standard, or laboratory standard, may be used for routine analytical comparison of batches of API and/or dosage forms in cases where a high-purity ARS is difﬁcult to obtain. The WS is generally speciﬁc to the phase of drug development in which it is being used. During early stages of drug development, the WS may be the only reference standard available for an NCE. The purity of a WS is typically very high (> 95%) but may be assigned without the same rigor as applied to the qualiﬁcation of an ARS. In fact, reference standards of higher purity at this early stage of drug development may not be preferred, as the existing impurities may be used to establish chromatographic system suitability. During later stages of drug development, the working standard is typically of higher quality and purity. In fact, qualiﬁcation of the material is usually based on identity and assay comparisons to the established ARS. The WS is often widely used in place of the ARS to conserve material. Ideally, the WS should be prepared by the normal synthetic route used for the manufacture of the API and subjected to further puriﬁcation if necessary. Authentic Materials: Authentic materials (AM) are reference standards, which are qualiﬁed for identity and approximate purity. The chromatographic purity of an AM need only be 80% or greater. They are not used in quantitative assays but are generally used to establish chromatographic system suitability and as identity comparators for spectroscopic assays. Authentic materials are usually available in small amounts and are frequently obtained by preparative chromatography.

III. LIFE CYCLE As every NCE brought forward into development is unique, the exact role that reference standards take for a speciﬁc drug candidate will vary but is generally keyed to drug development milestones. At any phase of drug development, the reference standard should be assessed versus its

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intended use and a balance should be struck between resource commitments, scientiﬁc judgment, and regulatory requirements. The following descriptions are not meant to be deﬁnitive in nature but are general practices.

A. Discovery Discovery is generally considered the phase of drug development where numerous lead compounds are generated that are active against an established biological target. The predominant reference standards, if needed during this phase, are AMs. Time and resource commitments render impractical the generation and full characterization of analytical reference standards for the multitude of NCEs. The principal analytical measurements made during this phase are relative in nature. The AMs generated during this phase are typically characterized for identity by NMR, IR, or mass spectrometry. Approximate purity of the AM is commonly achieved via high-performance liquid chromatography (HPLC) with UV detection; however, automated high-throughput procedures such as a chromatographic system connected to an array of detection modes14 may also be employed. Additionally, only small amounts of the AM are readily available from a ﬁrm’s compound distribution system.

B. Lead Declaration Lead declaration is generally considered the phase of drug development where formal preparations are made to introduce an NCE into clinical studies. Prior to this phase, a single NCE that is believed to be highly effective in treatment of the targeted indication has been identiﬁed in preclinical studies. The reference standard for analytical quantiﬁcation during this phase is typically a WS. Often the DS used in the dosage form candidate is frequently utilized as the WS, as it is readily available. Absolute versus comparative methods of analysis should be predominantly relied upon to determine the purity of the DS, because patient safety is especially imperative as the lead compound moves toward ﬁrst-in-human studies. Therefore, the widespread need for an ARS is minor during this phase of development.

C. Exploratory Development Exploratory development generally encompasses the investigational new drug (IND) submission and phase I stage in the drug development life cycle. It is during this phase of drug development that patient safety is demonstrated in clinical trials for the new chemical entity. It is also during this phase of drug development that an ARS is prepared. Information gained on the various forms of the DS from the lead declaration phase as well as from the ongoing DS stability studies should be critically scrutinized. Methodologies to fully characterize the proposed ARS should be developed and any nuances of the material should be explored. However, the reference standard for analytical quantiﬁcation during this phase is still predominantly the WS.

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D. Full Development Full development is generally considered as phases II and III in the drug development life cycle. It is during these phases that efﬁcacy is demonstrated for the desired indication and large population clinical trials are performed. It is also during these phases of drug development that the ARS reaches maturity in terms of scientiﬁc assessment. The physical form and appropriate storage conditions for the ARS have now been selected as a result of extensive characterization of the material by fully established analytical methodologies and an understanding of its inherent stability. Routine use of the ARS is implemented for analytical testing, which demonstrates ruggedness of the material, and the ARS is placed on a formal recertiﬁcation program. Procedures to generate bulk amounts are fully established by this point. Widespread use of a WS occurs in this phase only if there is a need to conserve the amount of ARS used and the WS has been fully certiﬁed against the ARS.

E. New Drug Application (NDA) Submission The testing and qualiﬁcation of reference standards should continue such that the necessary documentation (internal and external reports, certiﬁcates of analysis, stability reports, supporting raw data) is complete from both a regulatory and scientiﬁc standpoint at the time of the NDA ﬁling. A key analytical contribution to an NDA ﬁling is the methods validation package. A ﬁrm must provide all materials—including reference standards— necessary to perform the analytical methods that will be registered with the NDA.2 The FDA may, in fact, choose to verify the performance of the submitted methods within their laboratories. Some ﬁrms may choose to set aside the necessary reference standards at the time of ﬁling, while other ﬁrms may wait until the validation package is requested. A slow response to the FDA’s request for the validation package may delay approval of the application. It is therefore incumbent upon the project team to ensure that the necessary materials—reference standards, samples, columns, and special reagents if applicable—can be supplied to the FDA on very short notice. Additionally, care should be taken to ensure that the reference standards are within their validated shelf lives when supplied to the FDA.

F. Product Launch The drug development team must ensure that reference standards will be available to support the launch of a newly approved product once FDA approval is obtained. The quality control function is typically separate from the analytical development function in most ﬁrms, so a high level of cooperation and communication are critical. As launch approaches, the drug development team must work closely with the commercial manufacturing quality control unit to coordinate the supply of reference standards to the ﬁrm’s compound distribution system. It is useful to involve marketing forecasts to predict the approximate amounts of reference standards—based on projected batches—that will be necessary to support a worldwide launch.

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Grams of ARS Requested

Total ARS Requested

FIGURE 1 Atorvastatin Analytical Reference Standard Disbursements,1992^1997.

As an example, Figure 1 shows the disbursements for Atorvastatin ARS,15 the reference standard for LipitorÕ , in the years prior to and immediately after its launch in 1996 and gives an indication of the amounts of ARS required for a widely accepted prescription drug.

G. Product Maturation Several years after the successful launch of a new product, it is typical for North American pharmaceutical ﬁrms to become involved in the United States Pharmacopeia-National Formulary (USP-NF) revision process to allow inclusion of their API and formulations in the USP-NF. The USP-NF and their supplements are a compilation of monographs prepared under the authority of the Pharmacopeial Convention. The purpose of the USP-NF and supplements is to provide authoritative standards and speciﬁcations for materials and substances and their preparations that are used in health care or for the improvement or maintenance of health.16 The revision process— as outlined in the USP-NF—begins with ‘‘inquiries, comments and suggestions for revision in the USP-NF text’’ mailed directly to the USP.17 Participation in this process ensures consideration of the innovator’s methods, speciﬁcations, and reference standards for inclusion in the USP-NF. Approval for inclusion in the USP-NF is gained after consideration by an Expert Committee of the USP Council of Experts. If the Expert Committee so recommends, the proposed revision is published in the Pharmacopeial Forum (PF) for review and comments by interested readers of the PF. The comments and data submitted by interested readers are reviewed and addressed, and the Expert Committee then decides whether to recommend to the USP Council of Experts that the proposed revision should be adopted in the USP-NF. The development of USP Standards through the cooperation of industry ‘‘promotes uniform quality of drugs as an aid to the public health.’’18

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The USP typically obtains candidate ARS material for a newly approved article from the pharmaceutical innovator but may also source the material from other manufacturers. Occasionally, the USP may synthesize sufﬁcient material for use as an ARS. Once sufﬁcient material to meet the needs of the USP for several years is sourced, a collaborative study is performed between the laboratories of the USP, the innovator, and one or more neutral or interested parties to fully characterize the material. The data obtained from the collaborative study are then submitted to the USP Reference Standards Committee who reviews them to ultimately determine whether the Reference Standard is acceptable for its intended use in support of the relevant USP-NF tests.

IV. GOVERNANCE The requirements for the laboratory control of reference standards was embedded in the Code of Federal Regulations. Title 21 CFR Part 211 section 194 subsection 8b states ‘‘Complete records shall be maintained of any testing and standardization of laboratory reference standards, reagents, and standard solutions.’’ It falls to the individual ﬁrm to develop procedures to give inspectors the best possible assurance of compliance to the regulation. The central role of reference standards in the assurance of patient safety makes them a common focal point for regulatory inspections. Development of sound procedures and the strict adherence to such procedures is therefore critical. The pharmaceutical ﬁrm must deﬁne adequate qualiﬁcation of reference standards through one or more standard operating procedures. Reference standards SOPs typically fall into two categories: Reference standard control and use procedures and reference standard qualiﬁcation procedures.

A. Control and Use Procedures A Reference Standards Control and Use Standard Operating Procedure (SOP) would typically include assigned responsibilities and procedures to ensure that: .

.

.

A scope for the reference standards covered by the procedure has been defined. For instance, reference standards used for the calibration of equipment may specifically be excluded from the SOP. Reference standards enter the laboratories under adequate control. These procedures typically identify approved sources and specify the documentation (certificates of analysis, qualification reports) that must accompany the reference standard into the laboratory. The accompanying documentation must be available during a GMP inspection. All reference standards entering the laboratory are properly logged. Information typically logged is identification (compound number or name), lot number, storage conditions, retest date, and the analyst to whom the standard is assigned.

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.

. .

Reference standards are labeled such that their identity, potency, correct use, and correct storage can be assured at all times. Reference standard labeling must also ensure that laboratory personnel handling the material are properly informed of safety risks associated with potentially harmful chemicals. Reference standard users are kept informed of changes to the status of standards currently in use in the laboratory. These changes may include updated potencies based on new test results, the reporting of unusual results or any other results that might compromise the integrity of the data generated using the reference standard in question. The reference standard is removed from the lab and properly disposed of on or before the date of requalification or expiry. Corrections for solvents and moisture are correctly applied. The drying of the reference standard before use, if stated in the method, will eliminate residual solvent(s), unbound moisture, and sometimes bound moisture (depending on the drying conditions). The drying step is always included for hygroscopic compounds. On the other hand, drying can result in the loss of a hydrate or cause degradation in heatsensitive compounds.

B. Qualification Procedures To ensure the compilation of consistent, complete and scientiﬁcally defensible regulatory submissions, the qualiﬁcation of reference standards must be governed by a standard operation procedure. A reference standard qualiﬁcation SOP will typically assign responsibility for, and include procedures to ensure that .

. .

. .

. . .

A scope has been defined that specifically states what types of standards will be qualified according to the procedure and which types of standards are excluded. Approved sources for reference standards are identified. Reference standards are properly categorized for their intended use. A definition section that clearly links the type of reference standard to its intended use is critical. A minimum basis set of analytical tests is described for each type of analytical standard. A consistent, scientifically defensible process is followed when purity values are assigned. A clear rationale for the calculation of purity values as well as clear instructions on the use of purity values must also be included. Retest, requalification, and expiry dates are appropriately assigned. A well-defined retest schedule is applied to reference standards used for quantitative purposes. The test results are recorded in a consistent and traceable manner. These procedures may give details on the method for reporting results for submission to a regulatory agency, in the correct format

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for certificates of analysis, and even the correct storage area for electronic and paper copies of the supporting reports and raw data. The labeled potency of the reference standard remains valid from the time of first use through to the requalification or expiry date indicated. The project scientist must ensure that the stability of the chemical is understood and that the assigned requalification or expiry dates are scientifically defensible.

V. QUALIFICATION PROCESS The critical parameter in the discussion of reference standards is the process of qualiﬁcation. FDA guidance on this topic states that ‘‘A noncompendial standard should be thoroughly characterized to assure its identity, strength, quality and purity.’’3 It is therefore left to the pharmaceutical ﬁrm to establish ‘‘thorough characterization’’ for the ARS of each NCE. The overall qualiﬁcation process for an ARS includes characterization of the material, proper form selection, and assignment of purity. Some general considerations in this process include .

.

. .

The expectation that the tests and methods used to characterize an ARS will be different from, and more extensive than, those used to control the new drug substance The desirability of characterization methods which are not comparative (i.e. dependent upon the availability of a previously designated reference standard of the same drug substance) The necessity of some degree of blinding when testing is performed by an outside laboratory, in order to assure unbiased results The requirement that the results generated during characterization be cross-referenced with each other and the results achieve a full accountability of mass balance

Taken together, the data from a series of experiments must show in a scientiﬁcally defensible manner that the labeled purity and the identity of the reference standard are both accurate and appropriate for the reference standards intended use.19,20

A. Characterization Techniques An examination of the literature,10,21–24 authoritative guidances,6–9 and current industrial best practices, suggests that the analytical techniques in Table 1 be considered for the characterization of reference standards. Other techniques are occasionally employed but are not discussed here. These may include: particle size analysis,25 nephelometry, heavy metals analysis,26 surface area,27 bulk density,28 pH,29 dissociation constants, microbiological testing30 and other spectroscopic measurements (e.g., NIR, ﬂuorescence, CD, etc.). What follows is a discussion of the test methods commonly used to qualify reference standards and the pertinent information gained from these techniques. Frequently referenced textbooks have been cited and

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TABLE 1 Techniques Commonly Applied to the Qualification of Reference Standards Appearance (physical description) Visual inspection Optical microscopy Identification (proof of structure) Elemental analysis UV/Visible spectroscopy Infrared spectroscopy Raman spectroscopy X-ray diffraction High-resolution mass spectrometry Nuclear magnetic resonance spectroscopy Optical rotatory dispersion Purity Loss on drying Karl Fischer titration Residue on ignition Thermogravimetric analysis Differential scanning calorimetry High-performance liquid chromatography Ion chromatography Electrophoretic separations Supercritical fluid chromatography Thin-layer chromatography Chiral chromatography Gas chromatography Assay Titration Phase solubility analysis

can provide a broader scope of technical details. Many of these techniques are described in the United States Pharmacopoeia, the European Pharmacopoeia, and the Japanese Pharmacopoeia. Speciﬁc pharmaceutical applications of these techniques can also be found in other chapters of this book, volume 3 of this series,31 or current literature reviews.32 1. Appearance (Physical Description) Visual Inspection: The visible characteristics—color, texture, and morphology, as well as visible contamination—are important and sensitive

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measures of chemical purity. Substances that are reactive in the presence of light or moisture will nearly always undergo an easily observed color or texture change when exposed to these agents. Appearance is thus an important measure to be included in the testing of reference standards. Color chips are available33 to assist in describing the reference standard. Optical Microscopy: Optical microscopy deals with the examination of material and identiﬁcation of crystallinity under a microscope. A crude assessment of reference standard morphology, homogeneity, and birefringence, a qualitative aspect of light refraction by crystals, are easily observed using this technique. Crystalline particles will appear to change from bright to dark (or change colors) and noncrystalline (amorphous) particles will not change as the stage of a polarizing microscope is rotated.34 2. Identification (Proof of Structure) Elemental Analysis: The carbon, hydrogen, and nitrogen contents of reference standards may be determined by combustion analysis. Combustion analysis thus provides an orthogonal determination of the molecular formula and the purity of the reference standard. Poor agreement between the theoretical and experimental elemental compositions is generally an indication of an impure substance or a discrepancy between the theoretical and actual molecular formulas. Elemental analysis may also identify the presence of solvates and inorganic contaminants. Elemental analysis is typically performed in specialized laboratories under blinded conditions. Ultraviolet-Visible Spectroscopy: Ultraviolet-visible (UV-VIS) molecular absorption spectrophotometry (often called light absorption spectrophotometry or just UV-visible spectrophotometry) is a technique based on measuring the absorption of near-UV or visible radiation (180–770 nm) by molecules in solution.35,36 Reference standard characterization by UV-VIS spectophotometry includes determining the absorption spectra and the molar extinction coefﬁcient. These two spectral characterizations are used as identiﬁers of reference standards. Infrared Spectroscopy: Infrared (IR) spectroscopy deals with the interaction of IR radiation with matter. Most commonly, the spectrum is obtained by measuring the absorption of IR radiation (10–12,900 cm1).35,37–39 Because of the rich spectral information that can be obtained and multiple spectroscopic modes that do not require dissolution of the material, IR is the most commonly used identiﬁcation technique for reference standards. IR is also applied to characterize the presence of polymorphs and solvates. Raman Spectroscopy: Raman spectroscopy is based on the measurement of scattered electromagnetic radiation as a result of the irradiation of matter. Raman spectroscopy is considered complementary to IR spectroscopy, as the two techniques provide a complete vibrational picture of material. Raman spectroscopy is not as widely used for reference standard identiﬁcation purposes as IR spectroscopy because of the lack of familiarity. However, Raman spectroscopy is an extremely powerful tool in characterizing the presence of polymorphs.35,39

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X-ray Diffraction: When applied to single crystals, the technique is known as single crystal X-ray diffraction; applied to powders, the technique is known as X-ray powder diffraction (XRPD). Single crystal X-ray diffraction provides deﬁnitive structural information in terms of bond lengths and angles for reference standards, while X-ray powder diffraction yields information on the degree of crystallinity of the material as well as helping to identify polymorphs.38,40–42 Where suitable crystals can be obtained, single crystal X-ray diffraction provides the most deﬁnitive proof of structure for a reference standard. Mass Spectrometry: Mass spectrometry refers to the application of any of a number of mass-separative techniques to charged molecules in the gas phase. Techniques that are capable of mass resolution in the low parts per million (ppm) range are considered high resolution (HRMS)—the two most common instruments are magnetic sector and time-of-ﬂight mass spectrometers. Time-of-ﬂight mass spectrometry (TOFMS) has emerged as a highly accurate, relatively inexpensive technique for obtaining high-resolution mass spectra with errors of less than 5 ppm.43,44 Thus, HRMS is a powerful tool for conﬁrming the molecular formula of a reference standard by providing an accurate molecular mass. HRMS also provides orthogonal structural information to techniques such as NMR but may not be appropriate for nonvolatile molecules and molecules that are prone to fragmentation under mass spectrometric conditions. Nuclear Magnetic Resonance: 13C and 1H nuclear magnetic resonance (NMR) spectroscopy and the associated multidimensional experiments can provide deﬁnitive evidence for determining the structure of reference standards. Fully assigned NMR spectra are an integral part of the registration package for new chemical entities and their associated impurities. Solid-state NMR is also valuable for the identiﬁcation and monitoring of polymorphism in drug substances and products.40,45,46 Optical Rotatory Dispersion: Optical rotatory dispersion (ORD) is the measurement of the angle of rotation of the plane of linearly polarized radiation by an optically active (chiral) species. The dependence of the optical rotation on wavelength is termed the ORD. Speciﬁc rotation typically involves measuring the rotation of light at a ﬁxed wavelength under controlled temperature of a 1% reference standard solution.35,47 The speciﬁc rotation is used to conﬁrm the identity of a chiral reference standard. ORD can also be used to determine chiral purity but is generally limited by sensitivity. 3. Purity Loss on Drying: Loss on drying is a technique that determines the amount of volatile matter of any kind that is driven off under the temperature and pressure conditions speciﬁed. The technique is based upon accurate mass measurements, before and after drying. Ideally, the percent of material lost on drying should correlate to the total amount of solvent and moisture obtained by other measurements, when determining mass balance accountability for reference standard material. However, solvents/moisture that forms solvates/hydrates with the reference standard are often difﬁcult

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to quantify by this technique, as decomposition of the material occurs before the solvent/moisture is driven off. Karl Fischer Titration: Moisture content of reference standards is commonly and accurately determined by Karl Fischer (KF) titration. KF titration determines total water content, including both the free water and water of hydration trapped in the crystal structure. Coulometric KF-titration is the most sensitive application of the method and is normally used for the small amounts of water found in reference standards. A volumetric technique is available for samples containing large amounts of water. Interference is a relatively common problem in KF titration and thus, due caution must be exercised in the interpretation of KF results. Reagents have been designed to cope with interfering functional groups.48–50 Residue on Ignition: Residue on ignition (ROI) is a pharmacopeial method51 that complements chromatographic techniques by providing information on inorganic impurities in reference standards. The technique involves the charring, digestion, and ignition of a reference standard sample. The resulting residue is then weighed to determine the amount of inorganic impurities present as their sulfate salts. ROI can be coupled with spectroscopic techniques such as atomic absorption, ﬂame emission, and inductively coupled plasma (ICP) spectroscopy to provide identiﬁcation and quantiﬁcation of inorganic impurities. Thermogravimetric Analysis: Thermogravimetric analysis (TGA) measures the change in the mass of sample as the temperature is changed and is most commonly used to study the loss of solvents or other solid ! solid þ gas reactions. Therefore, as part of a reference standard characterization, TGA can often provide information regarding moisture and solvent levels, an indication if the material is a hydrate or solvate, and the rate of reaction Asolid ! Bsolid þ Cgas.42,52,53 Additionally, if a spectroscopic instrument (i.e., IR) is placed inline with the efﬂuent atmosphere, the identity of the solvent lost during heating from the material can often be determined. As with the other techniques of purity determination, the information gained with TGA should be compared with information from other reference techniques such as gas chromatography and single crystal X-ray diffraction. Differential Scanning Calorimetry: Differential scanning calorimetry (DSC) is a method that measures the difference in energy (heat ﬂux or heat ﬂow) between a reference and a sample. The result of a DSC analysis is a thermogram, a plot of temperature difference versus temperature and represents the enthalpies of various processes occurring in the heated sample, such as solvent loss, crystallization, polymorphism, and chemical reactions. Additionally, DSC is an absolute method and with proper calibration can be used to accurately measure the melting point and purity of the reference material.52,53 High-Performance Liquid Chromatography: High-performance liquid chromatography (HPLC) is the preeminent analytical separation technique in pharmaceutical chemistry that delivers the fundamental impurity information for reference standard qualiﬁcation. Numerous excellent reviews and texts are available54–58 as is a general chapter in the USP.59

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The predominant mode of HPLC, reversed phase, involves the separation of material based on the partitioning between a relatively polar mobile phase and a nonpolar stationary phase. Normal phase HPLC—nonpolar mobile phase and polar stationary phase—is considered an orthogonal technique to reversed-phase HPLC when qualifying reference standards. In fact it is common for the elution order to be entirely reversed when switching an analysis from reversed to normal phase. Therefore, highly nonpolar impurities can be easily characterized by normal phase separations. HPLC remains a powerful tool for the qualiﬁcation of reference standards because of the possibility of coupling the separation process with a myriad of complementary detection techniques. In 1998, ﬁve detection methods were reported to account for at least 95% of all HPLC analyses. The dominant detection techniques were UV-VIS, ﬂuorescence, refractive index, electrochemical, and conductivity detection.60 UV-VIS with diode array detection (DAD) has become increasingly common in pharmaceutical analysis owing to the development of sensitive and low-cost equipment. DAD detectors allow real-time collection of the entire UV-VIS spectrum of each peak in an HPLC chromatogram. DAD software packages can typically provide peak purity assessments and three-dimensional representations of chromatographic experiments. The peak purity algorithms available with these software packages are particularly useful in the qualiﬁcation of reference standards. The homogeneity of the peak can be determined61 by comparing the UV-VIS spectra at different points in the peak under study against reference spectra. The degree to which the two spectra match can be assessed mathematically to yield a numerical value or ranking for peak purity. The peak purity value is judged versus preestablished criteria that are indicative of an appropriate degree of purity. Care must be taken when using this technique as co-eluting impurities often have UV-VIS spectra that are nearly indistinguishable from the peak of interest. In fact, this is an inherent weakness in UV-VIS detection and highlights the importance of complementary detection techniques. Examples of other detection modes that are either commercially available or have been recently reported in the literature include evaporative light scattering, chemiluminescent, IR, Raman and inductively coupled plasma spectroscopy. New and improved detectors are continuously being developed and these new technologies are reviewed on a regular basis.61 Additionally, the rapid development of hyphenated techniques, such as liquid chromatography-mass spectrometry and liquid chromatographyNMR instruments, has rapidly advanced these techniques as tools for reference standard qualiﬁcation. The overriding consideration when qualifying reference standards is the importance of using complementary detection techniques. Ion Chromatography: Ion chromatography (IC) is a mode of HPLC in which ionic analyte species are separated on cationic or anionic sites of the stationary phase. The detection techniques largely fall under three categories: electrochemical, spectroscopic, or post-column reactions. In general, IC provides an orthogonal separation mechanism to traditional reversed-phase HPLC (RP-HPLC).54,63 This technique can be exploited to quantify ionic

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species such as polar impurities, residual metals or counter-ions of the main analyte.59 Electrophoretic Separations: The popularity of electrophoretic separations has exploded in the last decade with the advent of capillary electrophoresis. Capillary electrophoresis (CE) has many practical advantages over standard gel electrophoresis and has been broadly diversiﬁed into a family of specialized techniques. The most universal of these techniques is capillary zone electrophoresis (CZE) or free-solution CE. The principal of separation is based upon the charge-to-size ratio of the analytes, where the sample mixture in a free-ﬂowing background electrolyte (BGE) solution is exposed to both an electrophoretic and electroosmotic ﬂow in an open capillary via a high-voltage power supply. Additionally, most classical RP-HPLC detection techniques are compatible with CZE.63,64 CZE provides a completely orthogonal separation mechanism to traditional RP-HPLC. Similar to IC, this technique can be exploited to quantify ionic species in reference materials such as polar impurities, residual metals, or counterions of the main analyte. However, by adding selected modiﬁers to the BGE solutions, separations based on both RP and enantiomeric modes can supplement the charge-to-size mechanism. Supercritical Fluid Chromatography: Supercritical ﬂuid chromatography (SFC) is a column chromatographic technique in which a supercritical ﬂuid is used as a mobile phase. A supercritical ﬂuid is a gas or liquid brought to a temperature and a pressure above its critical point. Because of the unique properties of the mobile phase and the optional addition of organic modiﬁers, the SFC separation process is generally considered a blend of both gas chromatography (GC) and liquid chromatography (LC). In fact, the SFC column may either be a packed column, comparable to an HPLC column, or an open capillary column, comparable to a GC column. Detection is performed either on-line (i.e., UV-VIS) or after the expansion of the ﬂuid [i.e., ﬂame ionization detection (FID)]. SFC is generally performed in the normal phase (NP) mode and often NP-TLC (thin-layer chromatography) or NP-HPLC methodologies can be readily adapted to SFC methodologies.66,67 SFC generally provides an orthogonal separation mechanism to traditional RP-HPLC. Because of the similarity in the chromatographic measurement process to HPLC, this technique can be exploited to accurately quantify nonpolar impurities of reference materials. Thin-Layer Chromatography: Thin-layer chromatography (TLC) coupled with densitometric detection is a highly sensitive—though often ignored68,69—method for assessing the purity of reference standards. HP-TLC is an improved version of TLC that employs stationary phases of decreased thickness and particle size resulting in improved resolution over shorter elution distances. Components separated by TLC can be visualized by a number of techniques.70 Short-wave UV irradiation is one of the most convenient and universal of these detection techniques. Densitometers are available that measure the relative amounts of the separated components. In contrast to HPLC, all components in the sample mixture remain and are visualized on the TLC plate. TLC with densitometric detection has shown good linearity and limits of quantiﬁcation in the low nanogram range when

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coupled with post-chromatographic derivitization.71 TLC with densitometric detection has been directly compared with HPLC with favorable results.72,73 It is an ideal complementary chromatographic technique to HPLC because of the universal visualization techniques available and the often-complete accountability for all impurities. Chiral Chromatography: Chiral pharmaceutical active moieties are increasingly common. Therefore, the determination of enantiomeric purity is imperative for the qualiﬁcation of reference standards. The basis for the majority of enantiomeric chromatographic separations is the formation of a transient diastereomer, which is commonly performed either by using a chiral stationary phase or by adding a chiral selector to the mobile phase. Numerous chromatographic techniques including those discussed previously have been demonstrated to achieve chiral separations through one of these routes.74 Chiral separation technology has now matured to the extent that sensitivity to 0.1% of the minor enantiomer is routinely achieved. In general, the minor enantiomer is considered an impurity of the reference standard. Gas Chromatography: The role of gas chromatography (GC) in the qualiﬁcation of reference standards is primarily for the determination of organic volatile impurities (OVI) with FID, according to the USP.75 The polar nature and presumed poor volatility of most pharmaceutically important molecules relegates GC far down the list of techniques used in pharmaceutical analysis. Nevertheless, GC is an excellent analytical technique for special cases when volatile impurities are suspected to be present in a reference standard or for cases where a simple derivitization reaction may impart sufﬁcient volatility to a molecule to enable analysis. GC is complementary to loss on drying (LOD) and KF titration in the determination of total volatile impurities in reference materials. 4. Assay Titration: The determination of assay values for reference standards, counter-ions, or impurities can often be independently determined via titration. While titration assays generally have less selectivity in comparison to chromatographic methods, the advantages of a broad spectrum of classical titration techniques that exist for organic functional groups is often overlooked. The methodologies include not only classical potentiometric acid/base titrations but also nonaqueous, redox, indirect, precipitation, and derivatization titrations.76–79 Phase-Solubility Analysis: Phase-solubility analysis is the quantitative determination of the purity of a substance through the application of precise solubility measurements. Phase-solubility analysis is applicable to all species of compounds that are crystalline solids and that form stable solutions. Phase-solubility analysis is an absolute method that provides a totally independent assessment of purity and does not require a fully characterized reference standard or identity of impurities. Instead, this technique exploits the difference in solubility of the reference standard and its impurities. Ideally, the results from phase-solubility analysis should correlate quite closely with the sum of impurities from chromatographic analysis of the reference standard.80,81

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B. Form Selection During the course of extensive characterization of an analytical reference standard (ARS), attention should be given to gaining an understanding of the material’s full physicochemical proﬁle. The ARS should demonstrate excellent chemical stability under a wide range of storage conditions. This knowledge may be gained by challenging the hygroscopicity, and thermaland photostability of the proposed reference standard in a variety of closure/ container systems. This ensures that the appropriate salt, solvate, hydrate or polymorphic form of the active moiety as well as the closure/container system has been selected for the ARS. This up-front experimentation does not replace a rigorous and ongoing stability program of the ARS but ensures that surprises during development will be minimized. Ideally, it is preferred for the ARS to be of the same drug substance (DS) form as the material to be routinely identiﬁed and quantiﬁed. However, the form of the DS-ARS to be used for routine identiﬁcation and quantiﬁcation may be inherently unstable under the desired storage and testing conditions. The selection of an ARS in the same form as the DS is not a requirement but a preference because direct spectral comparisons cannot typically be applied for identiﬁcation when using an alternate form of the active moiety as an ARS. Additionally, during comparative assay calculations a molecular weight correction is required when the DS and the ARS are of different forms. To illustrate the ARS form selection process, two pharmaceutical examples of ARS form selection are provided. Indinavir sulfate is the API for Crixivan2, a speciﬁc and potent inhibitor of the HIV-1 protease used in the treatment of AIDS. Indinavir sulfate is produced as a crystalline ethanolate sulfate salt. If the material is stored in double polyethylene liners within ﬁber containers or repeatedly exposed to ambient conditions changes occur in both crystallinity and solvation. Using XRPD, KF, and RP-HPLC, conversion of the crystalline ethanolate to amorphous material or to a hydrate crystal form has been detected and degradation is observed. However, the material is stable if stored in a tightly sealed container impermeable to ethanol/moisture transport under an inert nitrogen atmosphere at a controlled room temperature.82,83 These storage conditions are not practical for a routinely used ARS. Therefore, the free base monohydrate form of indinavir sulfate was evaluated and selected as the ARS. This form of the API was demonstrated to be extremely stable under ambient conditions needed for routine analysis. Levothyroxine is another example of an active pharmaceutical ingredient with relatively poor stability that is available as a USP analytical reference standard in the free acid form, while the drug is formulated as the sodium salt in most commercial preparations.84 Levothyroxine is prone to extensive photochemical decomposition85 that is thought to be exacerbated by the facile ionization of the phenolic hydroxyl group.86 Supply of the USP analytical reference standard as the free acid provides a more stable form through suppression of the ionization of the phenolic hydroxyl.

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C. Assignment of Purity Once a thorough characterization of the reference standard has been completed, purity and assay values are assigned to the ARS. The purity, also known as potency or activity, is expressed on a theoretical and experimental basis. The theoretical purity (puritytheory) is the calculated amount (weight percent) of the API present in the DS based on the chemical structure. The puritytheory is used to calculate the assay value. Puritytheory % ¼ 100%

MWAPI MWDS

Where MWAPI is the molecular weight of the API, MWDS is the molecular weight of the DS. The experimentally determined purity (purityexp) or activity is the amount of the API present in the material being tested. For chiral drug substances, the achiral purityexp is the amount of both enantiomers in the sample, while the chiral purityexp is the weight percent of the desired enantiomer. The purityexp is used to calculate the assay value of the batch and is used for DP manufacture and for the calculation of dosages used for toxicological testing. X X Purityexp ð%Þ ¼ 100% ½%Related ½%Water X X ½%Residual Solvents ½%Other P where [%related] is the total amount P of related impurities typically determined chromatographically and [%other] is the total amount of inorganic components. The amount of each of the components subtracted in the above equation is expressed on a weight percent basis. In early phases of drug development, it is typically assumed that all related impurities have similar chromatographic response factors in comparison to the main component. In later phases of drug development, the amount of impurities is usually determined on a weight percent basis, using working standards. As the chromatographic area percent determination of related impurities does not account for 100% of the material present (i.e., excludes moisture, solvents, and inorganics), the following equation should be used to more accurately reﬂect purityexp if the level of related impurities is signiﬁcant (e.g. >1%) and they are not reported on a weight percent (w/w%) basis. P

½%Related 100 P P P 100% ½%Water ½%Residual Solvents ½%Other 100

Purityexp ð%Þ ¼ 100%

100%

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The assay value is the amount of the active moiety found in the material being tested as a percent of the theoretical value. The assay value is often reported on an ‘‘anhydrous, solvent-free’’ basis and should be consistent with the chromatographic area percent value. "

# Purityexp Assayð%Þ ¼ 100% Puritytheory 1 P P 100% 100% ½%Water %½Residual Solvents When assigning purity and assay values, some general considerations include: .

. .

.

The counter-ion should be expressed in terms of the un-ionized acid or base. A molecular weight correction should be applied (e.g., HCl equivalence). The purity and assay values are typically expressed with three significant figures. Residual solvent contents, water contents, residue on ignition contents, and process-related impurities below their method determination limit of quantitation (LOQ) or below 0.05%, whichever is the greater, are generally not included in the calculation. The LOQ should be 0.05% or less for residual solvents, water contents, and process-related impurity contents and 0.1% for residue on ignition contents. For drug substances defined as hydrates or solvates, an assay value consistent with the ‘‘anhydrous, solvent-free’’ basis can be calculated where the amount of water or solvents subtracted in the denominator is the difference between the measured and theoretical amounts.

In order to gain practical knowledge for these calculations, two examples are provided.

Case 1: DS Information: The DS is not a hydrate or solvate MW of compound X form Y ¼ 419.87 (an amine hydrochloride salt) MW of compound X ¼ 383.41 (free base) Sample characterization results:

Chloride Water Residual solvents

8.5 w/w% 0.3 w/w% < 0.05 w/w%

Residue on ignition (ash) Impurity A

0.1 w/w% 0.08 w/w%

Impurity B

0.3 w/w%

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Calculations: Puritytheory % ¼ 100%

383:41 ¼ 91:3% 419:87

HCl Equivalenceð%Þ ¼ 8:5%

36:46 ¼ 8:7% 35:45

Purityexp ð%Þ ¼ 100% ½0:3 þ 0:08% ½0:3% ½0:0% ½8:5% þ 0:1% ¼ 90:7% Assay ‘‘anhydrous; solvent-free’’ value ð%Þ 90:7 1 ¼ 100% 100% ¼ 99:6% 91:3 100% ½0:3% ½0:0%

Case 2: DS Information: The DS is a hydrate. The MW of compound Z form Q ¼ 219.67 (monohydrate, HCl salt) The MW of compound Z ¼ 165.19 (free base) Sample characterization results: Chloride

16.1 w/w%

Residue on ignition (ash)

0.3 w/w%

Water (Theoretical

8.4 w/w% 8.2 w/w%)

Enantiomer

1.0 w/w%

Residual solvents

0.5 w/w%

Impurity A

0.5 w/w%

Calculations: Puritytheory % ¼ 100%

165:19 ¼ 75:2% 219:67

Purityexp ð%Þ ¼ 100% ½1:0% þ 0:5% ½8:4% ½0:5% ½16:1% þ 0:3% ¼ 73:2%

Assay ‘‘anhydrous; solvent-free’’ value ð%Þ 73:2 1 100% ¼ 98:0% ¼ 100% 75:2 100% ½0:2% ½0:5%

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D. Bridging Standards The establishment of an ARS affords the opportunity to qualify new reference standards—both analytical and working—through bridging studies. A new candidate for an ARS can be analyzed against the previous ARS to give rise to a purity value expressed as a percentage by weight versus the qualiﬁed ARS. Care should be taken to reconcile the results of other tests against the results of the bridging study. Qualiﬁcation by bridging establishes a common reference point for subsequent batches and guards against potential drift in results that may arise from the combination of systematic errors present in all experiments.

VI. SUMMARY The intent of this chapter is to provide practical guidance on pharmaceutical reference standards, which are a critical aspect in all phases of drug research, development, and commercialization. The chapter’s key directions include providing clarity to the role, qualiﬁcation, and governance of reference standards used in the pharmaceutical environment. Reference standards serve as the basis of evaluation for both process and product performance and are the benchmarks for the assessment of drug potency for patient consumption. At the same time, the availability of reference standards and the degree to which they are characterized and governed is often dependent on the stage of the drug development process. Thus, an overriding theme is that at any phase of drug development, a reference standard should be assessed versus its intended use and a balance should be struck between resource commitments, scientiﬁc judgment, and regulatory requirements.

ACKNOWLEDGEMENTS The authors would like to acknowledge Dr. Steven Priebe, Dr. Brian Tobias, and Dr. Gerard Hokanson, Pﬁzer Global Research and Development, Ann Arbor, Michigan for their editorial review of this manuscript.

REFERENCES 1. Current Good Manufacturing Practice for Finished Pharmaceuticals, 21 CFR 211, April 1, 2001. 2. Guideline for Submitting Samples and Analytical Data for Methods Validation, Food and Drug Administration, Center for Drugs and Biologics, Office of Drug Research and Review (HFN-100), Rockville, MD, 1987. 3. Reviewer Guidance—Validation of Chromatographic Methods, Food and Drug Administration, Center for Drugs and Biologics, Office of Drug Research and Review, Rockville, MD, November 1994. 4. Terms and Definitions Used in Connection with Reference Materials 30, 2nd ed. 1992, International Organization for Standardization, Geneva, Switzerland, Ref. No. ISO Guide 30:1992 (E/F).

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5. Calibration in Analytical Chemistry and Use of Certified Reference Materials 32 1st ed. 1997, International Organization for Standardization, Geneva, Switzerland, Ref. No. ISO Guide 32:1997 (E). 6. Uses of Certified Reference Materials—Guide 33, 2nd ed. 2000, International Organization for Standardization, Geneva, Switzerland, Ref. No. ISO Guide 33:2000 (E). 7. Certification of Reference Materials/General and Statistical Principles–Guide 35, 2nd ed. 1989, International Organization for Standardization, Geneva, Switzerland, Ref. No. ISO Guide 35:1989 (E). 8. Guidance for Industry—Analytical Procedures and Methods Validation Chemistry, Manufacturing, and Controls Documentation. Food and Drug Administration, Center for Drug Evaluation and Research, August 2000. 9. Chemistry Reviews of DMF’s for Drug Substances/Intermediates (DSI), Manual of Policies and Procedures. Food and Drug Administration, Center for Drug Evaluation and Research, 1998. 10. Jenks, P. J. and Stoeppler, M. The deplorable state of the description of the use of certified reference materials in the literature. Fresenius J. Anal. Chem. 370:164–169, 2001. 11. Pauwels, J. and Lamberty, A. CRMs for the 21st century: new demands and challenges. Fresenius J. Anal. Chem. 370:111–114, 2001. 12. Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances. ICH Harmonised Tripartite Guideline (ICH Q6A), 1999. 13. Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients. ICH Harmonised Tripartite Guideline (ICH Q7A), 2000. 14. Taylor, E. W., Qian, M. G. and Dollinger, G. D. Simultaneous on-line characterization of small organic molecules derived from combinatorial libraries for identity, quantity, and purity by reversed-phase HPLC with chemiluminescent nitrogen, UV, and mass spectrometric detection. Anal. Chem. 70:3339–3347, 1998. 15. Jared, Brenda. Personal Communication. Compound Management, Pfizer Global Research and Development, Ann Arbor, Michigan. 16. United States Pharmacopeia Rev. 25 and National Formulary. 20th ed. Constitution, Article 1, Section 3 Name and Objects. p. xxiv, 2002. 17. United States Pharmacopeia Rev. 25 and National Formulary. 20th ed. Continuously Updated Standards and Specifications, p. xlviii, 2002. 18. United States Pharmacopeia Rev. 25 and National Formulary. 20th ed. Preface/Preample Technical Facts on Reference Standards. p. xlvi, 2002. 19. Impurities in New Drug Substances, ICH Harmonised Tripartite Guideline, ICH Q3A(R), February, 2002. 20. Impurities in New Drug Products, ICH Draft Consensus Guideline, ICH Q3B, October, 1999. 21. Ihnat, M. A synopsis of different approaches to the certification of reference materials. Fresenius J. Anal. Chem. 360:308–311, 1998. 22. Crowther, J. B. In Handbook of Modern Pharmaceutical Analysis, Ahuja, S. and Scypinski, S., Eds., Academic Press, San Diego, pp. 415–443 2001. 23. Webster, G. K. and Bell, R. G. Practical approaches to qualifying laboratory standard reference materials. Pharmaceutical Formulation and Quality, January/February, pp. 39–43 1999. 24. Boenke, A. Pure substances and a new generation of CRMs for chemical industry. Fresenius J. Anal. Chem. 360:388–392, 1998. 25. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h786i Particle Size Distribution Estimation by Analytical Sieving, p. 2044, 2002. 26. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h231i Heavy Metals, p. 1923, 2002. 27. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h846i Specific Surface area, p. 2072, 2002. 28. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h616i Bulk Density and Tapped Density, p. 1981, 2002. 29. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h791i pH, p. 2052, 2002.

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30. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h61i, Microbial Limit Tests, p. 1873, 2002. 31. Handbook of Modern Pharmaceutical Analysis, Vol. 3, Ahuja, S. and Scypinski, S., Eds., Separation Science and Technology Series. Academic Press, NY, 2001. 32. Gilpin, R. K. and Pachia, L. A. Pharmaceuticals and related drugs. Anal. Chem. 73:2805– 2816, 2001. 33. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h631i Color and Achromicity, p. 1995, 2002. 34. Byrn, S. R., Pfeiffer, R. R. and Stowell, J. G. In Solid-State Chemistry of Drugs, 2nd ed., SSCI, Inc., West Lafayette, Chapter 4, pp. 69–78 1999. 35. Ingle, J. D. Jr. and Crouch, S. R. Spectrochemical Analysis, Prentice-Hall, Inc., A Division of Simon & Schuster, Englewood Cliffs, NJ, 1988. 36. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h851i Spectrophotometry and Light Scattering, p. 2075, 2002. 37. Socrates, G. Infrared Characteristic Group Frequencies–Tables and Charts, 2nd ed., John Wiley & Sons, NY, 1994 38. Byrn, S. R., Pfeiffer, R. R. and Stowell, J. G. In Solid-State Chemistry of Drugs, 2nd ed., SSCI, Inc., West Lafayette, Chapter 8, pp. 111–117, 1999. 39. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h851i Spectrophotometry and Light Scattering, p. 2074, 2002. 40. Polymorphism in Pharmaceutical Solids, Brittain, H.G., Ed., Dekker, New York, 1999. 41. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h941i X-Ray Diffraction, p. 2088. 42. Physical Characterization of Pharmaceutical Solids, Brittain, H.G., Ed., Dekker, New York, 1995. 43. Guilhaus, M., Mlynski, V. and Selby, D. Perfect timing: time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry, 11:951–962, 1997. 44. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h736i Mass Spectrometry, p. 2029, 2002. 45. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h761i Nuclear Magnetic Resonance, p. 2034. 46. Padden, B. E., Zell, M. T., Don, Z., Schroeder, S. A., Grant, D. J. W. and Munson, E. J. Comparison of solid-state 13C NMR spectroscopy and powder X-ray diffraction for analyzing mixtures of polymorphs of neotame. Anal. Chem., 7:3325–331, 1999. 47. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h781i Optical Rotation, p. 2043. 48. MacLeod, S. K. Moisture determination using Karl Fischer titrations, Anal. Chem. 63:557–566, 1991. 49. Wieland, G. Water Determination by Karl Fischer Titration, Git Verlag GMBH, p. 17. 1987. 50. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h921i Water Determination, p. 2085. 51. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h281i Residue on Ignition. p. 1927. 52. Byrn, S. R., Pfeiffer, R. R. and Stowell, J. G. In Solid-State Chemistry of Drugs, 2nd ed., SSCI, Inc., West Lafayette, Chapter 5, pp. 81–82, 1999. 53. United States Pharmacopoeia Rev. 25 and National Formulary Rev. 20. 2002 General Test h891i Thermal Analysis, p. 2081. 54. Encyclopedia of Chromatography, Cazes, J., Ed., Marcel Dekker, Inc., NY, 2001. 55. Chromatographic Analysis of Pharmaceuticals, Adamovics, A., Ed., Chromatographic Science Series, vol. 49 & 74, John Marcel Dekker, Inc., NY, 1990/1997. 56. Snyder L.R. and Kirkland J.J. Introduction to Modern Liquid Chromatography, 2nd ed., John Wiley & Sons, Inc., NY, 1979. 57. Caude, M. and Jardy, A. In Chromatographic Science Series Volume 78: Handbook of HPLC, Katz, E., Eksteen, R. and Schoenmakers, P., Eds., Marcel Dekker, NY, pp. 325–363, 1998.

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7 SAMPLE SELECTION FOR ANALYTICAL METHOD DEVELOPMENT HUGH J. CLARKE AND KENNETH J. NORRIS Pfizer, Inc.,Groton,CT 06340

I. INTRODUCTION II. COMPONENTS OF THE KEY PREDICTIVE SAMPLE SET (KPSS) A. Counter-ions III. STEREOISOMERS IV. MATRIX COMPONENTS V. PROCESS-RELATED IMPURITIES (PRI’s) VI. PURPOSEFUL DEGRADATION SAMPLES A. Sample Screening Recommendations for Purposeful Degradation Samples B. Interpretation of Kinetic Purposeful Degradation Data C. Chiral Compound Screening Recommendations for Purposeful Degradation Samples D. Elimination of Degradation Samples from KPSS VII. STABILITY SAMPLES VIII. PHASE-SOLUBILITYANALYSIS IX. SAMPLE SELECTION STRATEGIES A. Experimental Considerations B. Sample Selection Strategies X. SUMMARY GLOSSARY REFERENCES

I. INTRODUCTION Analytical method development for the quantitation of low level impurities present in pharmaceuticals can be thought of as a three step process. 1. Sample set selection for analytical method development. 2. Screening of chromatographic conditions and phases, typically using the linear-solvent-strength model of gradient elution.1 3. Optimization of the method to fine-tune parameters related to ruggedness and robustness. This can be accomplished using a factorial optimization approach.2–5

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H. J. CLARKE AND K. J. NORRIS

FIGURE 1 Achiral method development process.

Figure 1 illustrates the primary method development process. Item 1, the identiﬁcation and selection of the sample set for method development is the focus of this chapter. Before beginning a method development experiment, the ﬁrst task of the chromatographer is to select the set of samples that accurately represents the purity and degradation proﬁles of the active pharmaceutical ingredient (API). Regulations set forth by the US Food and Drug Administration (FDA) and guidance documents published by the International Conference on Harmonisation necessitate that methods that measure the purity of a drug substance or drug product must be speciﬁc or selective for analytes.6–8 If a key degradant or impurity is omitted during method development, the resulting method may not resolve an important impurity. Therefore, the identiﬁcation of the set of compounds (degradants, impurities, and matrix components), which will be referred to as the essential component set, is critical for effective method development. Each compound may exist in puriﬁed form, but it is likely that many will be one compound in a mixture or solution. The set of samples that encompasses all the elements of the essential component set is referred to as the key predictive sample set, or KPSS. In the analysis of pharmaceutical molecules, there are two distinct elements of the essential component set: One element is speciﬁc for drug substance and the other is speciﬁc for drug product. The drug substance peak set includes process-related substances; therefore, it is process-speciﬁc. If the synthetic process changes, the component set may need to be updated. The drug product component set contains the drug substance set. The inclusion of the drug substance peak set for drug product applications does not necessitate the separation of all peaks present, but rather drug substance impurities must be tracked for potential interference with the drug product components of interest (degradants, preservatives, etc.). The drug product peak set is formula-speciﬁc, so formulation changes can require an update to the drug

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147

FIGURE 2 Key Predictive Sample Set (KPSS) concept.

product peak set. The components and samples, which minimally deﬁne the KPSS, are summarized in Figure 2 and Table 1. There may be other samples or stressed conditions in addition to those listed in Figure 2, which should be addressed on a case-by-case basis.

II. COMPONENTS OF THE KEY PREDICTIVE SAMPLE SET (KPSS) A. Counter-ions Counter-ions, usually small polar or ionic compounds, are routinely used to enhance the aqueous solubility and/or stability of the API. Because of their polarity, counter-ions are rarely resolved from the chromatographic solvent front in reversed-phase HPLC and have characteristically poor chromophores which makes detection difﬁcult. The counter-ion can be omitted from the achiral method development sample set with minimal risk when this holds true. Analysis of counter-ions is normally performed using ion chromatography.9,10 This assay is separate from the reversed-phase assay performed to measure organic impurity levels. Note: Benzoate, besylate, and tosylate salts are examples of counter-ions that are sufﬁciently retained and should be included in the method development set.

III. STEREOISOMERS Isomers are compounds that have the same formula as the parent, but different molecular orientation. Because of potential differences in therapeutic

148

TABLE 1 Component Selection Rules Separation priority

What

Why

Isomers

Need to determine whether the achiral method is specific for diastereomers or geometric isomers. If the method is not specific, chiral method(s) must be employed to determine isomeric purity.

MUST

Matrix components

For drug product applications, sample matrix is an important component of the KPSS. Typically, a representative placebo, placebo blend, or excipient ‘‘soup’’ is included in the KPSS for each formulation.

MUST

Antioxidants, flavours, and preservatives are commonly monitored for during stability. Isolated samples of these compounds are recommended for inclusion in the method development sample set, where applicable.

MUST

Antioxidants Coatings, dyes and inks Excipients (‘‘soups’’) Flavors Process-related impurities (PRI)

Any PRI > 0.05% in the finished goods (known or unknown) must be included.

Regulatory raw materials

1. PRI controlled at raw materials stage < 0.1% or > 0.1%

N/A/LOW WANT

Intermediates

2. PRI controlled at the in-process stage

WANT

Known and unknown synthetic by-products

3. PRI controlled at finished goods

MUST

Mother liquor components

The mother liquor of the last bond-forming step and first recrystallization are recommended for inclusion in the KPSS. Analysis of both these samples can reduce the KPSS versus that based on a single mother liquor.

H. J. CLARKE AND K. J. NORRIS

Preservatives

LOW WANT

2. Found in bond-forming liquor and first recrystallization liquor at < 10% of total impurities

WANT

3. Found in bond-forming liquor and first recrystallization liquor at 10% of total impurities

MUST

4. Have purging data to show it is controlled in the recrystallization scheme.

LOW WANT

Impurity grid components

The impurity grid acts as a hypothetical model to define what could potentially form during the drug substance synthesis. Since any material from this grid which actually forms will be a PRI or in the mother liquors, these are the least important component of the KPSS. If any of the impurity grid substances are included in the KPSS, they may need to be synthesized.

LOW WANT

Degradants Acid, base, light, oxidative, thermal, thermal humidity

Purposeful degradation experiments determine major degradation pathways and flags marker compounds for those pathways.

Stability sample components

1. Meets criteria for KPSS, i.e., present at a level of > 10% of total degradation.

MUST

2. Meets criteria for KPSS, not seen in real stability (6 months @ 40 C/75% RH or 2 years 30 C/60% RH)

LOW WANT

Stability samples act as a reality check for purposeful degradation and they can be used to determine if a kinetically favored secondary degradant is more likely in actual stability. 1. Seen in drug substance stability samples at > 0.05%, but not in purposeful degradation

MUST

2. Seen in drug product stability samples at > 0.1%, but not in purposeful degradation

MUST

7 SAMPLE SELECTION FOR ANALYTICAL METHOD DEVELOPMENT

Evaluation of liquors from two different lots is also desirable. 1. Found in bond-forming liquor but not in first recrystallization liquor

Because of availability and time constraints, some of the peaks or samples listed may not be included in the method development sample set. However, during validation these peaks should be considered for the specificity assessments if available.

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index and safety among stereoisomers, the FDA requires the evaluation of the API isomeric purity proﬁle. Applications for enantiomeric and racemic drug substances should include a stereochemically speciﬁc identity test and/or a stereochemically selective assay method. The choice of the controls should be based upon the substance’s method of manufacture and stability characteristics.11

Stereoisomers can be classiﬁed into two types: enantiomers and diastereomers. Enantiomers (mirror images) have identical physical and chemical properties and therefore are not separated on the conventional reversed-phase stationary phases. Their separation will not be discussed. Diastereomers are isomers which are not mirror images of the parent. They have slightly different physical and chemical properties and can often be separated on conventional stationary phases. There are two classes of diastereomers: optically active isomers when the API has two or more stereocenters and non-optically active geometric isomers, such as cis–trans, syn–anti, etc. Stereoisomers of chiral molecules must be included in the peak set. Either isolated diastereomers or racemic mixtures are preferred for the KPSS. If no isolated samples are available, a sample that contains an enriched level of the diastereomers may be used, but peak tracking may be hampered as diastereomers are challenging to separate from the API. These molecules are subject to elution order changes.

IV. MATRIX COMPONENTS In pharmaceutical analysis, the majority of matrix components (excipients) are present to improve manufacturing characteristics like bulk powder ﬂow, tablet hardness, or to enhance tablet disintegration.12 Other excipients are present in a formulation to preserve the active pharmaceutical ingredient13 (antioxidant, for example) or to act as an antimicrobial agent14 (methylparaben, for example). Still other excipients may be present to increase the palatability of the formulation15 (ﬂavors, for example). It may be necessary to include individual excipients and/or mixtures as part of the KPSS when analyte/excipient interferences occur. Preservatives and antioxidants are normally assayed for during release testing and stability evaluation of a formulation. Therefore, isolated samples of these excipients should be included in the KPSS in addition to the representative placebo or excipient ‘‘soup’’ samples to facilitate peak tracking. Inclusion of these samples is more critical for high-potency compounds, where the ratio of excipient to drug can be very large.

V. PROCESS-RELATED IMPURITIES (PRIs) A stability-speciﬁc-drug-substance-purity method must be able to separate any process-related impurity that is not purged or controlled prior to the ﬁnal step and all of the API degradation products. Ideally, one would

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like to develop a method that is capable of monitoring the quality of the API throughout the entire synthetic process (from raw materials to ﬁnished goods) and is also stability-speciﬁc (separates the API degradants). This method could be used to assess the quality and stability (where applicable) for the raw materials, isolated intermediates (in-process control testing), and ﬁnished goods. Because of polarity extremes of the various analytes and the sheer number of compounds that must be separated, the ideal method is rarely attainable and trade-offs must be made. For the ﬁnished product, impurities that are purged or controlled early in the synthetic process do not necessarily need to be included in the method development sample set (see Table 1). If a viable separation cannot be obtained for compounds that are purged or controlled, they may be dropped from the essential peak set. When a compound is dropped from the peak set, the scientiﬁc rationale should be documented. Table 1 provides general guidance on where trade-offs can be made by rating the compounds as a MUST for compounds that absolutely need to be separated, a WANT for compounds that may be present in ﬁnished goods, and a LOW WANT for compounds that only show up in early stages of the synthetic process and are controlled. Any process-related impurity, known or unknown, that has been observed in the ﬁnished goods at a level 0.05% must be included in the peak set. Experimental campaign bulk or in-process control samples (e.g., prior to recrystallization) are excellent sources of process-related impurities and are a vital component of the KPSS. Isolated fractions collected from a preparative or semi-preparative liquid chromatography (LC) system are also excellent KPSS samples. Sometimes small-scale synthesis of the impurity or degradant is possible and less time-consuming than the isolation techniques. During early stages of drug candidate development, raw materials and isolated process intermediates may be included in the ﬁnished goods essential peak set until the appropriate analytical and process-control strategies are implemented. Once the appropriate controls are in place as the candidate approaches commercialization, it may be possible to eliminate some of these components from the component set. An impurity grid can also be used to predict likely synthetic by-products. The grid is an impurity-tracking tool used to formulate the overall impurity control strategy for the API. Each synthetic step is tabulated with reactants and products. The synthetic product of each molecule is charted. If a material is not purged or not completely reacted, the impurity grid can predict potential synthetic by-products. By-products predicted by the grid may be included in the essential component set depending upon availability and at the discretion of the project analyst. Mother liquors from the key bond-forming step and last recrystallization can be used as surrogate challenge samples for the KPSS in the absence of purging data. The differences in impurity proﬁles of these two liquors can be used to identify potential synthetic by-products that do not purge and therefore must be controlled at ﬁnished goods. Screen the mother liquor samples using the current purity method and an orthogonal screen to reduce the risk of having missed any potential impurities before eliminating them

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FIGURE 3 Drug substance peak tracking example.

from the KPSS. The scientiﬁc rationale for this decision should be documented (see Figure 3). It is common upon scale-up that recrystallization steps will be eliminated from the synthetic route; therefore, it is prudent to include the mother liquor from the bond-forming step in the method development sample set even if there is evidence that the major synthetic by-products are being purged in the recrystallization steps.

VI. PURPOSEFUL DEGRADATION SAMPLES Representative challenged samples (typically 10–20% degradation) are a vital component of method development sample set.16 The generation of these key predictive degradation samples is beyond the scope of this text; however, general guidance on the screening and handling of purposefully degraded samples for use in the method development process is provided. For method development purposes, a reduction in the degradant peak set may be justiﬁed if the degradant(s) are not observed in the long term and accelerated stability samples (see Table 1). However, this data is usually not available until the time of regulatory ﬁling.

A. Sample Screening Recommendations for Purposeful Degradation Samples The purposefully degraded samples should be screened using the current purity methodology, or if a purity method is not available, a gradient of 5–95% organic in 60 minutes could be used. At a minimum, broad-spectrum (200–400 nm) photodiode array UV scans are recommended during the screening of these samples to minimize the risk of missing a degradant due to poor UV absorption. Peak purity assessments of API can then be made to identify co-elutions. Additionally, if a working standard or characterized lot of the drug is available, it can be used as an external standard throughout the screening runs to assess material balance for the challenged samples. This screening process is diagramed in Figure 4. Material balance assessments are critical in conﬁrming that degradants have not been missed and can identify drug/degradant co-elutions. A low bias in the material balance can be due to a number of factors. When performing solution degradation studies, low recovery can be due to poor solubility of

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(KPSS)

–

FIGURE 4 KPSS screening flowchart.

the drug or degradants. When analyzing purposeful degradation samples using UV detection, a low bias may be seen because of alteration of molecular chromophores in one or more degradation products. A low bias may be indicative of retention issue: highly retained or completely unretained (in solvent front) molecules not detected in the analysis. When a low material balance is observed, a thin-layer chromatography (TLC) screen is recommended to assure that no degradants have been missed in the highperformance liquid chromatography (HPLC) analysis.

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A high bias in the material balance is generally indicative of formation of a degradant with higher detector response relative to the API. In the case of UV detection, this would be a degradant with higher molar absorptivity than the API. Photodiode array (PDA) detectors can be used to examine the UV spectra of the parent peak and degradation products. Any degradants with UV spectra signiﬁcantly different from the parent will likely have different molar absorptivity, and hence, different UV response factors. High recoveries for the API itself, assay values > 103%, may be indicative of degradant/API co-elution. When this occurs, it is prudent to perform some additional orthogonal screening to separate the degradant from the API. In the case of one development candidate, a 120% high bias was observed in the oxidative degradation samples. The co-elution was detected from a PDA peak purity assessment. An orthogonal screen was performed on the questionable samples. The primary degradant was separated from the API on a phenylhexyl column run in the orthogonal screen. This had a signiﬁcant impact on the method development strategy for the candidate.

B. Interpretation of Kinetic Purposeful Degradation Data Once the sample screening has been completed for each study time point, the data is generally tabulated by peak identiﬁcation [compound identiﬁcation or relative retention time (RRT)] and area%. An easy way to compile these data is to start with the last time point (most degraded sample) and assemble the peak list working backwards to the initial time point adding to the peak list as necessary. Once the peak list (identiﬁcation) is complete, compile the area% values for each time point. This kinetic data is useful in identifying secondary degradants that would typically need not be included in the method development sample set. Table 2 shows an example for a degradation study of a drug candidate. This example shows that for this candidate, secondary degradation (double dehydration) was occurring at 24 h and beyond. The graph illustrates that the primary degradants at RRT 1.41 and 1.46 have reached steady state at 24 h and RRT 1.17 and 1.30 have ﬁrstorder growth after the 24-h timeframe.

C. Chiral Compound Screening Recommendations for Purposeful Degradation Samples For compounds with one or more stereocenters, it is prudent to screen the key samples (10–20% degradation timepoint) with the current chiral purity method to determine if the degradation pathway is stereospeciﬁc. From the achiral method development perspective, stereospeciﬁc degradation pathways will not affect the outcome of the method development process, but this information can affect the impurity control strategy for the compound.

D. Elimination of Degradation Samples from KPSS It is recommended to put the key degradation samples through an orthogonal reversed-phase and/or TLC screen to ensure that all the

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7 SAMPLE SELECTION FOR ANALYTICAL METHOD DEVELOPMENT

TABLE 2 Development Compound Degradation Kinetic Study Results @ 70 C

Sample Initial–Drug in water

Pull times

Parent compound

Rr 51.41

Rr 51.46

Rr 51.17

Rr 51.30

(h)

%area

(wt/wt)%

(wt/wt)%

(wt/wt)%

(wt/wt)%

100.0

ND

ND

ND

ND

70 C T0 H2SO4

0.0

100.0

ND

ND

ND

ND

70 C T1hr H2SO4

1.0

99.9

ND

ND

ND

ND

70 C T3hr H2SO4

3.0

98.4

0.14

0.05

ND

ND

70 C T7hr H2SO4

7.0

93.8

0.39

0.14

ND

ND

70 C T24hr H2SO4

24.7

83.0

0.76

0.28

0.45

0.49

70 C T96hr H2SO4

100.0

83.0

0.62

0.26

1.52

2.67

70 C T168hr H2SO4

168.8

77.3

0.60

0.28

2.37

4.55

NA

degradants have been accounted for (see Figure 4 for ﬂowchart). After screening if no unique peaks are present in the key challenge samples or the peaks present in those samples are covered by another sample in the KPSS, the challenge sample may be safely dropped from the method development sample set. The scientiﬁc rationale for this decision should be documented (see Figure 6).

VII. STABILITY SAMPLES Stability samples augment the peak set since they are more indicative of the actual degradation proﬁle under typical storage conditions than purposefully degraded samples. Any peak(s) which are observed in the accelerated challenge (40 C/75% RH 6 month or 50 C/20% RH 3 month) or long-term storage samples must be included in the essential peak set. It is critical to demonstrate speciﬁcity for these peaks (i.e., any peak > 0.05% and 0.1% of API for drug substance and product, respectively).

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4-Fluorothiophenol

FIGURE 5 Example elimination rationale form.

When long-term storage stability proﬁles become available, these proﬁles can be used to justify a reduction in the degradant peak set. The scientiﬁc rationale for this decision should be documented.

VIII. PHASE-SOLUBILITY ANALYSIS Phase-solubility analysis17 (sometimes referred to as phase equilibrium puriﬁcation) is the quantitative determination of the purity of a substance through the application of precise solubility measurements. At a given temperature, a deﬁnite amount of a pure substance is soluble in a deﬁnite quantity of solvent. The resulting solution is saturated with respect to the particular substance, but the solution remains unsaturated with respect to other substances even though such substances may be closely related in chemical structure and physical properties to the particular substance being tested. There are examples of the use of this technique in HPLC methods development18 and in the characterization of reference standards,19 but the

7 SAMPLE SELECTION FOR ANALYTICAL METHOD DEVELOPMENT

157

FIGURE 6 HCl salt impurity enhancement example.

technique can also be used to enrich saturated solutions with low-level impurities. The ability to track the key peaks from column-to-column and conditionto-condition is generally the most challenging aspect of the method development process. In particular, tracking impurities or degradants that are present at levels < 0.2% of the API can be difﬁcult, and peak misassignments are common. Photodiode array UV detection and mass spectrometry for tracking of these low level peaks may be of limited utility because of poor sensitivity, baseline noise, and ionization inefﬁciencies. For these cases, an impurity/degradant enhancement technique can be used to increase the levels present with respect to the API and thereby facilitate the peak tracking process. Of the impurity/degradant enhancement techniques available, the phasesolubility analysis technique requires minimal sample handling from the

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analyst and the observed enhancement factors can range from 5 to 10000 depending upon the solubilities of the drug and impurities. A saturated solution of drug substance (typically 1 gram in a few milliliters of solvent) is created, and the impurities present are concentrated in the solvent (supernatant) since they typically are not present at their saturation level. The magnitude of this concentration enhancement in general depends on the solubility of the API. Poor API solubility can result in signiﬁcant enhancement of the drug substance impurities. An example from a development project is shown in Figures 6 and 7. Saturated solutions of two salt forms of a development candidate, hydrochloride salt and citrate salt, were created in acetonitrile. The hydrochloride salt (Figure 6) was readily soluble (> 10 mg/mL) in acetonitrile, whereas the citrate salt (Figure 7) was only slightly soluble (0.04 mg/mL). The impurity enhancement factors were markedly different, 5 for the hydrochloride salt and 1000þ for the citrate salt. Even an increase in the impurity-to-drug ratio of 5 can make a signiﬁcant difference in terms of

FIGURE 7 Citrate salt impurity enhancement example.

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159

peak tracking. Impurities that are present at levels approaching 1.0% relative to the API are fairly easy to track from column to column and condition to condition. For cases where an impurity is present at low levels (< 0.5%), the phase equilibrium puriﬁcation technique is an easy way to concentrate the impurity to a readily trackable level. This technique can be used to concentrate multiple trace impurities in non-recrystallized bulk lots without resorting to the time-intensive semi-preparative fraction collection and/or isolation methods. When utilizing this technique, both the drug substance reference material and the enhanced supernatant from the saturated solution would have to be included in the method development sample set to insure that only the relevant peaks are being tracked during the screening process.

IX. SAMPLE SELECTION STRATEGIES The selection of the set of samples that best covers the impurity and degradation proﬁles of the API is critical to successful method development. For a given API there are several ways to design the method development sample set some of which are more advantageous, but incur more risk in terms of the ability to track peaks from column to column and condition to condition. This section will address method development sample set design strategies, experimental concerns, and trade-offs.

A. Experimental Considerations 1. Solution and Degradant/Impurity Stability The method development sample solutions need to be stable over the course of the screening runs, which are typically 24 hours or more depending on the number of samples in the method development set. Some instability can be tolerated as long as it does not interfere with the ability to track the relevant peaks. The chance for degradation and impurity interactions increases with solution complexity. Isolated or simple mixtures are less problematic from a solution stability standpoint, as degradation can readily be identiﬁed and generally does not interfere with the ability to track the few relevant peaks that are present. If little is known about solution stability or there is a reason to suspect that there may be an issue based on the chemistry, the use of a refrigerated autosampler and amber HPLC vials is recommended. A quick evaluation of solution stability over the course of 24 hours or at the end of each screening run can also be performed to identify stability issues and to insure that peak tracking is not compromised. Sample solutions should be stored at 2–8 C until solution stability has been established and for longterm storage 70 C should be considered. The sample solvent can be the source of instability and rapid degradation of the relevant impurity can occur. In the case of one development compound, an imine process-related impurity was a primary degradation product. The imine was stable in acetonitrile, but would readily hydrolyze in aqueous media. In fact, on-column degradation was observed when screening

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the imine under acidic conditions. It is important to consider compound/ solvent interactions whenever possible. 2. Solvent Selection The selection of an appropriate sample diluent can have a signiﬁcant impact on the ability to track peaks during the screening process. Since extreme gradient ranges (5–95% organic) may be employed during the screening process, solvent/mobile phase matching as a way to reduce system artifacts is not possible. Because of differences in solvent purity and mixing properties, the gradient artifacts will vary from diluent to diluent. Therefore, it is convenient to minimize the number of diluents used to generate the sample solutions, as respective blanks for each diluent should be included in the screening exercise. Solvent strength effects such as splitting of poorly retained species (small polar molecules) can occur if a strong solvent is used as the diluent.20 A solvent strength of 65:35 aqueous/organic has been found to work well for most applications. There are cases where a stronger solvent may be necessary because of poor degradant or impurity solubility. Generally, these compounds are retained longer and the diluent/mobile phase mismatch is not as severe, so peak splitting is usually not observed. Additionally, if methanol is used as a component of the diluent, a variable intensity displacement peak is commonly observed at screening wavelengths below 220 nm.21,22 If unexpected peak splitting is observed during the screening process, the diluent solvent strength for the sample in question should be investigated. 3. Handling of Fractions and Phase Equilibrium Supernatants Impurity enhancement techniques such as fraction collection and phase equilibrium puriﬁcation can be used to provide enriched samples for use in the method development process.23 When using the fraction collection approach, one or more cuts (fractions) of the chromatographic separation of a bulk lot or mother liquor are isolated. The excess solvent in these fractions is then evaporated to achieve the desired concentration enhancement. These fractions typically contain extraneous peaks because of the presence of salts in the mobile phase or sample degradation during the concentration step. The salts can be removed by extraction and/or a LC cleanup step. To insure that these extraneous peaks/artifacts are not identiﬁed as key peaks for separation, the original bulk lot or mother liquor should be included in the method development sample set. The same holds true for phase-equilibriumpuriﬁcation supernatants.

B. Sample Selection Strategies For a given API, there can be numerous ways to mix and match the available samples to construct the method development sample set. During early stages of development, only a few isolated intermediates and purposeful degradation samples may be available, making sample set selection relatively easy. Later in development, more isolated samples of the major degradants

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161

Individual Components

Cocktails or Complex Samples FIGURE 8 Sample selection trade-offs.

and process-related impurities will be available. The most difﬁcult task during chromatographic method development is accurately tracking the peaks from various columns or conditions.24–26 The use of isolated samples in the method development sample set reduces both the peak-tracking burden and the risk of peak misassignment, while increasing the run time of the screening process. As long as retention time drift is not occurring during method development, the beneﬁt of being able to unambiguously attribute the component outweighs the extra run time, since most screens are automated (See Figure 8). When the sample set is small, the use of isolated samples is strongly recommended, as peak attribution is generally the rate-limiting step in the method development screening process. When the sample set becomes large (15þ samples), the risk for retention time drifts to occur during the screening process increases and cocktailing should be considered to reduce the number of samples. Cocktails are mixtures of KPSS samples that combine two or more compounds to be separated into a single sample. This can save signiﬁcant time in the method development effort since the amount of injections necessary are reduced by the number of combinations. However, using complex cocktail mixtures can complicate the assignment of peak retention times. The following are guidelines for employing the cocktail technique: .

. .

The preparation of cocktail samples should be documented in a notebook, and that notebook ID should be used to name the sample for injection, along with a convenient naming convention. For example, ‘‘NB-12345-001 PD UV/PD thermal’’ could be used to define a mixture of two purposeful degradation samples into a cocktail sample. Stereoisomers should be kept separate from other samples, as they are easy to misassign. When possible, spike samples at different levels relative to peak response. Differences in peak areas of 30% should allow for

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.

.

discrimination between closely eluting samples. There are benefits to adding a component to more than one cocktail mixture, or overlapping. This allows you to control-chart the retention times and ensure that the HPLC system was well-equilibrated during the run. It also provides an assignable peak from one sample to another to act as a reference for polarity assessments of unknowns in the samples. The total number of tracked compounds in a cocktail sample should be limited by your confidence in correctly assigning peak IDs.

Drug substance lots with different impurity proﬁles can be combined for peak-tracking purposes, provided the impurity proﬁles are relatively simple and that the abundance of each impurity in the ﬁnal solution is sufﬁcient to obtain accurate photodiode array UV data.

X. SUMMARY Accurate quantiﬁcation of low level impurities is essential in deﬁning the quality of pharmaceutical products. To that end, a great deal of time is devoted to developing methods to meet these needs. The ﬁrst step of this development project must be to deﬁne and gather a set of samples containing any potential and actual impurities that need to be assessed by the purity method. With this set in hand, subsequent development experiments can assure that a method or methods can accurately and completely determine the purity of a pharmaceutical product. This chapter provides detailed guidance in selecting the set of samples that contain the compounds of interest that must be quantiﬁed at low levels for a pharmaceutical product. A list of potential components and their sources is provided. Guidance is given on sample-screening techniques and when to eliminate samples that are redundant or unnecessary. Finally, techniques are outlined to enrich and combine samples in order to minimize the sample set.

GLOSSARY API Active pharmaceutical ingredient. This is the therapeutic agent in the formulation. cocktail A solution comprised of two or more samples. The samples are mixed to increase the number of peaks tracked per injection. Cocktails are usually prepared in order to shorten method development timelines. impurity grid A table of the theoretical impurities that could possibly form during drug substance synthesis. For example, the table would show what would become of a raw material impurity after each step in the synthesis. matrix components All elements of the ﬁnished goods drug product including antioxidants, coatings, dyes, excipients, ﬂavors, inks, and preservatives. method development peak set All compounds necessary in a method development project to show that a method meets its speciﬁc business need. For a ﬁnished product purity assay, this could be all components of the

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essential component set. For an in-process purity determination, this could be a subset of the full essential component set. mother liquor The supernatant solution that is left over after recrystallization of bulk drug substance. Typically, mother liquors are excellent sources for impurities (synthetic by-products) that may be found in trace amounts in the crystallized bulk drug substance. orthogonal RP screen A series of LC experiments utilizing orthogonal reversed-phase columns and mobile phase conditions. The goal of the experiment is to resolve co-eluting peaks that may not be detected on a single chromatographic or mobile phase. PRI process-related impurity. A synthetic by-product of the manufacture of bulk drug substance. PRIs may be related to the API or to raw materials or processing agents used in manufacture. purposeful degradation Purposeful degradation studies of the drug substance or drug product include appropriate solution and solid-state stress conditions (e.g., acid/base hydrolysis, heat, humidity, oxidation, and light exposure, in accordance with ICH guidelines). Speciﬁed stress conditions should result in approximately 10–20% degradation of the drug substance or represent a reasonable maximum condition achievable for the drug substance. The speciﬁc conditions (intensity and length of time) will depend on the chemical characteristics of the drug substance. soup Drug product blend containing the proposed excipients and API at the maximum excipient/API range. This represents the worst-case example for investigation of drug product stability. stability-indicating methodology A stability-indicating method (generally, potency) must be able to separate the analyte (typically, the API) from the degradants, extractables, impurities, and matrix components. stability-speciﬁc methodology A stability-speciﬁc-drug-substance-purity method must be able to separate any process-related impurity that is not purged or controlled prior to the ﬁnal step and all of the API degradation products. A stability-speciﬁc-drug-product-purity method must be able to separate the API and its degradants from the matrix components, processrelated impurities, and extractables.

ACKNOWLEDGMENTS The authors thank Cheryl Bye for her contributions to this chapter.

REFERENCES 1. Snyder, L. R. and Dolan, J. W. The linear-solvent-strength model of gradient elution. Adv. Chromatogr. N.Y. 38:115–187, 1998. 2. McGraw, Joel D., Leonard, Jason A. and Madrak, M. K. Design of experimental approach to optimization of an HPLC separation. Abstracts of Papers, 222nd ACS National Meeting, Chicago, IL, August 26–30, 2001. 3. Mahesan, B. and Lai, W. Optimization of selected chromatographic responses using a designed experiment at the fine-tuning stage in reversed-phase high-performance liquid chromatographic method development. Drug Dev. Ind. Pharm. 27(6):585–590, 2001.

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4. Harang, V. and Westerlund, D., Optimization of an HPLC method for the separation of erythromycin and related compounds using factorial design. Chromatographia 50(9/10): 525–531, 1999. 5. Osborne, L. M. and Miyakawa, T. W. Use of experimental design in the optimization of HPLC methodology for the separation of stereoisomers. J. Liq. Chromatogr. Relat. Technol. 20(4):501–509, 1997. 6. FDA CDER Guidance: Analytical procedures and methods validation chemistry, manufacturing, and controls documentation. http://www.fda.gov/cder/guidance/2396dft.htm 7. ICH Q2A: Text on validations of analytical procedures, federal register, Vol. 60, March 1, 1995, p. 11260. http://www.ifpma.org/ich5q.html#Analytical 8. ICH Q2B: Methodology: federal register, Vol. 62, No. 96, May 19, 1997, p. 27463–27467. http://www.ifpma.org/ich5q.html#Analytical 9. Douglas, T. Gjerde, James, Fritz. S. Ion chromatography, 2nd ed. Heidelberg; NY: A. Huthig, xi, p. 283 1987. 10. Snyder, L. R., Glajch, J. L. and Kirkland, J. J. Practical HPLC Method Development, NY, Wiley, p. 341, 1988. 11. FDA’s Policy Statement for the Development of New Stereoisomeric Drugs. Publication Date: 5/1/92. http://www.fda.gov/cder/guidance/stereo.htm 12. Ainley, W. and Weller, P. J. Handbook of Pharmaceutical Excipients. American Pharmaceutical Association, 1994. 13. Papas, A. M. Antioxidant Status, Diet, Nutrition, and Health. CRC Press, Boca Raton, FL, 1999. 14. S.F. Bloomfield [et al.]. Microbial Quality Assurance in Pharmaceuticals, Cosmetics, and Toiletries. Halsted Press, NY, 1988. 15. Smolinske, Susan C. Handbook of Food, Drug, and Cosmetic Excipients, CRC Press, Boca Raton, FL, 1992. 16. Alsante, K. M., Friedmann, R. C., Hatajik, T. D., Lohr, L. L., Sharp, T. R., Snyder, K. D. and Szczesny, E. J. Degradation and impurity analysis for pharmaceutical drug candidates, In Handbook of Modern Pharmaceutical Analysis, pp. 85–172. Ahuja, S. and Scypinski, S., Eds., Academic Press, San Diego, CA, 2001. 17. USP 24 h1171i Phase-solubility analysis, The United States Pharmacopeial Convention, Inc 2001. 18. Rapado-Martinez, I., Garcia-Alvarez-Coque, M. C. and Villanueva-Camanas, R. M. Liquid chromatographic procedure for the evaluation of -blockers in pharmaceuticals using hybrid micellar mobile phases. J. Chromatogr. A 765(2):221–231, 1997. 19. Grdinic, V., Jaksevac-Miksa, M., Bezjak, A., Radaic, A. and Briski, D. Importance of factors for ruggedness test in phase solubility analysis. Eur. J. Pharm. Sci. 2(4):293–6, 1994. 20. Wilson, T. D. Sample solvent effects in an apparent chiral high-performance liquid chromatographic separation on -cyclodextrin. Pharm. Sci. Dep., Sterling-Winthrop Res. Inst., Rensselaer, NY, J. Chromatogr. 448(1):31–39 1988. 21. McCormick, R. M. and Karger, B. L. Role of organic modifier sorption on retention phenomena in reversed-phase liquid chromatography. Adv. Chromatogr. (Houston) 15:259– 73, 1980. 22. Dekany, I. and Nagy, L. G. Immersional wetting and adsorption displacement on hydrophilic/hydrophobic surfaces. J. Colloid Interface Sci. 147(1):119–28, 1991. 23. Blanchard, A. J., Alsante, K. M., Nickerson, B., Snyder and Kimberly, D. Extraction of low level impurities from tablets using accelerated solvent extraction. Abstracts of Papers, 222nd ACS National Meeting, Chicago, IL, August 26–30, 2001. 24. Strasters, J. K., Billiet, H. A. H., De Galan, L. and Vandeginste, B. G. M. Strategy for peak tracking in liquid chromatography on the basis of a multivariate analysis of spectral data. J. Chromatogr. 499:499–522, 1990. 25. Lankmayr, E. P., Wegscheider, W., Daniel-Ivad, J., Kolossvary, I., Csonka, G. and Otto, M. Recent advances in fuzzy peak tracking in high-performance liquid chromatography. J. Chromatogr. 485:557–67, 1989. 26. Dolan, J. W. Peak tracking. LC-GC 8(6) 1990.

8 SAMPLE PREPARATION METHODS FOR THE ANALYSIS OF PHARMACEUTICAL MATERIALS DAVID T. ROSSI AND KENNETH G. MILLER Pfizer Global Research and Development, Ann Arbor, MI 48105

I. INTRODUCTION II. SOLID-PHASE EXTRACTION (SPE) A. Suppliers B. Sorbents C. Cartridges D. Disks E. Extraction Process F. Nonconventional Techniques III. LIQUID ^LIQUID EXTRACTION (LLE) A. Theoretical Considerations in LLE B. Countercurrent LLE C. Liquid^Liquid Extraction Cartridges D. Emulsions E. Solvent Selection in LLE F. Drying Agents IV. SUPERCRITICAL FLUID EXTRACTION (SFE) A. Fundamental Properties of Supercritical Fluids B. Supercritical Fluid Extraction Instrumentation C. Static/Dynamic SFE D. Pumps Used for SFE E. Extraction Cells F. Modifiers G. Restrictors H. Collection Devices I. Pharmaceutical Applications of Supercritical Fluid Extraction V. ACCELERATED SOLVENT EXTRACTION (ASE) A. Sample Pretreatment B. Solvent Selection in ASE C. The Effects of Temperature in ASE

165

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D. The Effects of Pressure E. CycleTime F. Carry-over VI. CENTRIFUGATION A. Centrifugation for Small Molecule Sample Preparation VII. FILTRATION A. Membrane Selection B. Housing Selection C. Pore Size D. Filtration Device Size VIII. SUMMARY REFERENCES

I. INTRODUCTION Many samples in the pharmaceutical industry cannot be directly processed for chromatographic or spectroscopic characterization for a number of reasons. Biological or excipient-laden pharmaceutical samples are too complex and require a series of sample preparation, cleanup, and concentration steps. Sample preparation can be the most laborious, unreliable, and least reproducible step of the analysis. The characteristics of successful sample preparation steps are (a) that a homogeneous representative sample must be obtained, (b) the sample preparation must be performed without losing any of the target analytes for quantitative analysis, (c) the preparation should result in the sample’s being in the most ideal form for the chosen analytical technique while removing, and not cross-contaminating with interfering components, and (d) the preparation steps should dilute or concentrate so that the target analytes are in the correct concentration range for the chosen analytical technique. There are many literature references that describe sample preparation procedures for individual analytes and pharmaceutical samples, which are too numerous to reference here. Our aim instead is to discuss the characteristics of several widely used sample preparation techniques in their broadest sense and to take a more practical approach rather than a theoretical approach. The references for each section were chosen carefully and are recommended to give the reader a comprehensive link to speciﬁc topics. Several vendor and supplier Web sites are mentioned that contain detailed information. These sites were selected because of their usefulness, and we would encourage the reader to examine them for more speciﬁc information.

II. SOLID-PHASE EXTRACTION (SPE) The conventional process of solid-phase extraction involves passing a liquid sample over a solid surface in order to separate target analytes from the sample matrix. The attractive interactions of the analyte with the solid

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surface must be greater than the attractive interactions of the analyte with the sample matrix in order for retention and extraction to occur. The technique is based on the same principles as chromatographic separations (partitioning, adsorption, or ion exchange). The intent of this section is to serve as a practical guide to SPE sorbent and device selection, extraction steps, and expected performance.

A. Suppliers At the time of this writing, there are at least six main suppliers of solidphase extraction equipment and supplies. At least three of the suppliers offer technical guides and general solid-phase extraction literature.1–4 Table 1 lists the companies and their Internet sites. Thurman and Mills provide a comprehensive list of SPE product suppliers in their book Solid Phase Extraction (Wiley, 1998).5

B. Sorbents The retention mechanisms in SPE are similar to those of HPLC. The packings used in SPE are derivatives of the same materials used for HPLC and are subject to many of the same functional limitations and production problems.6,7 Table 2 summarizes the most commonly available sorbents for SPE. Typical sorbents are made of 40–200-mm irregularly shaped porous silica gel particles with pore diameters averaging 60 A˚ and ranging as high as 300 A˚. Bonded phases are often attached to the silica particles to extend and amplify their functionality. Carbon loading for typical bonded phases (C2 to C18, phenyl, cyclohexyl) ranges from less than 4% to about 17% carbon by weight. It has been reported that heavy loading of bonded ligands can block or ﬁll pores of the SPE sorbents, effectively creating a pellicular material.6 Chemically bonded silica gels with cyanopropyl, aminopropyl, and diol functional groups are also available and each has been used for normal-phase as well as reversed-phase separations. Polymeric-based packings are available and can be used for ion-exchange or reversed-phase applications. Crosslinked polymeric based packings have been combined with ion-exchange materials to create a more pH-stable stationary phase. Graphitized carbon TABLE 1 Solid-Phase Extraction Equipment and Suppliers Company

Internet site

Supleco (Bellefonte, PA)

www.Sigma-Aldrich.com

J.T. Baker (Phillipsburg, NJ)

www.JTBaker.com

3M Company (St. Paul, MN)

www.3M.com/Empore

Varian (Harbor City, CA)

www.Varianinc.com

Waters (Milford, MA)

www.Waters.com

Jones Chromatography (Lakewood, CO)

www.Joneschrom.com

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TABLE 2 Commercially Available Sorbents for Solid-Phase Extraction (compiled from refs. 1^5, 15,16) Sorbent

Typical mode of separation

Octadecyl

Reversed-phase

Octyl

Reversed-phase

Butyl

Reversed-phase

Propyl

Reversed-phase

Ethyl

Reversed-phase

Methyl

Reversed-phase

Cyclohexyl

Reversed-phase

Phenyl

Reversed-phase

Cyano

Normal-phase/reversed-phase

Amine (1 /2 )

Ion exchange/normal-phase/reversed-phase

Diol

Normal-phase/reversed-phase

Propyl amine

Ion exchange/normal-phase/reversed-phase

Silica gel

Adsorption/normal-phase

Alumina

Adsorption/normal-phase

Magnesium silicate

Adsorption/normal-phase

Graphitized carbon

Adsorption/normal-phase

Styrene-divinylbenzene

Adsorption

Quaternary amine

Ion exchange/normal-phase/reversed-phase

Carboxcylic acid

Ion exchange/normal-phase/reversed-phase

Propyl sulfonic acid

Ion exchange/normal-phase/reversed-phase

Benzenesulfonic acid

Ion exchange/normal-phase/reversed-phase

and styrene-divinyl benzene-based sorbents have been used widely for retention and extraction of aromatic-based compounds. Wide-pore sorbents having pore diameters of about 300 A˚ can utilize a size-exclusion mechanism for extraction. As an example of this, a 1,6 glucose polymer cross-linked with epichlorohydrin is available and has been used to desalt protein solutions.2 Magnesium silicate (FlorisilÕ ), silica gel, and aluminum oxide (alumina) are used as normal-phase sorbents and are available in a variety of packing grades. Primary, secondary, and quaternary amines, sulfonic and carboxylic acid, and cyano-based sorbents can be used in ionexchange modes. The pH and ionic strength of the sample solution and ion-exchange capacity of the packing are important operating parameters for ion-exchange extractions. It is common for SPE users to create their own packings by combining materials with different modes of separation. A common mixed-mode system is a weak cation exchange packing with a typical reversed-phase packing (e.g., C18). Variations of this mixed mechanism approach have been described as shielded hydrophobic phases, semipermeable surfaces, dual-zone phases, and internal-surface reversed phases.8,9 This type of methodology has been used

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extensively for sample cleanup with pharmaceutical compounds as the target analytes. Some packings not described in Table 2 utilize mechanisms that are more speciﬁc, such as afﬁnity solid-phase extraction.10 These packings are not generally available commercially but are highly speciﬁc for target analytes or classes of compounds. The mechanism is identical to performing an enzyme immunoassay where an analyte binds selectively to an active site on an antibody through multiple non-covalent interactions. In this case the antibody is attached, usually via an aliphatic chain, to a silica-based or carbohydrate (agarose) sorbent. The analyte is dislodged by upsetting the binding site and is eluted with buffer or organic solvent. A reversible denaturation of the antibody is a viable elution strategy. This approach is highly selective and is effective for peptides or other biopolymers.

C. Cartridges The basic design of SPE cartridges has changed little since its introduction in the late 1970s, although the cartridge format has now taken on a wider range of sizes. The typical SPE cartridge (see Figure 1) consists of an open syringe barrel containing a sorbent packed between porous plastic, stainless steel, or titanium frits. The dimensions of the sorbent bed are purposely miniaturized to minimize the difﬁculty in sample processing using gravity- or suction-aided sample ﬂow. The syringe barrels are constructed of polyethylene, polypropylene, glass, or metal. The open portion above the sorbent packing, generally referred to as the ‘‘sample reservoir,’’ is available with volumes from about 1–20 mL. Larger reservoirs with volumes up to about 100 mL are available and can be connected via an adapter to the inlet of an open syringe barrel device. The cartridge inlet can be shaped like a standard syringe or can be ﬂared to allow for easier sample introduction. Cartridges are also available with female Luer-lock ﬁttings at the inlet for easy connection and use with on-line applications. The standard outlet of the cartridge design is a male Luer-lock ﬁtting. Most open inlet designs have a Luer-lock – type ﬁtting at the outlet. Commercial cartridges are available with sorbent masses of about 25 mg to more than 10 g. The packing density of the cartridges varies widely and the cartridge beds are physically not very stable, which indicates that packing is not tightly controlled in the manufacturing process. The instability of the packing can result in heterogenous ﬂow through the sorbent, which reduces the effectiveness of the extraction. Typical processing ﬂow rates are usually between 3 and 30 mL/min. A cartridge containing about 500 mg of sorbent with a bed height of 6 mm will produce about 5–15 theoretical plates.11 As the primary function of SPE is retention and not separation, such low numbers of theoretical plates are not necessarily detrimental for its intended use. Most suppliers now include certiﬁcates of analysis for each lot of cartridges, which include some basic information on the silica gel such as average pore size, surface area, pore volume, surface pH, and average particle diameter. This information proves to be very useful because lot-to-lot variability for sorbent materials is high. It also provides a basis for comparison when switching manufacturers.

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FIGURE 1 A pictorial representation of solid-phase extraction, including conditioning, sample introduction, washing, and elution.

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D. Disks In the early 1990s particle-loaded membranes and particle-embedded glass ﬁber disks emerged as alternates to the SPE cartridge technology. These products were generically classiﬁed as disk technology for SPE and claimed the following advantages over cartridge format: (1) shorter sample processing times because of a larger cross-sectional area and decreased pressure drop, enabling higher sample ﬂow rates to be used, (2) decreased plugging by particles because of the larger cross-sectional area of the disks, (3) reduced channeling and improved kinetic performance resulting from the use of smaller sorbent particles and the greater mechanical stability of the disks, (4) the optimized use of bed mass resulting in a cleaner background and lower interferences in the isolation of the analytes of interest.6,11,12 Disks can be used for the same isolation techniques as cartridges. The disks or membranes are used essentially as ﬁlter paper except that the analytes to be isolated are extracted and retained by the membrane. Several suppliers sell specialized glass ﬁltration devices to make their use easier. SPE disks are available with the same sorbent functionality as cartridge packings and come in a variety of sizes ranging from 4 mm to 10 cm in diameter with 47 mm being the most popular for impurity isolation. The major difference between cartridges and disks other than format is particle size of the sorbent. The size of particles ranges from about 8 to 30 mm for disks. The large surface area and the small particle size presented by the disk format allows for rapid mass transfer and reduces problems associated with channeling within the bed. These characteristics make disk technology applicable to situations where large amounts of sample (e.g., water) need to be processed or where small volumes of conditioning and elution solvents would be beneﬁcial. Solid-phase extraction disks are available in several useful formats. The free disk format resembles ﬁlter paper and is treated in much the same manner. The syringe barrel format is similar to an SPE cartridge, but includes a single preﬁlter constructed of glass ﬁber pressed on top of the particleloaded membrane. The preﬁlter removes large particles associated with the sample matrix and allows for unimpeded passage of the sample through the disk. A miniature syringe barrel format is used for 96 well micro-titer plate applications and is useful when many small volume (< 500 mL) samples are to be processed. The ‘‘syringe tip’’ format replicates a typical syringe tip ﬁlter with Luer-lock ﬁttings and a polypropylene housing. The syringe tip ﬁlters are currently available in 13-mm or 25-mm diameters.

E. Extraction Process SPE is a sample preparation or sample clean-up technique used to separate compounds of interest from interfering matrix components. The actual process of SPE has been explained in as little as four steps— conditioning, sample introduction, washing, and elution. In reality it can be signiﬁcantly more complicated. In order to develop a useful method, the entire analytical scheme should be considered. A decision must be made to use a sample cleanup step, based on the requirements of downstream

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analytical techniques. Considerations should be given to the need for quantitative or qualitative analysis. The analytical technique to be used will affect decisions as to the quality and purity of the ﬁnal sample generated. Assuming these factors have been considered and SPE is deemed a viable choice for sample processing, the procedure for SPE can be developed. The ﬁrst considerations are for the target analyte and the sample matrix. Gaseous and liquid samples requiring no chemical modiﬁcation are generally the easiest to process. Solids and complex liquid samples, in which the target analytes are similar to or bound to the sample matrix can be challenging. Body ﬂuids and tissues are common sample matrices in which SPE has been widely applied. Fatty tissue, proteins, and salts have been handled successfully by homogenization, precipitation with organic solvents, and/or dilution with water. Reducing or increasing the solvent strength of the sample matrix depends on whether the target analytes are to be retained or washed through the SPE device and can be done by pH adjustment or addition of the appropriate co-solvent. The Handbook of Sorbent Extraction Technology4 provides a relatively comprehensive reference to modiﬁcations of common sample matrices. Knowing as much as possible about the physical and chemical characteristics of the analytes greatly enhances the chances of successful sample preparations. The solubility and polarity of the compounds of interest as well as the sample matrix determine what type of device and mode of interaction should be used. Most SPE supplier technical bulletins have useful ﬂowcharts for general sorbent selection. If the analyte is organic, the molecular weight is used to determine if size exclusion can be used as an extraction mechanism. If size exclusion is not appropriate, the solubility and polarity of the analyte can be used to determine what separation mechanism is suitable. Ionic water-soluble compounds are most readily handled by ion exchange while non-ionic or ion-paired compounds are managed by adsorption, reversed or normal-phase mechanisms depending on polarity. Ion-exchange sorbents are useful for trace metals and reversed-phase sorbents are useful for metal chelates. The characteristics of the sample, the matrix, and the required processing times determine whether a cartridge or disk is more suitable. The same basic procedures for conditioning, sample addition, washing, and elution apply to both cartridges and disks. Conditioning is required to wet the surface of the sorbent and to remove any impurities that may be present on the unused tube or disk. For reversedphase sorbents, an aqueous buffer with low concentration of organic solvent (e.g., 2% methanol) is typically used. For normal-phase sorbents, the packings are usually conditioned with the organic solvent in which the target analyte or sample exists. For ion-exchange sorbents, if the sample is in a nonpolar organic solvent, the sorbent should be conditioned with a solvent in which the target analyte or sample matrix exists. If the sample is in a polar solvent, a methanol buffer or acetonitrile buffer solution adjusted to the proper elution pH and ionic strength is normally used. The amount of conditioning solvent is usually one full column volume if using a tube or cartridge and about 5 mL for a 47-mm disk. Larger amounts of conditioning

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solvent will generally not affect the performance but can increase the performance in some poorly packed cartridges by redistributing the packing into a more uniform bed. The sorbent bed should not be allowed to dry before the sample is introduced, because buffer salts can precipitate and affect wetting of the stationary phase surface. A general recommendation is to leave just enough conditioning solvent so that the top of the frit or disk is covered. The extraction process using tubes or disks is similar to liquid chromatography (LC). Yet instead of a small injection plug of sample, the sample volume can vary from a few microliters to several liters. Samples are generally processed to become suitable liquid samples and poured quantitatively onto the top tube frit or disk as is or through a preﬁlter. Many commercial devices are available to aid with this sample introduction. Tubes can be processed individually by simply pouring the liquid sample on top of the frit or disk and allowing the solvent to drip through the packed bed by gravity. Other methods include the use of syringe plungers and single tube processors ﬁtted for the tube design and attached via a tube adapter or Luer-lock ﬁtting. Air or nitrogen lines can be attached to some tube processors to force the sample solution and elution solvents through the packing. Sample solution can be poured on top of a cartridge frit and placed in a centrifuge tube, then spun until the sample liquor is pulled completely through the packing. A vacuum ﬂask apparatus can be constructed for use with tubes or disks, using a holed rubber stopper, collection tube (e.g., test tube), a transfer line (e.g., large-gauge needle connected to the SPE tube), and house vacuum or water aspiration. Several companies manufacture vacuum devices that process multiple tubes. Typical designs process 6–24 tubes using an adjustable vacuum manifold, stainless steel or PTFE transfer lines, and glass test tubes as the collection devices. The sample matrix itself can contribute to the loss of retention capacity by not properly wetting the solid-phase surface. To reduce the risk of sample breakthrough via this mechanism, small amounts of organic solvent can be added (1–5%) to maintain properly wetted conditions. The general rule for retention capacity of silica-based sorbents is about 5% (w/w). In practice, the retention capacity is usually much lower before breakthrough can occur. One trick to test for breakthrough is to attach two or more tubes in series and apply the sample solution. The tubes are separated and processed individually to elute any retained analytes. If analytes are detected in more than the ﬁrst tube, breakthrough is occurring. The bed volume, ﬂow rate, sample matrix, or packing should be evaluated to eliminate breakthrough. Several researchers have addressed breakthrough in SPE, using liquid chromatographic theory.6,7,10–13 In some cases it is desirable to wash the sorbent bed after application of the sample matrix. If the desired analytes are purposely not retained on the SPE sorbent, a wash solution similar to the sample matrix is usually applied to ensure complete removal of the analytes of interest from the sorbent. The washing step typically uses similar volumes as the conditioning step. If the desired procedure is to retain the analytes of interest on the SPE packing and a wash step is necessary to remove sample matrix components, the solution

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selected should be strong enough to remove the interfering compounds but have minimal impact on the retained analytes. Usually, a similar but weaker solution of eluting solvent is used. Eluting the analytes of interest from the packing is customarily done with as small a volume as necessary to completely remove the analytes while leaving behind any interfering compounds that were retained during the wash step. Several smaller eluting volumes usually remove the analytes better than one larger volume. Depending on disk or tube size, eluting volumes range from around 100 mL to about 5 mL.

F. Nonconventional Techniques Solid-phase extraction devices and applications are evolving rapidly, and novel techniques that stretch the classical deﬁnition of SPE are becoming routine. Pawliszyn introduced solid-phase micro extraction (SPME) in 1989,5,14 and a commercial apparatus is available from Supelco (Bellefonte, PA). The SPME apparatus is merely a modiﬁed syringe that houses a fused silica optical ﬁber coated with an immobilized polymer ﬁlm. The ﬁber can be exposed for extraction and then retracted for insertion or removal from the sample vial or instrument. Both manual and autosampler devices are available and each can be adjusted for proper ﬁber depth. Several coatings are available with varying thickness including polydimethylsiloxane, polyacrylate, polydimethylsiloxane/divinylbenzene, and carbowax/divinylbenzene. In contrast to SPE, which is an exhaustive extraction approach, SPME will extract only a fraction of an available analyte, hence it is not suitable for the isolation of impurities and degradants in most applications.15 Extensions of silica ﬁber coating and derivitization have been described and theorized.16,17 Surface-phase extraction techniques involve coating or derivatizing the surfaces of labware or specialty devices and dipping or passing the liquid sample over the device. Simpson has suggested that a particle-loaded membrane or disk could be modiﬁed to become a solid-phase wipe or mop and that SPE sorbents could be placed into a permeable or semipermeable membrane to create a tea bag – like device in which target analytes could diffuse into.16 Although not yet applied to pharmaceutical impurity isolations, this could be the basis of future applications.

III. LIQUID^LIQUID EXTRACTION (LLE) Liquid–liquid extraction is an important sample preparation technique from both historical and practical perspectives.18,19 LLE has been in use for many decades as an analytical sample pretreatment to remove unwanted matrix components20 or to selectively extract components of interest from a mixture, thereby purifying and concentrating them for further workup. It is based on the principles of differential solubility and partitioning equilibrium of analyte molecules between two immiscible phases, usually aqueous and organic. Depicted in Figure 2, LLE initially involves pH adjustment of the original (aqueous) sample with an appropriate buffer. This pH adjustment is

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intended to neutralize the analyte molecules, making them more amenable to extraction into an organic phase. The next step in the process is the addition of an immiscible organic extraction solvent, followed by agitation (vortexing) to facilitate equilibrium partitioning of analyte molecules between phases. The phases are physically separated and the aqueous component is subjected to an additional extraction or, alternatively, discarded. If several extracts have been produced, the organic phases are combined, evaporated to dryness and, possibly, resuspended with a clean solvent, depending on the next step in the process. Back extractions into a secondary aqueous phase, once common, have largely become unnecessary when the higher selectivity of HPLC/tandem mass spectrometric detection is available.21 When performed in the organic chemistry laboratory, LLE is a preparative technique, performed manually and employing separatory funnels. When performed in the pharmaceutical analysis laboratory, LLE can be either preparative-scale (hundreds of milliliters), using a separatory funnel, or analytical-scale (microliters to a few milliliters) and performed using test tubes, vials, and glass or polypropylene pipets. Liquid–liquid extraction is a very good sample cleanup technique for nonpolar or moderately polar analytes that can be deionized in aqueous solution by simple pH adjustment. From a practical perspective, the best recoveries are obtained when an excess of organic solvent (3- to 10-fold excess) or multiple-step extractions are used. Multiple-step extractions have recently fallen into disfavor for routine use because they are time-consuming and labor-intensive. Method development for LLE is usually straightforward, as the approach can have a high probability of some success on the ﬁrst attempt. The technique can, however, be labor-intensive, and it does not provide good recoveries for highly polar or zwitterionic species unless ion-pair reagents are added to the

FIGURE 2 A pictorial representation of liquid^liquid extraction, including pH adjustment, extraction, phase separation, solvent evaporation and resuspension steps.

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sample, effectively making the analyte neutral. This approach is elaborate and often not worth the trouble, especially because other effective approaches for dealing with ionic compounds, such as ion-exchange SPE, are available. Automated liquid handling systems using a 96-well format have gone a long way toward making LLE an effective tool for microanalysis in situations such as drug discovery, where high sample throughput is required.21

A. Theoretical Considerations in LLE For an electronically neutral analyte that is partitioning between water and organic liquid phases, the equilibrium process can be described as the distribution KD ¼

½A org ½A w

ð1Þ

where KD is the distribution constant for the analyte A, partitioning between organic and aqueous phases. A large distribution constant indicates a preference for the organic phase over the aqueous phase, and a small KD indicates a preference for aqueous over organic. With a large KD and a rapid rate of transfer between phases, quantitative transfer from aqueous to organic phase can be made in a single extraction step. When a theoretical basis for LLE is developed, it is often assumed that KD is independent of concentration, yet strongly dependent on other variables such as pH, temperature, and ionic strength. At some point in the concentration range, however, the solubility limit of the analyte in one or both phases will be reached and relationship (1) will break down. One drawback of this effect is that unpredictable recoveries can occur when an extraction procedure is being scaled up. Exhaustive extraction involves the quantitative removal of a solute, while selective extraction involves the separation of two or more solutes from each other. In developing and/or applying LLE it is sometimes expedient to choose solvents and conditions to gain high recovery (exhaustive), then extend and improve the separation so that it becomes more selective. When Vorg milliliters of organic solvent is used to extract XA moles of the analyte of interest dissolved in V milliliters of aqueous sample, the distribution ratio, D, is deﬁned as D¼

CAorg ðXA YA Þ=Vorg ¼ CA YA =A

ð2Þ

where YA moles of A remain in the aqueous sample after a single extraction. The fraction of analyte remaining unextracted is given as w ¼

YA 1 V ¼ ¼ ð1 þ DðVorg =VÞÞ ðV þ DVorg Þ XA

ð3Þ

and is assumed to be independent of the initial concentration of analyte molecule.22 It can be seen from equation (3) that some of the ways to obtain

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a high recovery of the analyte (or a low fraction remaining) are to obtain a high D (generally through the selection of solvent, manipulation of pH or ionic strength), to make the ratio of Vorg/V large, or a combination of the two. In the development of an LLE, a common tactic is to optimize recovery by using a large volume of an extracting solvent with a smaller volume of sample to be extracted. This approach allows a nearly quantitative or exhaustive extraction with only a moderate distribution constant. If successive extractions are performed with fresh aliquots of organic solvent, after n extractions, the fraction remaining unextracted, n, can be represented as n 1 þ DV org w ¼ ð4Þ V There is no practical gain for n > 4 or 5 because n approaches zero asymptotically.22

B. Countercurrent LLE When the distribution ratio is not highly favorable, it is still possible to obtain a quantitative and selective separation through the use of a countercurrent liquid extraction approach. Although such approaches are no longer practical, having largely been supplanted by instrumental techniques such as preparative HPLC and continuous solvent extraction, countercurrent separations are conceptually useful. These approaches can be applied to preliminary separation of complex mixtures or in the isolation of compounds that do not perform well in LC because of undesirable interaction with the stationary phase (irreversible adsorption, denaturation, etc.). For these reasons, most applications of countercurrent separations involve the isolation of natural or biochemical products from plant or animal extracts. As will be described below, countercurrent extractions form the theoretical basis for LLE cartridges. Countercurrent separations are performed by the exposure of separated aqueous-phase samples with fresh, separate portions of organic phase in an experimental layout known as the countercurrent distribution23 or the Craig apparatus,24 shown in Figure 3. This ﬁgure depicts a countercurrent extraction of ﬁve sample containers, each containing 20 units of analyte dissolved in equal volumes of solvent. These samples are extracted sequentially, using ﬁve equal-volume aliquots of clean organic solvent. For the sake of simplicity, the distribution ratio is 1.0. The distribution ratio can be higher or lower than this, with proportionally better or poorer recovery of the analyte. At equilibrium, when a volume of aqueous phase is extracted with an equal volume of organic phase, 10 units of analyte will partition into the organic phase and 10 units will remain in the aqueous phase. If all aliquots (5V) of aqueous phase were combined and extracted with a single aliquot of organic phase (5V), and assuming no loss of material and completely efﬁcient phase separations, the recovery of analyte into the organic phase would be 50%.

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FIGURE 3 A pictorial representation of a 10 -step countercurrent extraction process for five aqueous samples and five organic aliquots.

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The right-hand vessel containing aqueous sample (bottom row) is extracted ﬁrst, using the left-hand vessel containing fresh organic solvent (top row). As the extraction proceeds, the organic phases are used to extract each of the aqueous samples in sequence, so that each organic sample is used for ﬁve extractions, and each aqueous sample is extracted ﬁve times. In order to accomplish this process, 10 extraction steps (involving 25 total extractions) are needed. Again, it is assumed that each extraction step is completely efﬁcient, with no loss of material. At the end of the extraction process, 75.4% of the analyte is recovered into the organic phase, thereby showing a signiﬁcant improvement in overall recovery for the countercurrent process relative to the single-step process (50% recovery). If this process were extended to the separation of multiple components using many extraction vessels, it is apparent that this approach could be used to separate multiple component mixtures, as long as the individual components had differing distribution ratios. The larger the difference in distribution ratio (D), the easier the separation becomes. The greater the number of vessels (analogous to the number of theoretical plates in the distillation or chromatography experiments) the easier the separation is to perform. The countercurrent model as shown here is not practical for real-world separations because the material losses sustained from ﬁve phase separation steps would make the real recovery closer to 50% than the predicted 75%. To make countercurrent extractions practical, they must be implemented in a ﬂow stream, cartridge, or other geometry, where material losses are not so devastating. One such approach is given below.

C. Liquid^Liquid Extraction Cartridges Historically, the difﬁculty associated with LLE has been the mechanical act of separating the phases.21 As the number of phase separations in an experiment increases, performing the separation becomes increasingly tedious and labor-intensive. A number of different approaches have been proposed for doing the separation of phases in LLE, including freezing the aqueous phase to facilitate organic phase removal, phase separation by ﬁltration through a silicon impregnated cellulose membrane, and direct separation of phases through automated solvent manipulation. An older approach that has only recently been commercialized and is gaining some popularity involves the use of LLE cartridges containing diatomaceous earth.25 In this approach, an aqueous sample is added to a small gravity-fed column packed with diatomaceous earth. The water, along with the analytes of interest and sample matrix components are retained by the diatomaceous sorbent, which can be unbuffered (pH 10) or buffered (pH 4.5 or 9) to offer pH control of the aqueous phase. Acidic compounds are extracted at pH 4.5, while basic compounds are extracted at pH 9. When organic solvent is added to the columns, the analytes partition between the aqueous and organic phases. The organic phase itself is poorly retained and can pass freely through the column and be collected. Additional fresh organic solvent can be added to the column so that, in essence, a countercurrent LLE system is employed. The cartridge LLE approach is useful because it facilitates the phase separations and

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because it can allow LLE to be adapted with automated SPE systems that are becoming well established.26 An added advantage of LLE cartridges is the ability to avoid emulsion formation. This problem is described below.

D. Emulsions An emulsion is a colloidal suspension of one liquid within another.27 Minute organic droplets are occasionally suspended in an aqueous solution after vigorous mixing of the two solvents, especially if any viscous or gummy material was present in either solution. Emulsions are problematic in LLEs because they can take a long time to separate out and allow the formation of a deﬁned phase boundary, thus facilitating a clean phase separation. Emulsions can be eliminated in several ways, including the addition of a small aliquot of concentrated salt solution (NaCl or KCl), a small amount of water-soluble detergent, or a small amount of a nonpolar organic solvent to the biphasic mixture. It is also sometimes possible to avoid emulsions entirely by ﬁltering the aqueous sample before extraction. This can remove the gummy material that leads to emulsion formation. Emulsions can also be minimized through clever solvent selection because they are less likely to occur with nonpolar solvents such as cyclohexane than with partially watermiscible solvents such as ethyl acetate. This, then, suggests a trade-off between emulsion potential and distribution coefﬁcient (and ultimately, analyte recovery).

E. Solvent Selection in LLE Liquid extraction methods are excellent for extracting drug impurities from drug matrices because they offer easy method development and effective sample cleanup. A wide variety of available organic solvents offer a range of solvating and partitioning properties, thereby allowing the selectivity of the extraction to be customized to the analyte. Some desired features of effective solvents for LLE include immiscibility with water, polarity to match the analytes of interest, high volatility for easy drydown, moderate viscosity for effective volumetric transfer, and lower density than the aqueous phase, so that the organic phase rises to the top and can be siphoned off. This last characteristic is not required in that denser-than-water solvents including methylene chloride and chloroform have been used effectively for numerous analytical-scale LLE. Table 3 gives a list of important physical properties for several of the more desirable organic solvents. Although liquid extraction methods have historically been difﬁcult to automate, recent advances in workstations designed to manipulate liquids in a 96-well format have made highly effective semi-automated approaches possible.21

F. Drying Agents Some water is soluble in all organic solvents to varying degrees. This solubility varies from trace amounts for hexane, heptane, and pentane to more than 3% (w/w) for a polar solvent such as ethyl acetate. Depending on

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TABLE 3 Important Physical Properties of Highly Desirable Liquid^Liquid Extraction Solvents Boiling pt (C)

Polarity

Density (g/ml)

Methyl ethyl ketone

80

4.7

0.80

Ethyl acetate

77

4.4

0.90

MTBE þ5%EtOH

58

3.2

0.75

Diethyl ether

35

2.8

0.71

MTBE

55

2.5

0.74

Butyl chloride

78

1.0

0.89

Pentane

36

0.0

0.63

the application, water can be an unwanted component in the sample extract. For example, if the next processing step in a chemical isolation is to remove the solvent by nitrogen drydown, then the presence of water will drastically increase the time required for this process. If the process must be conducted at low (< 40 C) temperatures because of analyte stability considerations, then the presence of a few tens of microliters of water will increase the solvent evaporation time from minutes to hours. If the extract is to be injected into a normal phase HPLC system, then the presence of water (a strong eluent in normal phase LC) will destroy the chromatographic separation. A drying agent is an anhydrous inorganic compound that acquires water of hydration when exposed to moist air or a wet solution. Drying agents can be used to remove water from the organic layer after a phase separation. The crystals of the salt are added directly to the organic layer in sufﬁcient quantity to make a thin layer at the bottom of the container. After a period of standing, the crystals are removed by ﬁltration or decantation. If the solution is very wet, the process can be repeated. Often the organic solvent goes from opaque or cloudy to clear as the drying agent acquires water. Common drying agents are given in Table 4,27 and are classiﬁed in terms of capacity, completeness, rate and use. Magnesium salts sometimes cause rearrangements with epoxides, as magnesium is a strong Lewis acid and should be avoided in these circumstances. Calcium chloride cannot be used with compounds containing nitrogen or oxygen because it forms complexes. Calcium chloride also absorbs methanol and ethanol and can be used effectively in this way. Sodium sulfate is a good all-around drying reagent, but may not completely dry the solvent.

IV. SUPERCRITICAL FLUID EXTRACTION (SFE) It can be argued that the ﬁrst supercritical ﬂuid extractions (SFE) were performed in 1879 when Hannay and Hogarth investigated the solvating capabilities of ethanol.28 However, it took roughly 100 years before supercritical ﬂuids made any signiﬁcant impact on industrial processes. The removal of caffeine from coffee beans was reported in the 1970s29 and led to

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TABLE 4 Solid Drying Agents that Can Be Removed by Filtration Acidity

Capacity

Completeness

Rate

Use

Magnesium sulfate

Neutral

High

Medium

Rapid

General

Sodium sulfate

Neutral

High

Low

Medium

General

Calcium chloride

Neutral

Low

High

Rapid

Hydrocarbons, halides

Calcium sulfate

Neutral

Low

High

Rapid

General

Potassium carbonate

Basic

Medium

Medium

Medium

Amines, esters, ketones, bases

Potassium hydroxide

Basic

–

–

Rapid

Amines only

Molecular sieves

Neutral

High

Extremely high

–

General

Anhydrous silica gel

Neutral

Medium

Extremely high

Rapid

General

a large increase in the number of published articles focusing on large-scale extractions using supercritical carbon dioxide. The development of commercial supercritical ﬂuid chromatographs and extraction systems escorted a slew of publications about analytical-scale work in the 1980s. In general, the use of supercritical ﬂuids for extraction was touted as having several advantages over traditional extraction techniques such as LLE. These included lower solvent cost, better selectivity through pressure and temperature modiﬁcation, and better solvating power because of ﬂuid properties. A complete understanding of SFE and its relation to other extraction methods cannot be made without some knowledge of the basic properties of supercritical ﬂuids and the basic principles of analytical SFE instrumentation. The purpose of this section is to give an introduction to the use of supercritical ﬂuids in analytical-scale extractions while focusing on the application of SFE to pharmaceutical analysis.

A. Fundamental Properties of Supercritical Fluids A typical phase diagram (pressure versus temperature) for a pure substance is shown in Figure 4. The regions where the substance exists as a single phase are bounded by curves indicating the coexistence of two phases, which determine the points of sublimation, melting, and vaporization. These curves intersect at the triple point (Tp) and represent the point where solid, liquid, and gaseous phases coexist in equilibrium. The liquid–gas line breaks at the critical point (Cp). The critical point is deﬁned as a point in the phase diagram designated by a critical temperature and a critical pressure where no liquefaction will occur with increasing pressure and no gas phase will be formed upon raising the temperature. These properties deﬁne the supercritical ﬂuid region and are a deﬁning characteristic of a substance. Table 5 displays the characteristic critical pressures, temperatures, and densities of some common solvents.30–35 The density of a supercritical ﬂuid depends on the pressure and temperature to which it is subjected. Critical densities of commonly

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FIGURE 4 Phase diagram of a pure substance. TABLE 5 Critical Properties of Representative Solvents Used in SFE (compiled from refs. 30^35) Solvent

Tc (C)

Pc (bar)

Density (g/mL)

CO2

31.1

72

0.47

N2O

36.5

70.6

0.45

NO2

158

98.7

0.27

Ammonia

132.5

109.8

0.23

Water

374.2

214.8

0.32

SF6

45.5

38

—

Helium

268

2.2

0.07

Hydrogen

240

12.6

0.03

Xenon

17

56.9

1.1

HCl

51

83.3

0.45

Methane

82

46

0.17

Ethane

32.3

47.6

0.2

Propane

96.7

42.4

0.22

Methanol

239

79

0.27

Ethanol

243

72

0.28

IPA

235.3

47.6

0.273

Chlorotrifluoromethane

28.8

214.8

0.58

Tetrahydrofuran

267

51

0.32

Acetone

235

47

0.28

Acetonitrile

275

47

0.25

used solvents used for SFE are typically between 0.2 and 0.8 g/mL. The high densities lead to strong ﬂuid–solute interactions similar to liquids. Table 6 compares the properties of supercritical CO2 and typical gas and liquid values.34 High diffusivity and low viscosity lend to the efﬁcient extraction capabilities of supercritical ﬂuids. The solvating power of

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TABLE 6 Comparison of Critical Properties of Supercritical CO2 and Typical Liquid/Gas Values (adapted from ref. 34) Density (g/mL)

Viscosity (g/cm-S)

Diffusion coefficient (cm2/S)

Gas

0.001

0.0002

0.1–0.4

Liquid

0.6–1.6

(0.2–3) 102

(0.2–2) 105

CO2 at Tc/Pc

0.47

0.0003

0.0007

supercritical ﬂuids at high density approaches that of a liquid. The maximum solubility in most liquids surpasses that of supercritical ﬂuids. Even though supercritical ﬂuids do not offer much advantage over liquid solvents as far as solvating power is concerned, they do have other beneﬁts. Mass transfer determines the rate at which extraction can be accomplished. Because solute diffusivities in supercritical ﬂuids are typically higher than those of liquid solvents by an order of magnitude, their mass transfer properties are more favorable. Other important physical chemical properties are polarity and dielectric constant. Water has a high dielectric constant (78.5 at STP), which would effectively mask ionic charges and lead to high solubility of ionic compounds. The dielectric constant of CO2 at 200 bar and 40 C is approximately 1.5, and CO2 is considered a very non polar solvent. As would be expected, polarity inﬂuences solubility for supercritical ﬂuids. Carbon dioxide has a dipole moment of 0.0 Debye, while the value for NH3 is approximately 1.5. Therefore, CO2 by itself is poorly suited for dissolving polar compounds.

B. Supercritical Fluid Extraction Instrumentation Supercritical ﬂuid extraction can be performed effectively with very simple systems. Figure 5 displays the basic components of an effective analytical SFE device. There are relatively few commercial suppliers of dedicated supercritical ﬂuid extraction instrumentation. Table 7 shows the companies that promote SFE instrumentation as of the writing of this chapter. Some of the more traditional instrument manufacturers such as Hewlett–Packard (7680T SFE), Dionex (SFE 723), and Supelco (SFE-400) have discontinued their SFE lines. Dionex has invested quite heavily into high-temperature/high-pressure solvent extraction devices, and this will be described in the next section. For most purposes, inexpensive and efﬁcient extraction units can be assembled using the basic components shown in Figure 5.

C. Static/Dynamic SFE Supercritical ﬂuid extractions can be performed statically or dynamically. During static extractions the extraction cell is pressurized and heated to the desired temperature. The supercritical ﬂuid remains in the extraction cell and

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FIGURE 5 Basic components of an analytical supercritical fluid extraction system (adapted from ref. 34).

TABLE 7 Commercially Available Analytical Scale SFE Instruments in the US

Applied Separations 930 Hamilton St. Allentown, PA 18011

Isco 4700 Superior, Lincoln, NE 68505

Durability, Inc. 1872 Pratt Dr., Suite 1630, Blacksburg, VA 24060 - 6344 (Isco-Suprex)

Spe-ed SFE 2

SFX 220

SFE-101

Spe-ed SFE 4

SFX 3560

Spe-ed SFE NP

FastFat HT

Thar Designs, Inc. 730 William Pitt Way, Pittsburgh, PA 15238 Prep and pilot scale systems

does not ﬂow through the restrictor. Extracted components are typically collected by a short dynamic extraction. A vent or shutoff valve must be placed between the extraction cell and the restrictor for static extractions. Dynamic extractions are performed by allowing the ﬂuid to continually ﬂow through the cell and are collected in some sort of collection device.30–35

D. Pumps Used for SFE Pumping liquids (e.g., in HPLC) generally requires less constraint than pumping ﬂuids. Pumps for SFE (or chromatography) must be able to withstand greater pressures (approximately 600 atm) and be able to maintain a constant ﬂow and pressure. It is highly desirable for a pump to be able to recover to full pressure quickly after pressurizing a cell. All of the commercially available instruments identiﬁed in Table 7 use syringe pumps. Syringe pumps have the advantage of supplying and maintaining a wide ﬂow and pressure range and can generally withstand pressures in excess of 600 atm, depending on design. They have the disadvantage of a ﬁnite volume and must be reﬁlled by the user. Reciprocating pumps have been used successfully, and

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HPLC pumps have been modiﬁed to behave as ﬂuid pumps. Probably the simplest and most affordable supply of high-pressure CO2 is that described by Westwood where a 10-mL cell was ﬁlled with 9 g of crushed dry ice and heated to 100 C. It provided 170 atm of CO2 for over 30 min.34

E. Extraction Cells The extraction cell is the heart of the SFE process. The characteristics of a good extraction cell include being capable of withstanding the necessary pressures and temperatures of the extraction (i.e., not leak), be capable of withstanding effects from samples, modiﬁers, or co-solvents; be easy to assemble/disassemble, ﬁll, and clean; and not contribute to carry-over issues. In early work, researchers commonly used modiﬁed HPLC ﬁttings or columns. The advent of commercial SFE equipment resulted in the development of analytical-scale high-pressure cells with ﬁnger-tight seals that eliminated the need for wrenches. The companies listed in Table 7 supply such cells. Keystone Scientiﬁc (Bellefonte, PA) and Thar Designs (Pittsburgh, PA) also supply analytical-scale SFE vessels. The general shape and size of cells used for SFE are cylindrical and contain between 1 and 20 g of sample within an approximate volume of 1–20 mL. Stainless steel is the most common material used for the housing and frits of SFE cells, while TeﬂonÕ / graphite are common seal and O-ring materials. Isco, Inc. (Lincoln, NE) has a patented cell design constructed of a high-temperature crystalline polymer.30–35

F. Modifiers Carbon dioxide is the most commonly used solvent for SFEs because it is nontoxic, available in high purity grades, and has achievable critical points. It is capable of dissolving and extracting nonpolar to moderately polar compounds but is not particularly useful for extracting polar species. Other solvents such as ammonia have better physical characteristics for extracting polar compounds; however, their toxicity, reactivity, or hazardous nature prohibit them from achieving more widespread use. The easiest approach to solving this problem has been to modify the solvating parameters of the extracting solvent by adding a co-solvent. There is a great deal of literature describing the effects of solvent-modiﬁed CO2 extractions versus pure CO2, and the reader is encouraged to research the literature for speciﬁc areas of interest. Unfortunately, there is relatively little published information between 1980 and 2000 that reports on the proper selection of a modifying solvent. A common-sense approach would be to begin with a solvent that is compatible with the target analytes and the supercritical solvent. The critical temperature of CO2 with a modiﬁer will be higher than that of pure CO2, and considerations as to the general parameters of the extraction may have to be adjusted (e.g., temperature). The actual mixing of a modiﬁer with a ﬂuid can pose a problem but is usually handled in one of the four ways. The simplest way is to add solvent

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187

directly to the extraction cell. The results of this can be signiﬁcant as the co-solvent can disrupt the sample matrix, effectively aiding in the release of target analytes as well as increasing their solubility in the supercritical ﬂuid. The term used for such an extraction is more appropriately named solventassisted SPE and closely resembles the principles of accelerated solvent extraction, described in the next section. When using a syringe pump, another and still less-expensive way to add a modiﬁer to the ﬂuid is to add a known amount of co-solvent directly to the pump chamber. The advantage of this technique is that it is simple and a constant supply of modiﬁed ﬂuid can be supplied to the extraction cell. A disadvantage is that the volumes and concentrations of the solvents in the pump chamber vary, which make it difﬁcult to predict the actual composition of the ﬂuid. Another potential problem is contamination of the pump chamber and seals with the co-solvent. A third, and more expensive method for modiﬁer addition, is to purchase the mixed solvent directly from the gas supplier. Custom mixes (e.g., CO2/ methanol or CO2/formic acid) are common but costly. The advantage is a more reliable and predictable solvent composition. The fourth method is to have a second pump, dedicated to supplying a constant amount of solvent, connected to the system. This is likely the most expensive design, but also very common, as most commercially available SFE instruments are designed in this fashion.30–35

G. Restrictors The restrictor controls the ﬂow rate of the SFE system. It is positioned after the extraction cell and ends in a collection device (off-line SFE) or in the injection port of another analytical device (on-line SFE). A shutoff valve is typically placed between the restrictor and extraction cell to enable static extractions to occur. A review of the literature indicates that the restrictor is one of the more problematic aspects of SFE. Restrictors are prone to plugging by ice formation, caused by expansion cooling of the supercritical ﬂuid at the outlet of the restrictor, or by extracted material from the sample matrix. The technology of restrictors as ﬂow-control devices in SFE has made signiﬁcant advances since initial descriptions30 and has redeﬁned restrictors as either ﬁxed ﬂow or variable ﬂow. Short pieces of small-diameter stainless steel tubing or fused silica (15–50 mm id) are examples of ﬁxed-ﬂow restrictors. Fixed-ﬂow restrictors have also been constructed from tapered fused silica or crimped stainless steel tubing.36,37 The beneﬁts of most ﬁxed-ﬂow restrictors are low cost and simple construction. Stainless steel has the beneﬁt of ﬂexible strength. Small diameter fused silica tubing can become very brittle and fragile when used with modiﬁers. Flow control is determined by the length and internal diameter of the tubing. Plugging that is due to ice formation can usually be controlled by keeping the temperature of the tip of the restrictor above 0 C. This can be accomplished simply by placing the collection device in a temperature-controlled water bath. Variable-ﬂow restrictors have the advantage of manual or computercontrolled heating and adjustment of internal diameter of the tubing. Applied

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D.T. ROSSI AND K.G. MILLER

Separations (Allentown, PA) and Isco (Lincoln, NE) have their own versions of variable restrictors, and these are included with their commercial SFE instruments. The cost of variable restrictors can range from hundreds to thousands of dollars.

H. Collection Devices Restrictors have been described as one of the more problematic aspects of SFE. Collection for on-line and off-line SFE has common issues, but on-line SFE is intimately dependent on the connected analytical device. Therefore, only off-line collection techniques with the end result of a chromatographycompatible sample will be summarized here. To have a successful and quantitative extraction, the ﬂuid of choice must solvate the analytes of interest, transport them from the sample matrix, through the extraction cell, tubing, and restrictor, and quantitatively deposit them in some sort of collection device while depressurizing and escaping as a gas. The most common method for analyte collection after SFE is bubbling expanded ﬂuid through a small volume of liquid solvent. This can be viewed as another extraction step in the sample preparation scheme. The proﬁciency of this method depends on many factors, including the solubility of the analytes in the solvent, the mass-transfer rate of the analytes to the solvent, the ﬂow rate (aerosol formation), and possibly the solvent volume (saturation level) if using a small volume of solvent. Because the expanding ﬂuid can rapidly cool the collection solvent, the collection vial is usually placed in a water bath. Numerous articles can be found on the use of sorbent materials such as silica, stainless steel beads, and common solid-phase extraction materials for collection of supercritical ﬂuid–extracted analytes.38–42 In this scenario the ﬂuid is usually allowed to expand into a gas, which is forced through a packed bed of solid sorbent material. The analytes are bound or simply deposited and cryogenically trapped onto the sorbent and are later eluted with appropriate solvents. The advantage of this technique is that further cleanup can be performed by carefully selecting the eluting solvent.

I. Pharmaceutical Applications of Supercritical Fluid Extraction Supercritical ﬂuids are widely used in manufacturing operations, however analytical-scale SFE has had a relatively modest impact on the isolation of pharmaceutical impurities. Pharmaceutical samples usually consist of complex matrices with polar target analytes, which lead to difﬁcult method development and optimization steps for SFE. Nevertheless, novel laboratory research has successfully used supercritical ﬂuids for the isolation of active pharmaceutical ingredients (API) and pharmaceuticals products. Roston and coworkers found that formic acid–modiﬁed CO2 effectively extracted an active enantiomer of misoprosotol, which was covalently linked to the drug product polymer matrix.43 Karlsson, Torstensson, and Taylor

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189

summarized and demonstrated that SFE could be used with success for extraction of polar substances in tablets, creams, ointments, and aqueous infusions.44 Bonazzi et al.45 extracted four imidazole antimycotic drugs from creams and quantitatively trapped them on a C18 sorbent. The precision of the assay was equivalent to a standard solid-phase extraction procedure and suitable for a quality control assay of commercial cream formulations. Roston and Sun46 showed improved precision and accuracy results for the extraction of an HIV protease inhibitor and benzophenone from animal feed with the use of an internal standard. Simmons et al.47 extracted 13-cis retinoic acid and its photoisomers from cream, gel, and beadlet dosage forms using methanol-modiﬁed CO2. Eckard and Taylor48 used 1-heptane sulfonic acid as an ion-pairing agent to extract pseudoephedrine hydrochloride from an inert surface. Khundker, Dean, and Jones49 compared SPE to SFE for the isolation of ﬂuconazole from animal feed. The extraction of polar drugs from chicken livers and meat products was demonstrated by Parks et al.51 and Ezzell et al.50 The general conclusion of both studies was that the solubility of the target analytes in supercritical CO2 does not alone determine the extent of the extraction from a complex matrix. The addition of modiﬁers played several roles including matrix breakdown and transport. Luque de Castro and coworkers reported for the ﬁrst time the extraction of vitamin D2 and D3 from multivitamins.52 In this study, the addition of methanol added directly to the sample cell drastically reduced recovery efﬁciency; however, diethyl ether increased recovery by 10%.

V. ACCELERATED SOLVENT EXTRACTION (ASE) Accelerated solvent extraction (ASE), also referred to as pressurized liquid extraction,53–55 is a relative newcomer to the battery of extraction techniques. This technique has been applied successfully to problems in environmental analysis56–58 and has recently begun to ﬁnd a few applications in pharmaceutical analysis as well. Some of these applications have involved biological samples59,60 and solid-dosage forms such as transdermal patches.61 As shown schematically in Figure 6, a solvent is selected for ASE with the relative solvating properties in mind. A volume of this solvent is pumped into a sample cell or ‘‘bomb’’ (1–100 mL) containing the sample to be extracted. Usually the volume of solvent added is equal to approximately two-thirds of the volume of the bomb. For a 1-g sample, a bomb of 25–50 mL is a useful size. A photograph of a typical bomb is shown in Figure 7. The bomb is moved via carousel to an oven; pressurized by addition of nitrogen and heated for a predeﬁned time (typically 10–20 minutes). After the prescribed time has elapsed, additional clean solvent is pumped into the bomb and the solvent is purged into a collection vessel with nitrogen gas. The extract is lyophilized, evaporated to dryness with nitrogen, or used directly in the subsequent analytical steps.

190

D.T. ROSSI AND K.G. MILLER

FIGURE 6 Schematic representation of an accelerated solvent-extraction system, showing pneumatic and fluidic pathways.

FIGURE 7 Stainless steel sample cells (5 -mL), or ‘‘bombs,’’ showing screw cap ends and sintered stainless steel frits.

A. Sample Pretreatment An ideal sample consistency for ASE is similar to that desired for liquid extraction, that being a dry, ﬁnely divided powder. To achieve this, ASE samples are often ground prior to placing in the bomb. To avoid particle aggregation, a dispersing agent such as Ottawa sand or diatomaceous earth is

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191

occasionally added to the sample. If the sample contains an appreciable amount of water, it is useful to choose a co-solvent system that is water miscible, such as acetone or alcohol. Although a conventional drying agent such as sodium sulfate, molecular sieves or silica may be added to the sample at this point, this represents an additional step in the extraction process and could be unnecessary. Two reasons why trace water is undesirable in the extraction process are the relative difﬁculty in performing a dry-down of the extract and the greater potential for degradation of the analyte by hydrolysis. Alternatively, one of the attractive features of ASE is the potential to minimize sample pretreatment prior to extraction. It is therefore desirable to omit as many of these steps as possible.

B. Solvent Selection in ASE A typical solvent or co-solvent system is selected based on the ability to solvate the analyte(s) relative to the undesired matrix components and the ease with which the solvent can be eliminated after extraction. Co-solvent blends are useful because the polarity (or other properties) can be tailored to that of the analyte(s). Traditional aprotic organic solvents are useful because they can be removed quickly and at low temperatures. Because of the dramatic increase in extraction efﬁciencies, solvents that have only moderate extraction properties at room temperature and atmospheric pressure can perform quite well under ASE conditions. Because organic-aqueous co-solvents can be used, it is often possible to prepare a solvent that can chemically neutralize the analyte molecule, thereby further facilitating the extraction. Dilute organic acids or bases can be employed for this purpose. Strong mineral acids are generally undesirable because they attack and destroy the stainless steel bombs or other instrument system components.

C. The Effects of Temperature in ASE Aside from solvent selection, temperature is one of the most important variables in ASE, as it dictates solvent viscosity, wetting properties and ultimately the equilibrium of analyte partitioning between matrix and solvent. Extraction efﬁciency and overall recovery will increase nonlinearly to a point and then level off. Higher temperatures are generally more efﬁcient unless thermal degradation of the analytes begins to occur. The temperature range of commercially available ovens can be precisely controlled from nearambient to 300 C or higher, but the higher temperatures may not be appropriate if drug degradation is observed. When developing an extraction method for a drug impurity and optimized recovery is desired, a good approach is to construct a graph of recovery versus temperature over 20 C intervals. A rule of thumb is to operate approximately 20 C below the temperature where degradation is ﬁrst observed and recovery begins to decline. An idealized recovery experiment that is hindered by thermal degradation is shown in Figure 8. Many environmental applications operate in the 75–125 C range, while pharmaceutical applications operate at lower temperatures because greater potential for degradation exists.62

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FIGURE 8 Graph showing relative recovery versus temperature for a thermally labile compound in accelerated solvent extraction.

D. The Effects of Pressure Increased pressure will allow the solvent to remain as a liquid (although it may be above the atmospheric pressure boiling point) and facilitate its transit through the extraction system. Changes in pressure will have little effect on analyte recovery when the applied pressure is well above the minimum required to maintain the solvent in liquid state. Typical ASE extractions work well between 1000 and 2000 psi.

E. Cycle Time All other variables being equal, a partitioned equilibrium for the analyte between the sample matrix and the extraction solvent is reached more quickly at higher temperature and pressure because the analyte solubilization kinetics are improved. Therefore, cycle time can be much shorter for ASE extractions relative to room-temperature/pressure-solvent extractions. If certain sample variables such as pore size or structure make rapid equilibrium questionable, it is simple to design a recovery versus extraction time experiment (the results of which are shown in Figure 9) so that variability and lower recovery due to a pre-equilibrium phase separation can be avoided. The desirable extraction duration is a trade-off between the recovery and the time required to achieve it and generally runs from 10 to 17 min. It is also possible to introduce fresh solvent into the cell over the course of the extraction or to add additional extraction cycles to the overall extraction process. As fresh solvent is added to the cell, extract is transferred into the collection vessel. This process helps to disrupt and shift the equilibrium toward quantitative completion of the extraction.53–55

F. Carry-over A big advantage of ASE is the ability to extract with reduced volumes of solvent relative to more conventional liquid extraction approaches such

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FIGURE 9 Graph showing percent recovery as a function of extraction time in accelerated solvent extraction.

as Soxhlet extraction.6 A 1-g sample can be extracted in less than 15 minutes using less than 10 mL of solvent using ASE, while this could require an overnight extraction using continuous Soxhlet. This low volume capability does, however, raise some concern about the carry-over performance of the instrumentation. Recently it has been shown with the Dionex ASE-200, that carry-over is minimal and manageable (< 0.13%) for non-ionic analytes.63 Unfortunately, cationic analytes occasionally adhere to metal surfaces (especially the sintered stainless steel frits within the extraction cells), and silation or special washing solvents, such as dilute organic acid, could be required to prepare the surfaces before or after an extraction.63 Compared to older, more-established sample extraction techniques, ASE is an infant. Although commercial instrumentation has been developed to the point where it can be effectively utilized for many difﬁcult tasks in isolation of drug-related substances, only a very few pharmaceutical applications have appeared to date.61 Reasons for this could be that the approach is a more efﬁcient, yet relatively risky alternative to SPE or LLE. Solvent heating is often involved in ASE and sometimes causes degradation of the compound to be isolated if the thermal degradation properties are not deﬁned. These properties are seldom deﬁned in the early stages of impurity isolation. ASE does, however, offer distinct advantages over roomtemperature, atmospheric-pressure techniques. The solvating power of heated, pressurized solvent far surpasses that of room-temperature, atmospheric-pressure solvent and the potential for automatically performing difﬁcult extractions with relative ease is high. As ASE becomes more common in pharmaceutical analysis laboratories and researchers better understand how to contend with the variable of thermal degradation, it is likely that ASE will become more commonly used and more widely applied than it is today for the isolation of impurities.

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VI. CENTRIFUGATION A. Centrifugation for Small Molecule Sample Preparation Centrifugation is a physical method for separating particles from each other in solution. Although it is widely used in biochemistry for the separation of various cellular fractions and components (also as whole cells, subcellular organelles, membranes, or large biomolecules), it also can be used effectively for separating small molecules contained in heterogeneous solution from suspended particulate matter. The centrifugation process, called sedimentation, is governed by Stoke’s Law, which describes the settling of a sphere in a gravitational ﬁeld.64 From the Stoke’s equation v¼

2r2 ðp m ÞG 9

ð5Þ

it can be seen that the sedimentation velocity ( ) is directly proportional to the radius (r) of the particle squared, the difference in density between the particle (p) and the medium (m), and the centrifugal force (G). Sedimentation velocity is inversely proportional to the viscosity of the medium (). The Stoke’s equation shows that the sedimentation rate is faster for large particles than for small particles, is faster in low viscosity media than in high viscosity ones, and is faster as the centrifugal force increases. The sedimentation rate is faster when the density difference between the particles and the medium is large. Centrifugal force (G) is calculated from the equation G ¼ !2 r

ð6Þ

where ! is the angular velocity (in radians per second, 2p in a complete revolution) and r is the effective radius of the centrifuge rotor. The calculated centrifugal force is often converted to relative centrifugal force (RCF) in units called x g. Relative centrifugal force can also be calculated directly from the equation RCF ¼ k rpm r

ð7Þ

where k is a constant (1.118 10-5), rpm is the rotor speed in revolutions per minute, and r is the effective radius of the rotor (in cm). This effective radius is not the actual radius of the rotor; it is the distance of the sample from the center of the rotor. For low-speed sedimentation applications, RCF of 2000–3000 g and rpm values of 2–3 k are typical. Regular glass centrifuge tubes can be used at these speeds and forces. For biological sedimentation applications, RCF of 10–20 k (ultracentrifugation) are typical. Special glass containers can be used at speeds up to 18 k rpms. At the highest speeds, polypropylene or nitrocellulose tubes are used so that tube shattering is

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avoided. Centrifuge rotors are subjected to great stress during centrifugation, and for this reason they are rated for maximum speeds at which they can be safely used.65 A technique called differential centrifugation is commonly used to fractionate particles into pellet and supernatant. The pellet is an aggregate of all sedimenting components, while the supernatant is a puriﬁed portion of the sample containing only the slowest sedimenting components. In this way, differential centrifugation is a puriﬁcation technique in which large particles are removed from the supernatant. Differential centrifugation65 is commonly used in work involving the isolation of impurities and/or degradants from tablets or other solid dosage forms because, although the active drug components and related substances are often extractable into a simple solvent such as water, alcohol, or acetonitrile, the excipients seldom are. Differential centrifugation can be used to remove these insoluble component particles from the sample preparation before a secondary extraction technique, such as solid-phase extraction, is applied. During the process of differential centrifugation, some of the analyte of interest is lost as it occludes with the pellet, making the yield of this technique less than perfect. Zone velocity centrifugation66 is a technique that can lead to higher yields because a separation gradient of the various analytes is formed. In contrast to differential centrifugation, this gradient technique displays a distinct band for each analyte. It is not often used for the separation of low molecular weight impurities from solid-dosage-form extracts because it is dependent on using a gradient material (solvent) that has a density lower than the analytes of interest, and this is not usually achievable or desirable in low molecular weight (< 2000 Da) impurities isolation.67 In addition to the relatively straightforward particle removal applications described above, ultracentrifugation is also effective at separating, purifying, and fractionating biological materials and for the study and physical characterization of macromolecules. These applications are beyond the scope of this chapter.

VII. FILTRATION Membrane ﬁltration is a widely used but narrowly understood technique for sample preparation in chemical analysis. This section has the goal of providing some basic information to aid in the use of ﬁltration tools with drug impurities. Many of the common sample preparation approaches described elsewhere in this chapter, such as liquid extraction, supercritical ﬂuid extraction, and accelerated solvent extraction are effective at removing the dissolved analytes of interest from the matrix while leaving behind many poorly soluble or insoluble matrix components. In contrast, ﬁltration is designed to remove these suspended particles from the extract prior to subsequent analytical steps. Unﬁltered samples can destroy the performance of a downstream analytical technique such as HPLC or optical spectroscopy.68,69 The choice of a ﬁlter is dependent on four parameters: the membrane material, the material used to house the membrane, the membrane pore size,

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and the device size or membrane diameter.70 The effects of these parameters are described below.

A. Membrane Selection The membrane material is selected on the basis of compatibility with the solvent being used and the chemical nature of the analyte. Membrane material is selected so that the analyte will not bind to it and so that analytical recovery will be as high as possible. The selection of material may also extend to the size and amount of particulate present in the sample. For example, certain materials such as sintered borosilicate glass are excellent for removing particles from ﬂuids. The potential for interactions of the membrane or ﬁlter housing with the sample components is also a consideration. For example, when protein analytes are being considered, a cellulose or cellulose acetate ﬁlter would be preferred over a synthetic polymer membrane such as nylon. For removal of large particles in aqueous samples from ground tablets, polysulfone membranes work well. When organic solvents are present and fewer particles need to be ﬁltered out, polypropylene or polyethyltetraﬂuoroethylene (PTFE) membranes can be effective. Table 8 lists the most common membrane ﬁlter materials and typical applications for them.

B. Housing Selection Housing material is generally either acrylic or polypropylene. Acrylic is suitable for aqueous samples. It offers fair to poor solvent resistance. Polypropylene offers high solvent and acid/base resistivity. Either of these housing materials offers extremely low extractables and low protein binding. Analyte binding to either membrane or housing material is difﬁcult to predict and must be empirically evaluated on an individual case basis. Some analytes such as polyamines will be very adsorptive, and their binding can be predicted by the extent of protein binding that is expected.

C. Pore Size Membrane pore size rating refers to the size of a speciﬁc particle to be retained by the ﬁlter with a speciﬁc degree of efﬁciency. Pore size will affect the ﬂow rate, back pressure, and life of the ﬁlter. Membrane pore size is outlined in Table 9 and selected on the basis of the applications requirements. The size and amount of particles to be ﬁltered from the sample can also affect the pore size selection. Most applications in the isolation of drug impurities and degradants would either require preﬁltration to remove large clumps of insoluble excipients or a ﬁltration/clariﬁcation step prior to HPLC, and would be chosen on these bases.71

D. Filtration Device Size The size (diameter) of the ﬁltration device is selected on the basis of the volume of liquid to be ﬁltered and, to some extent, the cleanliness of the

Membrane type

Application

Compatibility

Borosilicate glass

Prefiltration

For particle-laden samples. Prolongs membrane life and reduces filtration time.

Cellulose acetate

Clarification sterilization

Good for biological applications.

Nitrocellulose

DNA and RNA binding

Good for biological applications with higher protein-binding characteristics.

Regenerated cellulose

Particle filtration

Good for particle filtration.

Nylon

General filtration, sterilization

Displays some protein binding and good solvent inertness.

Polypropylene

HPLC sample preparation, general filtration

Good for both aqueous and organic samples. Has low protein-binding properties.

Polysulfone

General filtration, sterilization

Good flow-rate characteristics. Best for aqueous samples.

Polytetrafluoroethylene

Gas, air, and solvent filtration

Good for chromatographic applications and when protein binding is not an issue.

Organic solvent resistance

Protein binding

þþþþ

þþþ

[ [

þ

–

þ

þþ

[ [

þþþ

—

þþ

þ

[

þþþ

–

þ

–

þþþþ

þþ

Hydrophilicity

[

8 SAMPLE PREPARATION METHODS FOR THE ANALYSIS OF PHARMACEUTICAL MATERIALS

TABLE 8 Properties and Uses of Filtration Membrane Materials

þþþþ indicates strong solvent resistance or high protein binding þþ indicates moderate solvent resistance or protein binding – indicates low solvent resistance or protein binding — indicates very low solvent resistance or protein binding

197

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D.T. ROSSI AND K.G. MILLER

TABLE 9 Filtration Membrane Pore Sizes and Possible Applications

Application

Components for removal

Pore size (km)

Biological separations

Subcellular particles

0.1

Liquid sterilization and aseptic processing

Microorganisms

0.1–0.2

HPLC samples on < 3 mm columns

Particulates

0.2

General clarification

Particulates

0.45–1.0

HPLC samples on > 3 mm columns

Particulates

0.45

Solvent venting

Airborne particulates

0.2–0.45

Prefiltration

Macro particulates

0.8–5.0

TABLE 10 Relationship Between Filter Diameter and Volume of Fluid to Be Filtered Fluid volume (mL)

Membrane diameter (mm)

Fluid holdup volume (kL)

<2

4

<10

<10

13

<14

<10

13

<30

<100

25

<100

<150

32

<100

<200

47

<500

liquid. The relationship between volume and ﬁlter diameter is shown in Table 10. If a small volume sample were to be ﬁltered on an inappropriately large device, the results could be a low yield of the ﬁltrate. In contrast, a large volume of ﬁltrate could overwhelm and clog an unnecessarily small ﬁltration device. Hence, a critical selection of device size is desirable. Two additional properties of ﬁlters, which are less apparent but still important in ﬁlter performance, are the extractables and afﬁnity for the analyte. These ﬁlter properties are not as easy to predict for drug degradation products and impurities in that these compounds are often of unknown structure and poorly characterized. With regards to analyte afﬁnity, it is useful to conduct a recovery study to ascertain the extent to which an analyte adheres to the membrane. This is not always practical if the preﬁltrate is not suitable for direct chemical analysis or if the sample is precious. Extractables from ﬁlter membranes can be classiﬁed into two groups, inorganic ions and organic molecules. The organic components are residual components from either the polymerization process or a chemical modiﬁcation step. Filters are deliberately washed to remove these components, and the extent to which they remain in the membranes is

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generally small and manageable. Inorganic ions represent a more difﬁcult problem in that they are usually present in many of the wash solutions used to pretreat commercially available ﬁlters. If these ions, such as sulfate and nitrate are not problematic to the subsequent sample manipulations, they can be ignored. If it is desirable to exclude these ions, they can sometimes be eliminated from the ﬁlter by prewashing it with clean solvent or by discarding the ﬁrst few drops of the sample during the ﬁltration process.

VIII. SUMMARY This chapter provides practical guidance on the use of six widely used extraction and isolation techniques from the sample preparation perspective. The ﬁrst two sections, solid-phase extraction (SPE) and liquid–liquid extraction (LLE) deal with liquid sample types. The sections on supercritical ﬂuid extraction (SFE) and accelerated solvent extraction (ASE) focus mainly on solid samples, while the centrifugation and ﬁltration sections handle the suspensions. The theme of each section is that a successful sample preparation protocol should account for speciﬁcity and homogeneity as well as recovery and ﬁnal physical state of the target analyte. The ultimate aim is to produce a sample that is compatible with the intended analytical technique, so that the maximum information can be obtained.

ACKNOWLEDGMENTS Special thanks are given to Kyla Johnson and Will Clem of Pﬁzer Global Research and Development for help in preparing this manuscript.

REFERENCES 1. Supelco Technical Bulletin #T197910, Supelco Inc., Bellefonte, PA. 2. J. T. Baker Technical Bulletin, Solid Phase Extraction for Sample Preparation, Phillipsburg, NJ USA. 3. Varian Technical Bulletin, Solid Phase Extraction, Varian, Harbor City, PA. 4. The Handbook of Sorbent Extraction Technology, 2nd ed., Varian Inc., Palo Alto, CA 1993. 5. Thurman, E. M. and Mills, M. S. Solid Phase Extraction, Wiley, NY, 1998. 6. Poole, C. F. and Poole, S. K., Chromatography Today, Elsevier, Amsterdam 1991. 7. Miller, K. G. and Poole, C. F. J. High Resol. Chromatogr. 17:125, 1994. 8. Haginaka, J. Trends Anal. Chem. 10:17, 1991. 9. Unger, K. Chromatographia. 31:507, 1991. 10. Hage, D. S. Clin. Chem., 45:593, 1999. 11. Fernando, W. P., Larrivee, M. L. and Poole, C. F. Anal. Chem. 65:588, 1993. 12. Larrivee, M. L. and Poole, C. F. Anal. Chem. 66:139, 1994. 13. Seibert, D. S. and Poole, C. F. Chromatographia 41:51, 1995. 14. Seibert, D. S., Poole, C. F. and Abraham, M. H. Analyst 121:511, 1996. 15. Belardi, R. and Pawliszyn, J. J. Water Pollution Res. Canada, 24:179, 1989. 16. Simpson, N. J. K. Solid Phase Extraction Principles, Techniques, and Applications, Marcel-Dekker, NY, 2000. 17. Fritz, J. S. Analytical Solid Phase Extraction, Wiley-VCH, NY, 1999.

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18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

Rossi, D. T. and Wright, D. S. J. Pharm. Biomed. Analysis, 15:495, 1997. McDowall R. D. J. Chromatogr. 492:3, 1989. Rossi, D. T. Mass Spectrometry in Drug Discovery, chapter 6, Dekker, NY, 2002. Zhang, N., Hoffman, K. L., Li, W. and Rossi, D. T. J. Pharm. Biomed. Analysis, 22:131, 1999. Laitinen, H. A. and Harris, W. E. Chemical Analysis, chapter 23, McGraw-Hill, New York 1975. Craig, L. C. and Post, O. Anal. Chem. 21:500, 1949. Craig, L. C. Anal. Chem. 22:1346, 1950. http://www.varianinc.com/cgi-bin/nav?varinc/docs/spp/liqextract Rossi, D. T. and Zhang, N. J. Chromator. 885:97, 2000. Pavia, D. L., Lampman, G. M. and Kriz, G. S. Introduction to Organic Laboratory Techniques, p. 525f, Saunders, 1976. Hannay, J. B. and Hogarth, J. Proc. Roy. Soc. 29:324, 1879. Zosel, K. German Patent 1,493,190 1964. Taylor, L. Supercritical Fluid Extraction, Wiley, NY, 1996. Williams, J. R. and Clifford, A. A. Supercritical Fluid Methods and Protocols, Humana Press, Totowa, NJ, 2000. Ramsey, E. D. Analytical Supercritical Fluid Extraction Techniques, Kluwer Academic Publishers, Boston, MA, 1998. McHugh, M. and Krukonis, V. Supercritical Fluid Extraction, Butterworth-Heinemann, Boston, MA, 1994. Westwood, S. A. Supercritical Fluid Extraction and Its Use in Chromatographic Sample Preparation, Blackie Academic, London, UK, 1993. Charpentier, B. and Sevenants, M. Supercritical Fluid Extraction and Chromatography Techniques and Applications, ACS, Washington, DC, 1998. Dean, T. A. and Poole, C. F. J. Chromatogr. 600:175, 1992. Smith, R. D., Fjedsted, J. C. and Lee, M. L. J. Chromatog. 247:231, 1983. Yang, Y., Hawthorne, S. B. and Miller, D. J. J. Chromatog. A 699:265, 1995. Husers, N. and Kleibohmer, W. J. Chromatog. A 697:107, 1995. Furton, K. G. and Lin, Q. Chromatographia 34:185, 1992. Mulcahey, L. J., Hendrick, J. L. and Taylor, L. T. Anal. Chemistry 63:2225, 1991. Mulcahey, L. J. and Taylor, L. T. Anal. Chemistry, 64:2352, 1992. Roston, D. A., Sun, J. J., Collins, P. W., Perkins, W. E. and Tremont, S. J. J. Pharm. Bio. Anal. 13:1513, 1995. Karlsson, L., Torstensson, A. and Taylor, L. T. J. Pharm. Bio. Anal. 15:601, 1997. Bonazzi, D., Cavrini, V., Gatti, R., Bosellil, E. and Caboni, M. J. Pharm. Bio. Anal. 18:235, 1998. Roston, D. A. and Sun, J. J. Pharm. Bio. Anal. 15:461, 1997. Simmons, B. R., Chukwumerije, O. and Stewart, J. J. Pharm. Bio. Anal. 16:395, 1997. Eckard, P. R. and Taylor, L. T. J. Pharm. Bio. Anal. 15:63, 1997. Khundker, S., Dean, J. R. and Jones, P. J. Pharm. Bio. Anal. 13:1441, 1995. Ezzell, J. L., Richter, B. E. and Cross, R. F. J. Chromatog. Sci. 31:162, 1993. Parks, O. W., Lightfield, A. R. and Maxwell, R. J. J. Chromatog. Sci. 33:654, 1995. Luque de Castro, M. D., Gamiz, L. and Jimenez-Carmona, M. M. Chromatographia 51:428, 2000. Ding, W. H. and Fann, J. C. J. Chromatogr. 886:79, 2000. Suomi, J., Siren, H., Hartonen, K. and Riekkola, M. L. J. Chromatogr. 868:73, 2000. Benthin, B., Danz, H. and Hamburger, M. J. Chromatogr. 837:211, 1999. Dionex Technical Note 339, Determination of Organotin Compounds in Sediments Using Accelerated Solvent Extraction (ASE). Dionex Technical Note 341, Extraction of Base/Neutrals and Acids (BNAs) from LargeVolume Samples Using Accelerated Solvent Extraction (ASE). Dionex Technical Note 342, Determination of PCBs in Large-Volume Fish Tissue Samples Using Accelerated Solvent Extraction (ASE). Wang, G., Lee, A. S., Lewis, M., Kamath, B. and Archer, R. K. J. Agric. Food Chem. 47: 1062, 1999.

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60. Draisci, R., Marchiafava, C., Ferretti, E., Palleschi, L., Catellani, G. and Anastasio, A. J. Chromatogr. 814: 187, 1998. 61. Dionex Technical Note 327, Extraction of Nitroglycerin from Transdermal Patches by Accelerated Solvent Extraction 62. Dionex Technical Note 206, Investigations of Thermal Degradation During Accelerated Solvent Extraction. 63. Dionex Technical Note 207, Investigation of Carryover or Cross-contamination in the Dionex ASE-200. 64. Hsu, H. W. Separations by Centrifugal Phenomena, Wiley, NY, 1981. 65. Rickwood, D., Centrifugation: a Practical Approach, Oxford, Washington, DC, 1984. 66. Graham, J. M. Biological Centrifugation, Oxford, Washington, DC, 2001. 67. Schachman, H. K. Ultracentrifugation in Biochemistry, Academic Press, NY, 1959. 68. Cheremisinoff, N. P. Liquid Filtration, Butterworth-Heinemann, Boston, MA, 1998. 69. Matteson, M. J. and Orr, C. Filtration: Principles and Practices, Marcel Dekker, NY, 1987. 70. http://www.pall.com/catalogs/filterbook/ Filtration and Separation Products for the Laboratory. 71. Rushton, A., Ward, A. S. and Holdich, R. G. Solid-Liquid Filtration and Separation Technology, Weinheim, NY, 2000.

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9 ISOLATION METHODS I: THIN-LAYER CHROMATOGRAPHY PAMELA M. GORMAN AND HONG JIANG Pfizer Global Research & Development Division,Groton,CT 06340

I. INTRODUCTION TO THIN-LAYER CHROMATOGRAPHY (TLC) A. History B. Fundamentals of TLC C. Why UseTLC? II. TLC APPLICATIONS IN PHARMACEUTICAL INDUSTRY III. TLC METHOD DEVELOPMENTAND VALIDATION A. Drug Substance Method Development and Validation B. Drug Product Method Development and Validation IV. IMPURITY ISOLATION AND CHARACTERIZATION BY TLC A. TLC-Specified Impurities B. Known Impurities C. Unknown Impurities D. Summary of Impurity Isolation and Characterization byTLC REFERENCES

I. INTRODUCTION TO THIN-LAYER CHROMATOGRAPHY (TLC) Thin-layer chromatography (TLC) is one of the most popular and widely used separation techniques because of its ease of use, cost-effectiveness, high sensitivity, speed of separation, as well as its capacity to analyze multiple samples simultaneously. It has been applied in the disciplines of biochemistry,1,2 toxicology,3,4 pharmacology,5,6 environmental science,7 food science,8,9 and chemistry.10,11 TLC can be utilized for separation, isolation, identiﬁcation, and quantiﬁcation of components in a mixture. It can also be utilized on a preparative scale to isolate a particular component. A large variety of TLC apparatus is commercially available.

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P. M. GORMAN AND H. JIANG

A. History Pioneer work in thin-layer chromatography to isolate and analyze medicinal compounds was performed by Izmailov and Shraiber on unbound alumina as early as 1938.12 However, E. Stahl introduced the term ‘‘thinlayer chromatography’’ in 1956, which was considered the beginning of modern TLC.13 Since the 1960s, commercialization of precoated TLC plates and automation of sample application and detection have made it accessible to all laboratories. A number of valuable texts have been written about the history of TLC.14–20 The most recent one is reviewed by C. F. Poole.12

B. Fundamentals of TLC In TLC, the sample is applied as a small spot or streak to the marked origin of stationary phase supported on a glass, plastic, or metal plate. The sample solvent is allowed to evaporate from the plate that is then placed in a closed chamber containing a shallow pool of mobile phase at the bottom. The mobile phase moves through the stationary phase by capillary forces. The components of the mixture migrate at different rates during movement of the mobile phase through the stationary phase. When the mobile phase has moved an appropriate distance, the plate is removed from the chamber and the solvent front is marked. Mobile phase is evaporated from the plate by drying at room temperature, by forced air ﬂow, or in a heated oven. If the components of the mixture are not naturally colored or ﬂuorescent, a detection reagent is applied to visualize the bands. Sometimes more than one detection technique is used to ensure the detection of all components in the mixture. The Rf value is a convenient way to express the position of the substance on a developed chromatogram. It is calculated as follows: Rf ¼ distance of component from origin/distance of solvent front from origin. A variety of sorbents have been used as the stationary phase in TLC, including silica gel, cellulose, alumina, polyamides, ion exchangers, chemically modiﬁed silica gel, and mixed layers of two or more materials, coated on a suitable support. Currently in the pharmaceutical industry, commercially precoated high-performance TLC (HPTLC) plates with ﬁne particle layers are commonly used for fast, efﬁcient, and reproducible separations. The choices of mobile phase range from single component solvent systems to multiplecomponent solvent systems with the latter being most common. The majority of TLC applications are normal phase, which is also a complementary feature to HPLC that uses mostly reverse-phase columns. The migration of each component in a mixture during TLC is a result of two opposing forces: capillary action of the mobile phase and retardation action of the stationary phase. Both forces contribute to achieve differential migration of each component. Developed TLC plates can be detected by various means, based on the nature of the sample. They could be nondestructive (UV/densitometer), destructive (derivatizing agents), or the combination of both. The results can be documented by photography and saved electronically for archiving and future reference.

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C. Why Use TLC? Today, while HPLC is widely used for separation and quantiﬁcation, TLC remains a valuable and commonly used separation technique because of its features complementary to HPLC. The majority of TLC applications use normal-phase methods for separation, whereas reversed-phase methods dominate in HPLC. Some of the most important features of TLC compared to HPLC are brieﬂy discussed here. 1. Open format of stationary phase and evaluation of the whole sample—In TLC separation, a mixture is applied to the stationary phase followed by development. It is an open system from separation to detection. In contrast to TLC, HPLC is a closed-column system in which a mixture is introduced into the mobile phase and solutes are eluted and detected in a time-dependent manner. There are times that TLC reveals new and unexpected information about the sample, while that information is lost in HPLC by retention on the column, because of strongly sorbed impurities, early elution, or detection. In addition, TLC has little or less contamination with a disposable stationary phase while in HPLC the column is used repeatedly. 2. Simple sample preparation—Samples for TLC separation often involve fewer cleanup steps because every sample is separated on fresh stationary phase, without cross-contamination or carry-over. Even strongly absorbed impurities or solid particles in samples are not of much concern. This would be a disaster for HPLC separation, leading to column buildup, decay, and eventually destroying the performance. 3. High sample throughput—The simultaneous but independent application and separation of multiple samples in TLC results in higher sample throughput and less time consumption, as well as lower costs. HPLC cannot compete with TLC in terms of the number of samples processed in a given time period. 4. Flexible and versatile dissolving solvent and mobile phase—The choice of the sample solvent is not as critical in TLC as in HPLC because it is removed by evaporation before development of the plate. On the contrary, in HPLC the dissolving solvent chosen must be compatible in terms of composition and strength with the column and mobile phase. The same logic applies to the TLC mobile phase that is completely evaporated before detection. Therefore, the UV-absorbing properties, purity, or acid and base properties of the mobile phase are not as crucial as with HPLC. In addition, there is less solvent waste in TLC than in HPLC. 5. General and specific detection methods21,22—In TLC, many different detection methods, including inspection under short- and long-wave UV light and sequential application of a series of compatible reagents, can be employed on a developed TLC plate. Most well-established and routinely used HPLC detection methods still use UV, which cannot always capture all the eluates from column separation.

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6. One-dimensional multiple development and two-dimensional development—Multiple development through one or two dimensions can be applied to separate certain components in sequence, with detection at each step. This gives a theoretical increase in the capacity of the spots, so it is ideal for the separation of mixtures with a large number of components. In addition, it is a useful tool to confirm the purity of a given component. Though hyphenated HPLC could serve as a multiple separation technique, TLC takes the lead in this area by its faster separation and choice of different mobile phases and detection methods through each run. The comparison of TLC and HPLC has been described in a number of publications by a number of authors.18,23–29

II. TLC APPLICATIONS IN PHARMACEUTICAL INDUSTRY Sherma’s biennial review9 on planar chromatography shows that the greatest number of new TLC and TLC-densitometry methods were published for pharmaceutical analysis, including applications identiﬁcation, purity testing, assay, stability testing, and content uniformity testing of drug products, intermediates, and raw materials, as well as analysis of drugs and their metabolites in biological samples. TLC testing is relied upon because of its simplicity, ﬂexibility, speed of analysis, and unique detection methods on both a qualitative and quantitative basis. Often synthetic intermediates, process-related impurities, and degradation products do not contain chromophores that can be detected by the UV detector. One can utilize other detection methods in conjunction with HPLC or some other separation techniques to monitor the impurity. But none of them is as simple, fast, and straightforward as TLC. Therefore, these impurities are often speciﬁed by TLC, especially when there is a functional group that can be easily detected with certain derivatizing agents in TLC. Sometimes impurities elute at the solvent front in the HPLC method. It can be difﬁcult to monitor and quantitate them, and changes to the HPLC mobile phase or column may not be able to adequately resolve them. On the other hand, sometimes impurities elute very late in the HPLC run, which can leave them undetected. One advantage of TLC is that it is an open system. Samples are evaluated as a whole, whether they remain on the origin or ride with the solvent front. This is not always the case in HPLC. Thin-layer chromatography plays an essential role in the early stage of drug development when knowledge about the impurities and degradants in drug substance and drug product is limited. It is often used as an orthogonal technique to HPLC to ensure quality of the pharmaceuticals. TLC has been established in almost all pharmacopoeias worldwide. The ﬁrst stand-alone analytical method by TLC was described in the European Pharmacopoeia of 1974 (Ph. Eur. 1), in which TLC was speciﬁed for the identiﬁcation of 23 drugs. Figure 1 shows how TLC is involved at different stages of drug development. After a new drug entity has emerged, HPLC methods are

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FIGURE 1 Different stages whereTLC is involved in drug development.

needed for potency and purity analysis. At the same time, an orthogonal TLC method is also developed and validated for drug substance and drug product. If there is an unknown impurity observed by TLC during release testing, isolation and characterization would be required to identify the impurity. If HPLC is unable to quantify the impurity, TLC will remain as the primary testing method to quantitate this particular impurity until an alternative method with more accuracy is developed. In this chapter the applications of TLC in a pharmaceutical environment such as method development and validation for drug substance and drug product, and impurity isolation and characterization by TLC are discussed.

III. TLC METHOD DEVELOPMENT AND VALIDATION Method development of TLC has been thoroughly discussed in the recent books by Elke Hahn-Deinstrop, and Bernard Fried and Joseph Sherma.30,31 The most recent review article by Colin Poole and Neil Dias also provided certain guidance on method development in TLC.32 While the focus of this chapter is not on TLC method development, there are a few points that need to be stressed regarding method development from a regulatory perspective. First and foremost, use of qualiﬁed/calibrated equipment is part of the GMP compliance. In other words, all instruments must have updated Instrument Qualiﬁcation (IQ), Operational Qualiﬁcation (OQ), and Performance Qualiﬁcation (PQ) according to the company’s SOP (Standard Operation

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Procedure). The lab environment in which testing is to be conducted should also be GMP compliant. Last but not least, lab personnel must have gone through regular GMP training in order to document their ability to work effectively in a GMP facility. While densitometric detection is becoming more popular for quantitation in TLC, the work presented in this chapter is based on visual detection that can be routinely used as an acceptable alternative for semiquantitative purposes.

A. Drug Substance Method Development and Validation Once a new drug candidate has been identiﬁed, a TLC method is needed as an orthogonal tool with respect to HPLC methods. Similar to HPLC method development, certain criteria must be met: 1. Demonstration of adequate resolution and selectivity between drug substance and known or unknown impurities. 2. Determination of specified impurities at an appropriate level (for example: LOD (limit of detection) ¼ 0.05%, LOQ (limit of quantitation) ¼ 0.1%). 3. Determination of drug substance at an appropriate level (for example, LOD ¼ 0.05%, LOQ ¼ 0.1%) if it is to be used for quantitation of impurities. 4. Adjustment of Rf 0.50 for drug substance. 5. Demonstration of a stability-indicating assay through purposeful degradation studies. 6. Demonstration of ruggedness and reproducibility. The International Conference on Harmonisation (ICH) guideline outlines the following four types of analytical procedures to be validated: (1) identiﬁcation tests; (2) quantitative tests for impurities content; (3) limit tests for the control of impurities; (4) quantitative tests of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product. In the pharmaceutical industry, TLC testing falls between categories (2) and (3). The characteristics to be validated are speciﬁcity, detection limit, quantitation limit, and low-level linearity. TLC method development and validation can be performed rapidly in accordance with the current guidelines.33 However, the example of a validation procedure described here is based on semiquantitation by visual examination. All the examples in this chapter utilize HPTLC plates. 1. Specificity The purpose of this test is to demonstrate that immediate precursors and other known impurities are resolved from the drug substance and do not interfere with any unknown impurities existing in the drug substance bulk. All impurities should also be separated from any TLC-speciﬁed impurities. Figure 2 shows a drug substance band separated from all known impurities. The nominal concentration of the compound is determined in the method development phase according to the requirement of LOD and LOQ.

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FIGURE 2 Specificity.

The resulting Rf values are recorded for the drug substance and each impurity that is evaluated. It should be emphasized that all impurities show adequate resolution from the drug substance band and TLC-speciﬁed impurities. 2. LOD/LOQ/Low-Level Linearity for Drug Substance This is done to demonstrate the low level linearity of drug substance, which is used to quantitate impurities at certain levels. In most cases, quantitation of an impurity at 0.1% is required. It is imperative that the validation data demonstrate a sufﬁcient LOD and LOQ. An example in Figure 3 shows the LOD at 0.05% and LOQ at 0.1% for a drug substance. Here, a nominal concentration of drug substance at 25.0 mg/ml and a serial dilution of this solution to 0.5, 0.4, 0.3, 0.2, 0.1, and 0.05% relative to nominal concentration are made to demonstrate LOD and LOQ, as well as linearity. It has to be mentioned that sometimes the shape of drug substance band is somewhat compromised in order to fulﬁll the requirement of LOD and LOQ. 3. LOD/LOQ/Low-Level Linearity for Specified Impurities in Drug Substance The LOD and LOQ are determined for the speciﬁed impurity based on ICH Impurity Guidelines and/or regulatory requirements. It is demonstrated in the same manner as described in the section above. An example of a speciﬁed impurity (due to its carcinogenicity) is given in Figure 4.

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FIGURE 3 LOD/LOQ/low-level linearity for drug substance.

FIGURE 4 LOD/LOQ/low-level linearity for aTLC-specified impurity.

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4. Stability on Plate/Stability in Solution The intent of this test is to ﬁnd any degradation products that arise from sample solution and sample on the TLC plate over a period of time. In our lab, these studies are performed by weighing 5 samples and labeling them as 0, 1, 2, 3, and 4 hours. .

.

.

.

.

.

At time zero, solvent is added to the 4-h sample and is immediately applied to the plate for the time on plate studies. It is identified as the 4-h sample, and the solution is set aside to complete the stability-insolution study later. An hour later, the second sample is prepared and applied next to the 4-h sample for the stability-on-plate study, marked as the 3-h sample, and the solution is set aside. Two hours later, the third sample is prepared and applied next to the other two samples for the stability-on-plate study, marked as the 2-h sample, and the solution is set aside. Three hours later, the fourth sample is prepared and applied next to the other three samples for the stability-on-plate study, marked as the 1-h sample, and the solution is set aside. Four hours later, the final sample is prepared and applied next to the other four samples for the stability-on-plate study, marked as the 0-h sample, and the solution is set aside. This plate can now be developed as the stability-on-plate experiment. Immediately after the final solution is prepared, the five solutions are applied to a new plate that is developed immediately. This is for the stability-in-solution study.

Once developed, the plates are examined to determine if any degradation has occurred during the time course, either on plate or in solution. Figure 5 presents an example of stability-on-plate validation in which two new bands show up after 1 hour on the plate. If degradation is observed in the ﬁrst time point (1 hour), a subsequent experiment can be conducted as described above, but with time points as 60, 45, 30, 15, and 0 minutes. Sometimes an existing band in the drug substance will grow in intensity with time, or a new band appears with time. Both are indicative of some type of degradation occurring. If evidence of degradation in either experiment exists, it should be speciﬁed in the test procedure. 5. Post-Development DryingTime This test is conducted to determine the proper amount of time a TLC plate should be dried after development. It is often found that a range of drying time is acceptable. However, a plate that has not dried long enough sometimes shows an adverse effect when sprayed by a derivatizing agent. If a derivatizing agent is being employed as the detection method, four identical plates are prepared with the following concentrations of drug substance: 0.05, 0.1, 0.2, 0.3, 0.4, 0.5%, and nominal concentration. After development, the ﬁrst plate is dried for 60 min in a room-temperature, aircirculating oven (or equivalent), and a photograph is taken. The remaining

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FIGURE 5 Stability on plate.

three plates are dried for 45 minutes, 30 minutes, and 15 minutes respectively. Each plate is photographed to document potential changes as a result of inadequate drying time. The appropriate time range of drying time is determined based on the results. 6. As Is Plate/Prewashed Plate/Prewashed Plate 24 h ago The intent of this study is to demonstrate the effects of using a TLC plate straight from the box (as is), and then comparing this to one that has been prewashed in methanol, dried, and used the same day, as well as one that was left in a dry, covered TLC tank, and used 24 hours later. Since prewashing plates takes time, if there is no advantage to prewashing the plate, this step can be eliminated once the validation work is complete. By the same token, if several TLC plates were prewashed and dried, then set aside in a dry, sealed TLC tank, and used the next day, this can also be utilized as a time-saving step. An example is shown in Figure 6. An HPTLC plate is prewashed by adding 10 ml methanol to a 10 10 cm developing chamber, inserting a HPTLC plate, and developing to approximately one inch from the top of the plate. After development, allow the plate to dry in a 100 C aircirculating oven (or equivalent) for 20 minutes, then cool to ambient temperature prior to use. This plate is set-aside in a clean dry tank for 24 hours. The same procedure is repeated for a new plate, 24 hours later, such that one will be prewashed and dried for 24 hours, and one will be freshly prewashed and dried. A third plate is taken from the box and untreated (used as is). All three plates are used to develop the following

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FIGURE 6 As is plate (top)/prewashed plate (bottom left)/prewashed plate 24 hours ago (bottom right).

sample solution of drug substance: 0.05, 0.1, 0.2, 0.3, 0.4, 0.5%, and nominal concentration. Plates are developed and dried, and observations are recorded. The example in Figure 6 shows the as is plate gives a streak below the solvent front (might interfere with any potential impurities) while the other two prewashed plates provide a clean appearance. Therefore, the prewashed plate is preferred. Plates are usually prewashed and used the same day to avoid any undesirable effects. If densitometric detection is to be utilized, it is a necessity to prewash TLC plates with solvent (usually methanol), to eliminate background noise that is usually acquired from the packing material that TLC plates are stored in. 7. Saturated Chamber/Unsaturated Chamber The purpose of this study is to determine what effect, if any, is seen when a saturated environment is utilized in comparison to an unsaturated environment. As in the example (Figure 7), two plates are prepared with the following sample solutions of drug substance: 0.05, 0.1, 0.2, 0.3, 0.4, 0.5%, and nominal concentration. A developing chamber is prepared by placing blotter paper in one trough, covering this blotter paper with 5.0 ml of developing solvent, and adding another 5.0 ml of developing solvent to the other trough, then immediately placing the HPTLC plate to be developed. This would

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FIGURE 7 Saturated chamber (left)/unsaturated chamber (right).

FIGURE 8 Blotter paper (left)/no blotter paper (right).

constitute the unsaturated chamber. Another developing chamber is prepared in the identical manner except the lid is then replaced on the developing chamber, and the tank is allowed to equilibrate for 10–15 min. This constitutes the saturated chamber. Once the equilibration time has been reached, the HPTLC plate is developed. The example in Figure 7 shows that the saturated chamber gives a less tailed drug substance band than the unsaturated one. Generally speaking, the saturated environment allows a more consistent environment for TLC plate development. It also shows less development time compared with an unsaturated environment. Therefore, the saturated tank is usually employed in TLC. However, there are also situations where an unsaturated chamber is the preferred choice for plate development. 8. Blotter Paper/No Blotter Paper The intent of this experiment is similar to the previous experiment involving saturated and unsaturated tanks. TLC tanks are usually lined with appropriate-sized blotter paper to aid in saturation of the TLC tank. An example is shown in Figure 8. Two plates are prepared with the following sample solutions of drug substance: 0.05, 0.1, 0.2, 0.3, 0.4, 0.5%, and nominal concentration. A developing chamber is prepared by placing blotter paper in one trough, covering this blotter paper with 5.0 ml of

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developing solvent, and adding another 5.0 ml of developing solvent to the other trough, then the tank is allowed to equilibrate 10–15 min before the HPTLC plate is developed. This would constitute the chamber using blotter paper. Another developing chamber is prepared in the identical manner, except that the developing solvent is added to an empty chamber, containing no blotter paper. The lid is then replaced on the developing chamber and the tank is allowed to equilibrate for 10–15 minutes. This constitutes the chamber using no blotter paper. Once the equilibration time has passed, the HPTLC plate is developed. The example presented in Figure 8 shows that development without blotter paper not only pushes the drug substance band toward the solvent front, but also gives poorer linearity than the one with blotter paper. In general, blotter paper is usually used in the TLC tank unless there are adverse reasons that appear in the validation studies to utilize an unlined TLC tank. However, there are also situations where a chamber without blotter paper is the preferred choice for plate development. 9. Spraying/Dipping Traditionally, TLC plates have been sprayed with derivatizing agents, without much consistency because of operator dependence. In addition the technique is difﬁcult to master. Manufacturers of TLC equipment have now developed an immersion device that allows the TLC plate to be dipped into a pool of derivatizing agent, and a timer can be adjusted for the length of exposure, as well as the speed. This has now made spraying plates a less attractive option than dipping plates. However, the dipping chamber cannot be used for some derivatizing agents. Results are compared and a decision made whether dipping the plate is an acceptable alternative to spraying the plate for the given derivatizing agent. Since the immersion device is more reproducible and there are fewer hazards associated with dipping plates than spraying plates, dipping plates is the preferred choice. 10. Two-Dimensional Study This test is done to rule out any TLC artifacts that can occur from the sample applied to the origin not moving at the same rate as the drug substance upon the ﬁrst pass in the mobile phase. It is often utilized to rule out TLC procedure–related bands, as described later in this chapter (Impurity Isolation and Characterization by TLC). A plate is spotted, not streaked, with an appropriate amount of sample at a nominal concentration (typically 10– 15 mL). This is done in several applications of about 3–5 units each to prevent diffusion. It is spotted on the lower left corner, at the origin, and this lane is labeled as ﬁrst pass. The plate is developed in the normal fashion. Once dried, the plate is rotated 90 counterclockwise, another spot is applied in the lower left corner, labeled as second pass, and developed in the same manner. The result shows any TLC artifacts present in the second pass. Figure 9 shows such an example. A band observed that is initially at the origin in the ﬁrst development has the same Rf value as the drug substance in the second development. It is labeled as a TLC procedure–related band.

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FIGURE 9 Two-dimensional study.

11. Ruggedness/Robustness It is considered good practice to have 2 different individuals and, if possible, 2 different laboratories run the method to ensure consistency and reproducibility as part of the validation. 12. Validation Summary A summary page is generated upon completion of the method validation. This gives the user a quick reference of where to ﬁnd the data and the conclusions of each experiment. It also alerts the analyst to any potential issues, such as degradation. Table 1 is an example of validation summary for drug substance.

B. Drug Product Method Development and Validation After the drug substance TLC method has been developed and validated, method development for drug product is relatively straightforward unless the dose strength is very low (for example, 1.0 mg or less per tablet), and/or the formulation is somewhat unusual (lotion, lyophile, etc.), and/or a suitable extraction solvent cannot be readily found. The drug substance method is a logical starting point for the drug product method development. It is common that the same method can be used by modifying the extraction process, and most of the validation work from drug substance can also be utilized under appropriate justiﬁcation. As a result, the validation protocol is shortened to reﬂect the work already conducted for drug substance.

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TABLE 1 Summary of TLC Method Validation for Drug Substance Experiment Specificity

Notebook reference #12345 pp. 11–15

Results Precursors are resolved from drug substance Precursor 1

Rf ¼ 0.18

Precursor 2

Rf ¼ 0.33

Drug substance

Rf ¼ 0.52

Impurity 3

Rf ¼ 0.78

LOD/LOQ/low-level linearity of drug substance

#12345 pp. 16–19

LOD ¼ 0.05% LOQ ¼ 0.1%

LOD/LOQ/low-level linearity of specified Impurity

#12345 pp. 20–22

LOD ¼ 0.05% LOQ ¼ 0.1%

Stability on plate/stability in solution

#12345 pp. 23–27

Drug substance is stable in solution and on the silica plate up to 1 hour.

Post-development drying time

#12345 pp. 28–31

Recommended drying time is 0–60 min in a room-temperature oven.

As is/prewashed/prewashed 24 hours ago plates

#12345 pp. 31–35

Prewashed plates are recommended.

Saturated chamber/ unsaturated chamber

#12345 pp. 36–39

Plate developed in a saturated chamber is more consistent than the one in an unsaturated chamber.

Blotter paper/no blotter paper

#12345 pp. 40–42

Plate developed in tank lined with blotter paper is more consistent than the one without blotter paper.

Spraying/dipping plates

#12345 pp. 43–45

Plate dipped with Dragendorff’s spray yields similar result to the one sprayed with the same detection reagent.

Two-dimensional

#12345 pp. 46–48

The origin band is a TLC procedure–related band.

Any given drug product usually has a number of strengths associated with it. They can be evaluated by bracketing, i.e., studying the highest and the lowest strengths. A typical validation protocol for a drug product method is discussed in detail next. 1. Separation of Drug Substance from its Excipients The purpose of this test is to ensure that the excipients are resolved from the drug substance, and do not interfere with any other related known or unknown impurities. The ﬁrst thing is to establish a proper extraction solvent and a method to extract the active ingredient from the formulation. It is very likely that by now the solubility information of the drug product is available. The process may involve a series of steps to ensure full extraction from the

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FIGURE 10 Separation of drug substance from its excipients.

excipients. Here is an example of how to extract the actives from tablets: Wrap a tablet in a piece of weighing paper, crush with a hammer, and quantitatively transfer all contents to a suitable container. To this container, add an appropriate amount of extraction solvent (methanol) and stir for 10 minutes. Sonicate the solution for 10 minutes, transfer to a centrifuge tube, centrifuge at 3000 rpm for 10–20 minutes, and ﬁlter the ﬁnal solution as needed. To demonstrate that no excipients interfere with the drug substance, a TLC plate is prepared like the one on the left in Figure 10, where the drug substance is extracted from its formulation, compared with that of a placebo (a synthetic blend is prepared according to the formulation order if no placebo is available at the time of method development and validation). The in-going bulk lot of drug substance is weighed at the proper concentration, diluted in the extraction solvent, and applied to the same TLC plate for analyses. Should any excipients in the placebo interfere with the drug substance, another TLC plate will be prepared containing each individual excipient extracted in the same manner and the same concentration as in the placebo. As an example shown in Figure 10 on the right, excipient no. 6 is responsible for the dark band above the drug substance band. Once this has been identiﬁed, it will be referred to in the method as a known excipient. Excipients, placebo, tablet, and drug substance should all appear consistent with the same intensity since they are all prepared at the same concentration. 2. LOD/LOQ/Low-Level Linearity for Drug Product As indicated in the validation section for drug substance, the purpose of this test is to demonstrate that low-level linearity can be routinely achieved for drug product evaluation. If the extraction solvent for the drug product is the same as the dissolving solvent in the drug substance method, there is no need to repeat this validation test. However, if the extraction solvent has changed, this validation test must be repeated to demonstrate desired LOD and LOQ. In general, it is expected that an LOD ¼ 0.05% and an LOQ ¼ 0.1% would

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FIGURE 11 LOD/LOQ/low-level linearity for drug product.

be reproducibly demonstrated by the method, as the example shown in Figure 11. 3. LOD/LOQ/Low-Level Linearity for Specified Impurities in Drug Product (Spiking Experiment) When TLC is used to track a speciﬁed impurity in the drug substance, it is likely to be applied to the subsequent drug product method. A plate must be prepared to show that required levels of LOD and LOQ can be achieved for the speciﬁed impurity in the presence of excipients. This is known as a spiking experiment. The speciﬁed impurity is prepared and spiked into the vial of the drug product at different levels, then applied to the TLC plate to ensure it can be extracted to meet the requirement of LOD and LOQ. If no speciﬁed impurities are being tracked by TLC, this validation test can be excluded. 4. Stability on Plate/Stability in Solution If the same solvent is used for both the drug product method and drug substance method, there is no need to repeat this validation test unless there is a concern that any of the excipients might cause any instability of the drug substance. However, if the extraction solvent has changed from the drug substance dissolving solvent, this needs to be validated in the same manner as that of the drug substance discussed earlier. It is always a good

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practice that when all sample preparations are made, the solutions are immediately applied to the TLC plates. This test is conducted to alert the analyst for any potential degradation issues resulting from the extraction solution or the TLC plate. 5. Recovery Plate The ﬁnal validation test is done to demonstrate that no drug substance was lost from the extraction process, as this is a purity test. In a typical extraction process, as described above, the sample is crushed, extraction solvent is added, the sample is then stirred, sonicated, centrifuged, and ﬁltered. In some cases, the drug substance will adhere to the ﬁlter. In this event, a study of numerous types of ﬁlters ensues to ﬁnd one that yields 100% recovery. In the example shown in Figure 12, the drug substance is weighed, and solutions of 0.5, 0.3, and 0.1% of nominal concentration are made and applied to one half of the TLC plate as the control solutions. In addition, the same dilutions are made from one extracted tablet and applied to the same plate. Once the plate is developed, the two 0.1% standards are compared to each other to determine if they have the same visual response in intensity. The same comparison is made for the 0.3% and 0.5% standards. If all three standards appear to have the same intensity as their respective controls, then we state that the tablet has been extracted completely. If the standards are not the same in intensity and it is apparent that some drug substance is being retained by the ﬁlter, then another type of ﬁlter or possibly centrifugation alone would be examined until full extraction can be achieved.

FIGURE 12 Recovery on plate.

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TABLE 2 Summary of TLC Method Validation for Drug Product Experiment

Notebook reference

Results

Separation of drug substance from excipients

#12345 pp. 55–58

No excipients interfere with the drug substance

LOD/LOQ/low-level linearity of drug substance

#12345 pp. 59–62

LOD ¼ 0.05%

LOD/LOQ/low-level linearity of specified impurity (spiking experiment)

#12345 pp. 63–65

Stability on plate/stability in solution

#12345 pp. 66–72

Drug substance is stable in solution and on the plate for 4 hours.

Recovery plate

#12345 pp. 73–77

Drug substance is fully recovered from this extraction process

LOQ ¼ 0.1% LOD ¼ 0.05% LOQ ¼ 0.1%

6. Validation Summary A summary page is generated upon completion of the tablet method validation. This gives the user a quick reference of where to ﬁnd the data, and the conclusions of each experiment. It also alerts the analyst to any potential degradation. An example of a method validation summary for drug product can be found in Table 2.

IV. IMPURITY ISOLATION AND CHARACTERIZATION BY TLC A validated TLC method is used to evaluate drug substance or drug product for release or stability purposes. Usually there are three kinds of impurities observed from a TLC analysis: TLC-speciﬁed impurities, known impurities, and unknown impurities.

A. TLC-Specified Impurities As mentioned earlier, some of the synthetic starting materials, intermediates, precursors, and known potential degradants cannot be detected by a UV detector that is routinely used in HPLC analysis because of lack of a chromophore, or it may elute at the solvent front or elute very late in HPLC run. Alternatively, TLC is used to quantify these impurities. An example of these unique situations would be one in which guanidine hydrochloride (Figure 13), a potential degradation product, needs to be monitored in one of the drug candidates. By looking at the structure of guanidine, it is obvious that regular UV detection would not be suitable to evaluate this impurity. Since it is a potential degradant, TLC was selected because of its simplicity in

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sample preparation among other reasons. The detection method chosen for this compound was starch-potassium iodide because of its sensitivity to amines. In this case, levels of detection down to 0.1% are readily achieved (Figure 13).

B. Known Impurities The knowledge and availability of known impurities (process-related impurities and known degradation products) vary depending on the stage of development of the drug substance/drug product. As part of the method validation, the Rf values of synthetic starting material, intermediates, and precursors, as well as known degradants are also assessed. Impurities observed during the evaluation of a drug substance/drug product are ﬁrst checked against the validation data to see if any of them match the known impurities. If so, further TLC analysis with both sample and known impurities are run in parallel to determine if the impurity in the sample is indeed the known impurity. In the example shown in Figure 14, an impurity is observed during a stability study that is later conﬁrmed by TLC as a known impurity.

FIGURE 13 Quantitation of guanidine hydrochloride byTLC.

FIGURE 14 Confirmation of a known impurity by TLC (leftan impurity observed during a stability study; rightconfirmed byTLC as a known impurity).

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C. Unknown Impurities Often the impurities observed from TLC analysis are unknown. In this case, identiﬁcation of the impurities may become necessary, depending on the level of impurities (ICH Impurity Guideline). Questions one should ﬁrst ask are, Is the impurity real or an artifact? Is it salt-related? If the answers are No to these two questions, isolation and characterization are required to identify the impurity. 1. Is the Impurity Real or an Artifact? In analyzing samples in a cGMP environment, numerous things should be ruled out in impurity identiﬁcation, such as equipment malfunction, glassware contamination, etc. A thorough investigation should be conducted to ensure whether the impurity is real or an artifact. Tailing of the drug substance band in TLC often occurs with acidic and basic compounds. This can be minimized by suitable choice of pH or by the use of inorganic or organic salts during method development. However, during release testing a 2-D TLC experiment can conﬁrm if a band at the origin is drug substance residue or a real impurity. This test is done in the same manner as described in the validation of drug substance section. The result will yield a diagonal line for the ﬁrst sample applied. If the origin band from ﬁrst development rises to the same Rf as the drug substance as shown in Figure 9, this indicates the origin band is indeed drug substance, i.e., it is a TLC procedure–related band, or an artifact. If the origin band does not move at the same distance as the drug substance on the second development as the example shown in Figure 15, further investigation is required to identify this impurity. 2. Is the Impurity Salt-Related? Often drug substance exists as a salt to improve its solubility and bioavailability. The salt form changes sometimes during drug development. It is important to be aware of the impact of the type of salt on the TLC analysis result. An example in Figure 16 shows that Rf of the corresponding salt matches that of the impurity band in a drug substance lot. In some situations, the salt, when bound to the drug substance, can have a different Rf value than when the salt is applied to the plate itself. In the example shown in Figure 17, a spike experiment is conducted to show that the salt’s Rf value changes when in the presence of the drug substance. Therefore, the impurity band is identiﬁed as salt related. It is also important to mention that the salt does not always remain at the origin, as seen in the example shown in Figure 17. Salts can migrate anywhere on a TLC plate, depending on the salt itself and the mobile phase used as demonstrated in this example. 3. TLC Impurity Isolation and Characterization Once it has been determined that an impurity is real, i.e., it cannot be called a TLC artifact, the related salt, a known process-related impurity, or a degradation product, then it has to be isolated and correlated to the relevant

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FIGURE 15 2-D TLC to confirm that the origin band is an impurity.

FIGURE 16 Salt confirmation byTLC.

HPLC system. There are two approaches to the isolation and correlation of bands seen in TLC assays: (1) Isolate impurity band from TLC ! reassay on HPLC system; (2) isolate impurity from HPLC ! reassay on TLC method. The isolation and characterization by TLC is often required at the early stages of drug substance synthesis when a synthetic route is still below optimization.

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FIGURE 17 Rf of salt changes with drug substance.

In the ﬁrst situation, the band in question that is viewed in the TLC system would be isolated, extracted, reapplied for conﬁrmation, and analyzed by HPLC for correlation and conﬁrmation. The following example gives a detailed description of how this is done in our laboratory. To isolate the band in question, an 80-mm streak of the drug substance, typically at 25.0 mg/ml concentration, is applied to a 10 10 cm silica plate, usually at a volume of 200 ml, in an effort to overload the silica plate such that more of the impurity can be isolated. This process is repeated for 10 HPTLC plates or so. The plates are then developed according to the method and the impurity marked with a pencil under UV 254 nm if this is the method of detection. An example of an LP (less polar relative to drug substance) impurity and an MP impurity (more polar relative to drug substance) to be isolated is shown in Figure 18. Once the 10 plates have dried, the bands in question are scraped from the silica plate using a metal spatula and collected in separate containers. Approximately 5–10 ml of extraction solvent (usually the sample solvent in the relevant TLC method) is added to the container. The solution is stirred for about 10 min, sonicated about 10 min, and centrifuged at 2500 rpm for 10 minutes. The resulting clear solution is transferred to another suitable container and concentrated under a stream of nitrogen to approximately 0.5 ml of volume. This concentrated solution is then reapplied to a new TLC plate to conﬁrm the TLC isolation of the impurities. The remainder of the solution is then evaporated to dryness under nitrogen. HPLC sample solvent is used to reconstitute the isolated impurity that is analyzed by a respective HPLC method to determine if the impurity correlates to any impurity previously seen by HPLC analysis. In this case, both LP and MP bands correlate with the impurities in HPLC analysis. Therefore, these two impurities will be monitored by HPLC. In addition, the

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FIGURE18 Scaleup forTLC !HPLC correlation (top, scale-up isolation; bottom left, scraping of impurity bands; bottom right, confirmation of isolated impurities).

isolated impurity can be sent for MS and NMR analysis to determine the structure as needed. A blank TLC plate is prepared in the identical manner without drug substance. Silica from this plate is scraped off and extracted by the same procedure as the impurity bands. The blank sample is also analyzed by HPLC to rule out any anomalies from extraction process. This impurity isolation process (TLC ! HPLC) is commonly utilized in our laboratory. However, the process is tedious and lengthy for extraction from the silica plates, and timing must be coordinated such that the sample is immediately analyzed by the HPLC system to rule out any potential degradation. Sometimes, the band in question is very close to the drug substance or another band, making it difﬁcult to extract for TLC ! HPLC. An alternative is to correlate an impurity by collecting the impurity from HPLC and reapplying it to the TLC system (HPLC ! TLC). It is worth mentioning that whenever it is possible, scale-up is always helpful to collect impurities for identiﬁcation purposes. This also applies to HPLC ! TLC correlation studies. A preparative column or analytical column can be used to collect these samples with a fraction collector. Such an example is shown in Figure 19. Two impurities were observed during TLC testing. A correlation experiment is needed to conﬁrm if these two impurities

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FIGURE 19 HPLC ! TLC correlation (top, HPLC purity profile; bottom,TLC correlation of impurities separated from HPLC).

were identical to the ones observed from HPLC analysis. This time an HPLC ! TLC correlation is designed since the LP band is right above the drug substance band, which made it difﬁcult to scale up and scrape the impurity on TLC plates. The two impurity peaks were collected from HPLC runs, and subsequent TLC analysis conﬁrmed that they were the same two impurities.

D. Summary of Impurity Isolation and Characterization by TLC Figure 20 summarizes how impurity isolation and characterization is performed by TLC. If the correlation experiment shows an impurity match between TLC and HPLC, usually HPLC is used to monitor this impurity. On the other hand, if no match is found, TLC will continue to be the primary quantiﬁcation method for this particular impurity. In summary, TLC analysis plays a fundamental role in the drug development process. It is involved in release testing and stability study of

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FIGURE 20 Summary of TLC impurity isolation and characterization.

identity and purity of active pharmaceutical ingredient (API) in both drug substance and drug product. Especially at early stages of drug development, TLC’s complementary properties to HPLC make it an essential part of the quality control for pharmaceuticals goods.

REFERENCES 1. Abidi, S. L. Separation procedures for phosphatidylserines. J. Chromatogr. B. 717(1–2): 279–293, 1998. 2. Lawton, L. A. and Edwards, C. Purification of Microcystins. J. Chromatogr. 912(2): 191–209, 2001.

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3. Bhushan, R. and Martens, J. Separation of amino acids, their derivatives and enantiomers by impregnated TLC. Biomed. Chromatogr. 15(3):155–65, 2001. 4. Reddy, M. V. Methods for testing compounds for DNA adduct formation. Regulatory Toxicology & Pharmacology. 32(3):256–63, 2000. 5. Alemany, G. et al., Thin-layer chromatographic determination of catecholamines, 5-hydroxytryptamine, and their metabolites in biological samples—a review. J. AOAC Int. 82(1):17–24, 1999. 6. Roda, A., Piazza, F. and Baraldini, M. Separation techniques for bile salts analysis. J. Chromatogr. B. 717(1–2):263–78, 1998. 7. Porter, J. K. Analysis of endophyte toxins: fescue and other grasses toxic to livestock. J. Animal Sci. 73(3):871–80, 1995. 8. Oka, H., Ito, Y. and Matsumoto, H. Chromatographic analysis of tetracycline antibiotics in foods. J. Chromatogr. 882(1–2):109–33, 2000. 9. Sherma, J. Thin-layer chromatography in food and agricultural analysis. J. Chromatogr. 880(1–2):129–47, 2000. 10. Muthing, J. High resolution thin-layer chromatography of gangliosides. J. Chromatogr. A. 720(1–2):3–25, 1996. 11. Bereznitski, Y., et al. Thin-layer chromatography—a useful technique for the separation of enantiomers. J. AOAC Int. 84(4):1242–1251, 2001. 12. Poole, C. F. Planar chromatography at the turn of the century. J. Chromatogr. A. 856(1–2): 399–427, 1999. 13. Stahl, E. The historical development of the method. In Thin layer Chromatography (E. Stahl, Ed.) pp. 1–6, Springer-Verlag, Berlin, 1969. 14. Pelick, N., Bolliger, H. R. and Mangold, H. K. The History of Thin-layer Chromatography, In Advances in Chromatography (J.C. Giddings and R.A. Keller, Eds.), Marcel Dekker, NY, pp. 85–118, 1966. 15. Stahl, E. Thin-Layer Chromatography, Springer-Verlag, NY, 1969. 16. Heftmann, E. History of Chromatography. In Chromatography—A Laboratory Handbook of Chromatographic and Electrophoretic Methods (E. Heftmann, Ed.), pp. 1–13. Van Nostrand Reinhold, NY, 1975. 17. Kirchner, J. G. History of TLC. In Thin Layer Chromatography—Quantitative Environmental and Clinical Applications (Touchstone, J.C. and Rogers, D., Eds.), pp. 1–6. Wiley-Interscience, NY, 1980. 18. Jork, H. and Wimmer, H. Thin-layer Chromatography—History and Introduction. In TLC Report a Collection of Papers. GIT Verlag, Darmstadt 1986. 19. Wintermeyer, U. The Root of Chromatography: Historical Outline of the Beginning to Thin Layer Chromatography, GIT Verlag, Darmstadt, 1989. 20. Berezkin, V. The discovery of thin layer chromatography. J. Planar Chromatography.-Med. TLC. 8:401–405, 1995. 21. Jork, H., et al. Thin-Layer Chromatography—Reagents and Detection Methods. Vol. 1a: Physical and Chemical Detection Methods: Fundamentals, Reagents I, Weinheim: VCH, pp. 464, 1990. 22. Jork, H., et al. Thin-Layer Chromatography—Reagents and Detection Methods. Vol. 1b: Physical and Chemical Detection Methods: Activation Reactions, Reagent Sequences, Reagents II, Weinheim, VCH, pp. 496, 1994. 23. Borman, S.A. HPTLC: taking off. Anal. Chem. 54:790A-4A, 1982. 24. Coddens, M. E., et al. Quantitation in high performance TLC. LC Magazine, Liquid Chromatography and HPLC. 1:282–289, 1983. 25. Costanzo, S. J. High performance thin-layer chromatography. J. Chem. Educ. 61: 1015–1018, 1984. 26. Fenimore, D. C. and Davis, C. M. High performance thin-layer chromatography. Anal. Chem. 53(252A–266A), 1981. 27. Geiss, F. Fundamentals of Thin Layer Chromatography, Alfred Huthig Verlag, Heidelberg, pp. 5–8, 1987. 28. Maugh, T.H. II. TLC: the overlooked alternative. Science 216:161–163, 1982. 29. Sherma, J. Comparison of thin layer chromatography with liquid chromatography. J. Assoc. Off. Anal. Chem. 74:435–437, 1991.

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30. Fried, B. and Sherma, J. Thin-Layer Chromatography. In: Chromatographic Science Series, Vol. 81 (J. Cazes, Ed.), Marcel Dekker, NY, Inc. pp. 499, 1999. 31. Hahn-Deinstrop, E. Applied Thin-Layer Chromatography. Wiley-VCH, Weinheim, pp. 304, 2000. 32. Poole, C. F. and Dias, N. C. Practitioner’s guide to method development in thin-layer chromatography. J. Chromatogr. A. 892(1–2):123–142, 2000. 33. Renger, B. Contemporary thin-layer chromatography in pharmaceutical quality control. J. AOAC Int., 81(2):333–339, 1998.

10 ISOLATION METHODS II: COLUMN CHROMATOGRAPHY MARKGUINN, RONALD BATES, BENJAMINHRITZKO, TERI SHANKLIN, GLENN WILCOX, AND SAM GUHAN Pfizer Global Research and Development,Groton,CT 06340 I. INTRODUCTION II. BACKGROUND III. STATIONARY PHASES A. Achiral Stationary-Phase Functional Groups B. Chiral Stationary Phases IV. EQUIPMENT A. Chromatography Equipment Setup B. Online Detection V. SCREENING A. Achiral Impurity Isolations B. Chiral Impurity Isolations VI. DEVELOPMENT OF PREPARATIVE METHOD VII. SCALEUP OF PREPARATIVE METHOD A. Column Sizing B. Flow Rate C. Particle Size VIII. SUMMARY REFERENCES

I. INTRODUCTION With the high quality standards required of pharmaceutical products, there is a great need to determine the structure of impurities present in the ﬁnal drug substance. These impurities are present at extremely low levels, and analytical characterization could by itself be a technical challenge. In general, analytical chemists run a series of assays that utilize different binding modes (orthogonal assays) to conﬁrm that they have sufﬁciently characterized the sample and identiﬁed all the impurity peaks (if any)—and to also ensure that peaks that may co-elute using one technique may be visible using an orthogonal method. Analytical high-performance liquid chromatography (HPLC) is the most common technology employed for this analytical characterization. Once the sample has been analyzed, the impurity may need

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to be isolated (for structural characterization, etc.). While techniques such as LC-MS may give an indication of the compound identiﬁcation from the drug substance assay, the deﬁnitive proof is always obtained from an independent analysis of the isolated compound. Because of the relatively low levels of the compound (and also in many cases their close resemblance to the main compound), conventional separation techniques are usually not successful. The resolving power of chromatography (and in many cases HPLC) would be needed for this isolation. This chapter details the use of column chromatography for the isolation of impurities. It discusses the various options that are available for stationary phases and analytical detectors, as well as the current equipment available for this work. The choice of puriﬁcation techniques (prep HPLC, low-pressure silica, etc.) will also be discussed. Finally, a procedure (or a ‘‘How to’’) is laid out for both analytical methods development, as well as scale-up to preparative columns.

II. BACKGROUND For the purposes of this chapter, it is assumed that the analyst has an analytical assay that identiﬁes the impurity/impurities of interest. In general, this is a reversed-phase HPLC assay. Once this assay is in place, the following background information needs to be gathered prior to designing an isolation procedure: .

.

.

.

.

Are there any other analytical protocols available from the analytical chemist? The more information about a compound, the easier it will be to develop a purification procedure. For example, in addition to the reversed-phase method, are there thin-layer chromatography (TLC) methods available for this compound? This could help in deciding whether a silica column may be the appropriate tool for this separation. What is the time frame in which this compound is needed? In many cases, this factor is critical to deciding what separation procedure to use. Since this is not a protocol that needs to be commercialized, a nonoptimum process that meets the material requirement in a timely fashion is the most important factor. How much material will be required and for what purpose? If material is needed just for structural identification, < 100 mg may suffice; however, if a reference standard is needed, this may require gram quantities. What is the purity of the isolated compound that is required? If purity requirements are < 95%, then more options are available for developing the isolation techniques. However, if > 99% is required, the separation will be more challenging. What is the nature of the feed sample? Is the desired impurity eluting near the peak of interest? What is the level of the impurity in the feed solution? What is the solubility of the feed in different standard mobilephase solutions/solvents?

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Is there any data available on the structural nature of the compound? For example, if it is known that a charge is present on the compound of interest, this could be utilized to design an ion-exchange separation.

Once this data is available, we are ready to proceed to the next step— choosing an optimum stationary phase for this separation.

III. STATIONARY PHASES Liquid chromatography involves the use of a liquid mobile phase and a solid stationary phase. The solid stationary phase is also called the packing material and is available in a large variety of sizes and functionalities. Stationary phases are classiﬁed by their particle and pore diameters as well as the functionality attached to the noninteracting backbone. Typical particle diameters range from 1.5 to > 300 mm, with analytical particles being < 10 mm, high-performance preparative particles ranging from 10 to 30 mm, medium-pressure particles ranging from 30 to 90 mm, and low pressure resins (the so-called big beads) ranging from 100 to 300 mm. The different sized particles provide different separation efﬁciencies as well as different pressure drops. The pressure drop across a packed bed is inversely proportional to the square of the particle diameter and directly proportional to the viscosity of the solution. Thus, smaller particles typically provide higher efﬁciencies but at higher pressure drops. There are several types of particle backbones used in chromatography. By far the most common solid support (or backbone) is silica; although alumina-, zirconia-, and polymer-based backbones are also used. The solid support in most instances is chosen or coated to be noninteracting, e.g., a hydrocarbon chain immobilized onto a coated silica solid support structure, but there are instances when the solid support provides the functionality used to separate solutes, e.g., underivatized silica and unmodiﬁed polystyrene divinylbenzene. Typically, stationary phases are classiﬁed by their nominal particle size (diameter) into two groups: high performance and medium to low pressure. High-performance packing materials have diameters that are < 30 mm, and the chromatography resulting from the use of high-performance stationary phases is termed HPLC. Medium- to low-pressure particles are signiﬁcantly larger than 30 mm. The former group utilizes highly efﬁcient small particles, and the latter employs larger, less-efﬁcient beads. Particles are further classiﬁed according to the mode of separation performed by the stationary phase. The two main types of packing materials for small molecule separations are chiral and achiral: separating either based upon chirality or other chemical properties. These will be explained in more detail below.

A. Achiral Stationary-Phase Functional Groups Since the advent of liquid column chromatography, the desire of every chromatographer has been for a better stationary phase. The most common stationary phases for liquid chromatography are porous, rigid spheres (usually manufactured from silica, SiO2). Irregular or nonspherical particles

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are occasionally used but to a much lesser extent. Irregular particles are cheaper than corresponding spherical particles, but they are not as efﬁcient or robust as their spherical counterpart—often having large quantities of ‘‘ﬁnes’’ that plug frits and result in decreased productivity. The chemistry of silica is well known,1 and it is possible to manufacture silica in many different sizes. Silica particles are also very strong, commonly able to withstand pressures in excess of 100 bar. Unfortunately, uncoated silica dissolves readily in basic (pH > 7.5) aqueous mobile phases, thus limiting the effective pH range. Recently, some manufacturers have begun coating the silica particles to prevent the dissolution and to extend the available pH range into the alkaline region. Other particle backbones used in chromatography include zirconia, alumina (Al2O3), and polymers. Zirconia and alumina are manufactured and used similarly to silica, but to a lesser extent. The polymer-based supports are manufactured from highly crosslinked porous polymers, such as styrene divinylbenzene or polymethacrylate. The polymer-based supports offer a large acceptable pH range (often 1–13), but they do have the tendency to swell with different solvent systems and typically cannot tolerate high pressures. Stationary phases used for achiral, small molecule separations are often further separated into two major classiﬁcations: normal and reversed phase. Normal-phase chromatography uses a polar stationary phase with a less polar mobile phase, while reversed-phase chromatography operates using the exact opposite conditions. Additionally, there are several functionalized stationary phases that can operate in either normal or reversed phase, e.g., cyano and amine resins. 1. Normal PhaseUnderivatized Silica For the most part, normal-phase chromatography utilizes an underivatized silica particle with a mobile phase consisting of an organic solvent (such as heptane, ethyl acetate, or methylene chloride) and an alcohol (methanol or ethanol) or other polar organic solvent. The range of possible solvent combinations is very large, but it is important to ensure that the desired solute is stable and at least somewhat soluble in the mobile phase. Additionally, the type of detection being used is also of concern since some solvents, like ethyl acetate and toluene, have very high UV cutoffs. The solutes are separated by differences in their polarity based on dipole–dipole interactions between the solute and the silanol groups on the stationary phase in the presence of the mobile phase. The solutes elute in order of increasing polarity, i.e., the most polar compounds bind strongest to the silanol groups on the resin and elute after the less polar compounds. As mentioned above, silica is the most common stationary phase, and as such, it is available in a host of shapes and sizes. Underivatized silica is most commonly seen in one of three sizes: low-performance, ﬂash, or HPLC. Lowperformance silica stationary phases have large diameters (and often with a large particle-size distribution) and are packed into low-pressure glass or stainless steel columns. HPLC silica is typically 10 mm particles that are packed in ﬁnely polished stainless steel tubes. Flash silica particles are 30–70 mm and have low enough pressure drop that laboratory nitrogen

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pressure can be used to pass mobile phase through the packed column. Flash silica also comes delivered in prepackaged cartridges that ﬁt into a FLASH puriﬁcation system sold by Biotage. Since the functionality of most silica stationary phases is the same, the selection of the mobile phase is very important during the screening and method development of a normal-phase silica separation. As mentioned above, typical mobile phases involve a nonpolar solvent like heptane and a polar solvent like ethanol, but the combination of solvents is almost endless. A good screening (using TLC or analytical HPLC) should examine numerous solvent combinations utilizing different types of solvents, such as THF instead of alcohols, ethyl acetate or methylene chloride instead of heptane and should also examine the use of additives, e.g., acetic acid or triethylamine, to adjust the pH to either suppress or enhance interactions with the stationary phase. More information on the method development is provided in Section V. 2. Reversed Phase Reversed-phase chromatography most often operates using a silica backbone derivatized with a covalently bound nonpolar moiety (often an alkyl chain). Additionally, after binding of the nonpolar group, another chemical step is often undertaken to cap the free silanol groups on the silica backbone (with an agent such as trimethyl chlorosilane) to promote stability of the stationary phase and prohibit the possible polar interactions between the silanol groups and the solute.2 The nonpolar groups range from alkyl chains to phenyl derivatives. The alkyl chains can range from 1 to 30 carbon atoms, with 8 (C8) and 18 (C18) atom chains being the most popular. Solute partitioning in reversed-phase chromatography is based on nonspeciﬁc hydrophobic interactions (van der Waals forces); the elution of solutes is in order of decreasing polarity, i.e., the most polar components elute ﬁrst. By orders of magnitude, reversed-phase chromatography using a C18 packing material is the most common form of liquid chromatography. The most common mobile phases used for reversed-phase chromatography involve combinations of either methanol or acetonitrile and buffered water (like phosphate buffered, pH 7). There are other organic solvents that are used in conjunction with buffered water in reversed-phase chromatography, e.g., other alcohols or THF, but these are less commonly used. Similarly to normal phase, pH can be used to increase/decrease the interactions between the solutes and the stationary phase. Common additives are triﬂuoroacetic acid and diethylamine. As with normal-phase chromatography, the mobile phase must be chosen carefully. Generally, it is understood that the mobile phase should have a low viscosity, be UV transparent, and be completely miscible. In addition to the silica-based, reversed-phase particles, polymeric particles are available that utilize a hydrophobic stationary phase, e.g., cross-linked polystyrene divinylbenzene. In these polymeric stationary phases, the backbone of the particle provides the opportunity for hydrophobic interactions. Although these packing materials are available in highperformance particles, the particles cannot withstand the high pressure that silica particles can and thus are typically used in medium- to low-pressure operations. The polymeric stationary phases usually have a series of aromatic

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rings that separate solutes based upon p–p interactions. The hydrophobic polymeric resins use the same mobile phase combinations as the silica-based hydrophobic phases, e.g., acetonitrile and buffered water. 3. Combination There are also stationary phases that effectively partition solutes in either reversed-phase or normal-phase mode. These stationary phases are typically silica particles derivatized with cyano, diol, or amino functional groups. Particles with a cyano-functionality separate based on polarity utilizing nitrile interactions between the stationary phase and the solute. The amino group of typical amino stationary phases interacts primarily with anionic and organic acid portions of the solute. Diols utilize hydroxyl interactions similar to underivatized silica but offer a slightly different selectivity. These and other bonded-silica phases offer alternatives to underivatized silica, but they are used much less frequently. The mobile phases employed with these stationary phases are the same as used in standard reversed-phase or normal-phase chromatography.

B. Chiral Stationary Phases The packing materials described above separate chemical entities by exploiting chemical differences, e.g., hydrophobicity. Another class of stationary phases separates molecules based on chirality; this is accomplished using a silica particle derivatized with a chiral moiety. There are several classes of chiral stationary phases including helical polymers, brushlike functional groups, protein/peptides, and inclusion complexes. Each of these is described in more detail below. Some manufacturers produce chiral stationary phases that operate either in reversed-phase or normal-phase mode, and some chiral stationary phases can be used in both modes. As with other stationary phases, there are numerous manufacturers of chiral stationary phases. However, contrary to C18 and other achiral packing materials, each manufacturer of chiral stationary phases typically offers unique phases with completely separate selectivities. 1. Helical Polymers Helical polymers include amylose and cellulose derivatives, polymethacrylates and polyacrylamides. The most common of this group are the amylose and cellulose derivatives, with the functional groups attached to the monomers offering different chiral selectivities. Chiral Technologies is the major manufacturer of these polysaccharide-based stationary phases, with more than 10 types of packing materials available, including ChiralPak AD (amylose based) and ChiralCel OD (cellulose based). Chiral Technology offers versions of their packing materials that can be used in either reversed phase or normal phase, but not in both. Careful selection of mobile phase is required for use of the coated (noncovalently bound) stationary phases, since speciﬁc solvents (like ethyl acetate and methylene chloride) will dissolve the polysaccharide functional groups. Typical mobile phases for the polysaccharide-based packing materials in normal-phase mode utilize

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combinations of heptane and alcohol (isopropanol or ethanol). Additives, such as triﬂuoroacetic acid or diethylamine, can be added to the mobile phase to reduce nonspeciﬁc binding. 2. ‘Brushlike’ The brushlike chiral stationary phases were developed by Regis Technologies and William Pirkle to exploit p–p interactions between the analyte and the chiral stationary phase.3 The functionality of the stationary phase involves several phenyl rings and sites for hydrogen bonding arranged in such a way to provide chiral recognition. Regis’ chiral stationary phases utilize a covalent linkage between the chiral functional group and the silica backbone; and contrary to the coated materials offered by Chiral Technologies, the Regis stationary phases can withstand strong organic solvents such as methylene chloride, ethyl acetate or toluene, and thus allow for a greater ﬂexibility in the choice of mobile phases. 3. Protein and Peptide One of the original chiral stationary phases involved the immobilization of bovine serum albumin (BSA) on a silica backbone, with the chiral selectivity resulting from the chirality of the amino acids on the immobilized protein. BSA-immobilized columns are still used today, but their use is mostly restricted to analytical assays. Recently, the immobilization of small polypeptides and derivatized amino acids has become popular. Regis Technologies and Astec are two manufacturers of such packing materials. Regis offers several stationary phases using derivatized amino acids, e.g., leucine and phenylglycine. Astec’s two main chiral stationary phases involve the immobilization of a macrolide polypeptide, e.g., vancomycin and teicoplanin. Both manufacturers utilize a covalent linkage to the silica backbone, thus allowing for the use of the full range of solvents in either reversed- or normal-phase mode. 4. Inclusion Complex These stationary phases separate enantiomers on the basis that one isomer ﬁts in the ‘‘pocket’’ and the other does not. In this fashion, the relative speed of the isomers is different, and separation results. There are three main types of inclusion chiral stationary phases: -cyclodextrin, -cyclodextrin, and -cyclodextrin. From these three native cyclodextrins, several derivations can be made to alter the selectivity of the inclusion complex, including formation of acetates, esters, and carbamates. Astec produces all three native cyclodextrin stationary phases as well as several derivatized phases (called the Cyclobond series), and as with their macrolide polypeptide phases, they are covalently bonded.

IV. EQUIPMENT A. Chromatography Equipment Setup A typical two-solvent column chromatography setup is shown in Figure 1. Two solvents, A and B, can be withdrawn by a pump, with a valve to control

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FIGURE 1 Two-solvent chromatography system.

the relative amount of each. The column is downstream from the pump. The pump enforces a net positive ﬂow in the downstream direction from the injection loop. The column can have ﬂow in the horizontal or vertical direction (upﬂow or downﬂow) depending on the type of separation. The bed is packed with porous particles. Frits, usually installed at the inlet and outlet of the column, allow ﬂuid to pass in and out of the column while keeping the solid packing material in place. A distributor at the column inlet can reduce the radial differences between ﬂow vectors. A detector is commonly placed downstream from the column as close as possible to the outlet. The eluant (ideally with separated components eluting at different times) is collected downstream from the detector. 1. Low-Pressure Systems Low-pressure liquid chromatography is generally considered to be any chromatographic separation operated at a pressure of 10 atmospheres or less. Low-pressure chromatography systems consist of low-pressure columns, usually glass, plastic, or stainless steel, packed with stationary phases of a relatively large particle size (up to hundreds of microns in diameter). Connecting tube can be made of PTFE or stainless steel. Gradients in the mobile phase can be achieved with two constant-ﬂow pumps feeding to a

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mixing chamber positioned upstream from the column. A wide variety of liquid chromatography media used in pharmaceutical applications are available for low-pressure applications. Often these phases have silica-based or polymeric supports; examples include media that can partition solutes through bonded functional groups (C8, C18, CN), size exclusion, and ion exchange or ion exclusion. 2. High-Performance Systems High-performance chromatography operations are carried out anywhere from ten up to several hundred atmospheres. In high-performance systems, columns and frits are often made of stainless steel. Connecting lines can be made of PEEK or stainless steel for analytical or small-scale preparations (gram quantities). For larger preparative separations (kilogram scale), the connecting lines are typically made of stainless steel. To use mixed solvents or gradients in a high-pressure operation, the inlet valve in Figure 1 often is set to open and close according to a timing scheme that can apportion solvents A and B in either an isocratic or gradient mode.

B. Online Detection Finding an online detection method is an important step in carrying out any analytical or preparative chromatography operation. In a preparative operation, a reliable online detector can be used to determine at which time the desired product is eluting from a chromatography column. An ofﬂine analytical HPLC method with small injection volumes can be used to conﬁrm the composition of cuts collected from a preparative run. Ideally, an analytical HPLC method provides an acceptable separation of the mixture components, displays peaks that can be assigned to known components, and gives a detector response that is a linear function of analyte concentration. Using isolated standards and calibrating the analytical detector for the concentration of each component is a good way to know the mass of each component eluting from a chromatography column, when such standards are available. Ultraviolet (UV) spectroscopy, mass spectrometry (MS), refractive index (RI) detection, and electrochemical detection (ECD) are common online monitoring techniques for analytical chromatography. UV and RI are regularly used for monitoring preparative operations as well. To employ MS or ECD in a high-ﬂow scheme, usually a side stream must be diverted from the main eluate line via a ﬂow splitter so that what passes through the detector has a ﬂow rate that is acceptable for an analytical-scale system. 1. Ultraviolet/Visible (UV/Vis) Spectroscopy Ultraviolet/visible spectroscopy is one of the most common methods for online monitoring of analytes from a chromatographic column. This type of detector is fast (many data points can be taken every second) and sensitive (parts per million concentration levels can register a signiﬁcant response). When two or more compounds elute through the ﬂow cell at the same time, the UV signal is typically the superposition of the signals from each

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component. UV/Vis spectroscopy can be used for analytical or preparative chromatography. When calibration standards are not available, it is sometimes acceptable to estimate purity and composition as percent UV peak area, although this estimation can be subject to signiﬁcant error if the extinction coefﬁcients of the various components differ. 2. Mass Spectrometry (MS) A powerful and relatively common supplement to UV spectroscopy is mass spectrometry. In a mass spectrometer, particles injected into a sample chamber are nebulized, or broken up, into smaller molecular groups and are separated instantaneously according to their molecular masses. The method can potentially give the molecular weight of a compound injected at a given time, and the characteristic fragmentation pattern can be used as a ﬁngerprint for compound identiﬁcation. 3. Refractive Index (RI) Detection While the technique of refractive index detection is less sensitive than UV or MS detection, it represents yet another useful, highly developed, quantitative online detector. RI detectors can perceive the difference in refractive index between a reference solvent and a solution with one or more analytes. Because of the comparative nature of the signal, a reference solution is necessary and gradient operations can be carried out only with very precise controllers to ensure that the reference cell and the sample cell have the same mobile-phase composition at any given time. 4. Electrochemical Detection (ECD) Electrochemical detectors are sensitive and inexpensive. They can detect some compounds, such as macrolides, mercaptans, and hydroperoxides that can be difﬁcult to observe by UV absorption. In an electrochemical detector, a constant voltage is applied across a sample cell and a reference cell separately; the difference in electrical current generated in the two cells is the basis of the signal. Not every compound can be detected by ECD— cyclic voltammetry can be used to determine which compounds are detectable by ECD, as well as the best voltage to use. Because of the need to generate a current, solvents in the mobile phase must be conducting (sometimes introducing a salt or buffer is sufﬁcient); nonpolar mobile phases cannot be used.

V. SCREENING The ﬁrst task is to set up and verify the analytical method that has been used to detect the impurity of interest. This method will provide the basis for decision making in the screening process. The next step is to obtain a sample that has the greatest enrichment of the impurity possible. Development of a preparative chromatography separation is in many ways analogous to that for an analytical separation, and many texts are available detailing the development of analytical methods, e.g., Practical HPLC Methods

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Development, Snyder, Glajch, and Kirkland4. However, it is important to note that the goal of the screening process is to ﬁnd a system with the greatest resolution between the impurity of interest and its closest eluting neighbors, not to ﬁnd a system that resolves all the components in the sample. This often leads in a different direction than analytical assay development would.

A. Achiral Impurity Isolations For achiral impurities, the screening normally starts with the analytical assay. If the stationary phase is available in either large columns or bulk and the impurity is well resolved from the other components in the mixture, this might be a good system for preparative use. If the analytical system is not available on a larger scale, screening columns with the same mechanism of separation (C18 or cyano) that are available in sufﬁcient sizes should be identiﬁed, and then the mobile phases suitable for those columns should be examined. The ideal preparative mobile phase would have no additives. If mobile-phase additives are required for separation, then use of a volatile additive (i.e., triﬂuoroacetic acid, acetic acid, triethyl amine, etc.) is preferred over a nonvolatile additive (phosphate buffers) if removal of the mobile phase by evaporation or boiling is required. Finally, oxidizing buffers (perchloric acid, etc.) should be avoided if possible because of safety considerations with their use in large-scale processes. Often it is necessary to make adjustments in solvent strength and separation selectivity when developing a preparative LC separation. In many cases, only slight adjustments of one or two parameters will be required to obtain the desired resolution for the peak of interest. Examples in a reversed-phase LC system include adjustments of the organic solvents at 5–10% increments, changes in pH of an aqueous buffer solution, and column temperature. If the analytical system (assuming it to be a reversed-phase HPLC assay) shows promise, further reﬁnement can be accomplished quickly by making slight adjustments to the mobile phase. If a gradient operation is to be used, further separation of the peaks can be accomplished simply by decreasing the slope of the gradient. If the separation is to be run isocratically, the peaks can be slowed down (thus retained longer on the column) by decreasing the percentage of organic solvent in the mobile phase. Conversely, increasing the organic solvent content of the mobile phase will decrease the retention of the solutes on the column. It is important to note, however, that solely increasing or decreasing the strength of the mobile phase typically does not increase the resolution between the components; it only alters the retention of all the components. The resolution is frequently affected by adjusting the pH of the mobile phase, however; and occasionally simply substituting one buffer for another alters the resolution and retention. A thorough examination of the effect of pH on the retention/resolution can be achieved by examining three or four pH values (high, neutral, medium low, and low). It is also important to know the pH range under which the column is stable. If the resolution between the impurity and other components in the mixture is too low to use the analytical system preparatively (again assuming that this analytical system is a reversed-phase assay), then other stationary

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phases should be examined. One common phase is silica. Since this material separates based on polar H-bonding interactions, it provides an orthogonal technique to the reversed-phase assay. The screening for silica separations is normally done on TLC plates and often starts with several single-solvent systems (see Chapter 9). The goal of the initial experiments is to get a feel for where the compound of interest elutes. If the compound does not move from the origin, then the polarity of the solvent is increased either by moving to a more polar solvent or by adding a more polar solvent and creating a mixed solvent system (see Practical High-Performance Liquid Chromatography5, Table 5.1 for a list of solvent strengths in silica chromatography). Once the solute moves from the origin, how much retention is observed? Retention factor (Rf) is calculated by measuring the distance from the origin that the compound of interest has traveled, divided by the distance from the origin that the solvent front has traveled. For the screening, an Rf of about 0.2–0.3 is targeted so that the compounds will be moderately retained in column chromatography. If the retention is acceptable, then is separation of the desired compound from other compounds observed? What are the shapes of the bands? Very often, molecules containing basic functional groups will require the solvent system to include an organic modiﬁer (such as diethylamine, triethylamine, acetic acid, etc.) that competes for the stationary phase sites responsible for band tailing. After a system has been found that gives resolution of the desired impurity, conﬁrmation of identity and purity can be obtained by scraping the band off the TLC plate and extracting into a solvent. The solvent is then injected onto the analytical HPLC assay. One good method of detection on TLC plates is the use of iodine. After the plate has been run and dried, it is placed in a closed container containing iodine. The iodine vapor reacts with double bonds in the compounds to form brown complexes. If iodine doesn’t work, then other staining methods will have to be examined6 (ideally the staining method should be reversible so that the compound identity can be conﬁrmed by HPLC after the plate has been run and stained). After the selection of a solvent system that gives an optimal separation with the desired Rf , a silica column (either hand-packed or a prepacked ﬂashtype cartridge) is equilibrated with the mobile phase. The sample is then injected, and the efﬂuent is either monitored by UV or fractions are collected. The loading on the silica column will be related to the difﬁculty of the separation; typical loadings are in the range of 20 to 40 g of silica for each gram of feed sample. Often when the column experiments are done, small adjustments in the mobile phase are necessary to get the column system fully optimized. If an adequate separation is not obtained by the use of either the reversed-phase analytical method or the silica method, then other stationary phases are scanned for their separation capability (CN, C8, C4, phenyl, chiral, etc.). Often a scouting gradient can be employed to screen columns by HPLC.7 For reversed-phase systems (aliphatic and CN columns), a gradient of acetonitrile and water can be set up, beginning with 100% water and ending with 100% acetonitrile over approximately 30 column volumes. Of the columns that show some peak separation, the gradient can usually be

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converted to an isocratic condition or at least a truncated gradient system. To ﬁnd an initial isocratic condition, the average peak retention time can be calculated as the sum of the ﬁrst and last peak retention times, divided by two. At this point the solvent composition in the gradient can be used as a starting isocratic point. The ﬁrst isocratic runs should be performed for all systems showing some peak resolution. To help ensure that the peak in the preparative system is the desired impurity, the UV proﬁles of the peak versus the desired impurity can be compared if a photodiode array detector is used in the analytical and the preparative systems. The suspected peak of interest can also be collected to conﬁrm the peak identity by use of the analytical HPLC or other chemical analysis. Many alterations can be made to the above systems in an effort to further resolve the peak of interest. Water can be exchanged for buffers at any pH compatible with the stationary phase. Generally, both a low and high pH should be screened. Alternate solvent systems having the same solvent strength can be examined (for example, substitution of methanol or THF for acetonitrile). See Table 1 for rough solvent equivalence or Snyder and Kirkland,8 Chapter 6 for a more thorough discussion of solvent selectivity and strength. If the reversed-phase systems do not provide an adequate separation, then a screen is done on stationary phases that work in normal phase mode (CN, diol, amino, chiral). The screen is run in a fashion similar to the reversed-phase screen, except that the starting solvent is nonpolar and the strong eluent is the polar solvent in this case.

B. Chiral Impurity Isolations For situations in which the impurity is an enantiomer, the choice of stationary phase will be limited to chiral stationary phases. For a typical screen, the four common Daicel phases (Chiralcel OD, OJ, and Chiralpak AD, AS) are tried ﬁrst. The initial screen for these columns is done with TABLE 1 Equivalent Solvent Strength for Binary Solvent/ Water Mixtures for Reversed-Phase Chromatography Acetonitrile in water

Methanol in water

THF in water

0

0

0

10

16

7

20

27

15

30

38

22

40

50

30

50

60

37

60

70

45

70

77

51

80

85

58

90

93

66

100

100

72

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mixtures of ethanol or isopropanol in hexane or heptane. If resolution is found in this screen, the system can be optimized by examining other solvents; however, the choice of solvents is limited by the chemical compatibility of these phases. If one of these phases does not resolve the desired compound, then screening moves to other phases such as the Regis brush-type phases, the Chirobiotic peptide phases, or cyclodextrin-based phases, as described in Section III. B.

VI. DEVELOPMENT OF PREPARATIVE METHOD Separation of the desired material from interfering bands in the sample is the primary objective when developing a preparative chromatographic method. The main objective of almost every chromatographic separation is to optimize system resolution, speed, and capacity, otherwise known as sample load. Focusing on any one of these three parameters will require certain concessions from the other two. Sample load is usually the parameter compromised when developing an analytical separation, because peak recovery is not the primary goal of the separation. On the other hand, when component isolation is the main objective, emphasis should be on maximizing sample load, while speed and resolution will be compromised. It is advisable to invest more time exploring how selectivity varies with experimental conditions, because preparative separations can require large periods per run. Every effort should be made to perform loading studies with the same particle size and column length available at the preparative scale, as well as the same lot of feed material as the preparative separation. Analytical HPLC separations performed with sample loads of < 1 mg/g of column packing material usually do not alter column effectiveness. Preparative separations involve sample loads > 1 mg/g of stationary phase, and in many cases, the higher loading results in reduced selectivity and efﬁciency. Separation selectivity is of more importance in preparative chromatography than in analytical separations. Resolution (Rs) is affected by several chromatographic variables demonstrated in the following equation:7 pﬃﬃﬃﬃﬃ Rs ¼ 14 ð 1Þ N ðk0 =ð1 þ k0 ÞÞ where k0 ¼ (t t0)/t0; t0 ¼ time for a peak eluting in the void fraction of the column; t ¼ time for component of interest to elute; ¼ k0 of desired compound/k0 of nearest neighbor; N ¼ number of plates. Selectivity and retention are, for all intents and purposes, free of the effect of ﬂow rate as long as mass-transfer rates are not limiting. Resolution is relatively predictable for columns operated at loads of < 1 mg/g of packing. In overloaded columns the large sample size signiﬁcantly affects column equilibrium, and parameters such as plate count (N), retention (k0 ), and selectivity () change as overload varies. Deliberately overloading a column while maintaining an adequate separation decreases both selectivity and retention while increasing throughput. To begin an overload study, the feed solubility will need to be determined. The feed material should be dissolved in mobile phase at increasing

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concentration until it begins to fall out of solution. A concentration that is 80% of the maximum solubility at room temperature is typically used to load the column. If the solubility in the mobile phase is relatively poor (< 5–10 mg/mL), then alternate feed solutions should be tried (i.e., dissolving in the strong eluent, use of another solvent). However, the effect of the change in solvent will have to be determined as the peak shapes often can be signiﬁcantly affected, and in extreme cases, the feed solution can precipitate on the column frits leading to high back pressure. Once a feed solution has been determined, the analytical-scale preparative system can now be run under increased loading. The injection volume should be increased in a stepwise manner, and the resolution of the desired component from the nearest eluting peak(s) must be monitored. Often the UV absorption is over the reporting limit at higher loadings, and a wavelength off from the maximum absorption should be used to monitor loading studies. It is common to perform loading studies with several stationaryphase/mobile-phase combinations to determine the most efﬁcient system. Systems can be compared by their throughput. Throughput is the maximum system loading in mass per column volume, divided by the run time for that system. The peak of interest from each system should also be collected during these loading studies, and analyzed to ensure that it is indeed resolved to the desired purity. To isolate relatively small amounts of the desired impurity at high relative purity, the sample load should be increased until the base of the peak of interest begins to overlap with an adjacent peak(s). This methodology will give the highest purity with highest recovery of the peak of interest without having to worry about fractionating the product peak and recycling partially pure fractions. To isolate more material than can be done with an analytical column, simply scale up the separation based on the preparative column diameter as described in Section VII to allow for larger sample mass introduction. In the separation just described, the capacity of the system is limited by the resolution required. If larger amounts of puriﬁed material are needed than can be separated using the technique above, the column loading will have to be increased beyond the touching bands. The degree of peak overlap will increase with increasing sample load. By collecting eluant fractions through the region of the peak of interest, one can collect a greater amount of material in a shorter period. However, collecting material in this ‘‘heart-cut’’ portion of the chromatography will provide the desired component at lesser purity than that of the same component collected at the loading limit mentioned earlier and at a lower yield. Collecting subfractions through the heart-cut range can provide solutions that contain the component of interest at rather high purity. Subfractions that do not meet the purity requirement can either be discarded or repuriﬁed, depending on the scarcity of the feed material. Employing heart-cut methodology allows for added ﬂexibility, especially when the component of interest is hidden within a matrix of several peaks. For exceptionally difﬁcult separations, it may be necessary to isolate the desired impurity as a mixture from the ﬁrst column. It may be possible to repurify the mixture on the same column with a second pass, or alternatively, it may

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be necessary to screen this mixture for alternate columns that can provide resolution between the remaining components. Having to deal with only one or two interfering peaks will greatly increase the chance of success for isolation of the desired band. After the desired bands are collected, it is likely that undesired components remain on the column. Obviously, if these materials elute rather quickly from the point at which the desired bands were collected, it is best to continue elution without increasing the strength of the solvent. This will avoid the need for equilibration before the next sample injection. In many cases, signiﬁcant undesired components from the feed matrix elute at extreme concentration and/or retention. Increasing the ﬂow rate can sometimes resolve this situation; again, undesirably high back pressure or an unnecessary increase in solvent usage can limit this. A better method to clear the column of undesired material is to sharply increase the solvent strength and then follow with a period of equilibration with the desired mobile phase. The column is then ready for another injection of the feed matrix or a second pass of the pooled material collected from the heart-cut fractions.

VII. SCALEUP OF PREPARATIVE METHOD A. Column Sizing Once loading studies have been performed, scale-up can take place. First, the amount of feed material needed to yield the desired quantity of impurity must be determined. An HPLC assay or other chemical assay can be used to determine the potency of the feed with respect to the desired impurity. If an assay is not available, the impurity potency can be approximated from analytical peak collection. Additionally, an estimation of the recovery yield (percentage of impurity present in feed that is recovered from the separation) can be obtained from a mass balance on the high-loading peak collection experiments. Off-cuts, fractions collected from the column that contain the impurity but do not meet purity, can often be recycled into the feed for the next run, increasing the recovery yield. The required preparative column volume can then be calculated as follows: V¼

ðM=PÞ=Y LC

where V ¼ prep-scale column volume (L); M ¼ mass of impurity needed (g); P ¼ potency of column feed (%); Y ¼ recovery yield (%) as determined in loading studies; L ¼ maximum loading determined in loading studies (g feed/L column); and C ¼ number of cycles to be run. In order to maintain the same level of resolution and run time seen at the analytical scale, the column length should be held constant if possible. The column diameter can then be scaled to obtain the desired column volume. rﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 4V DP ¼ L

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10 ISOLATION METHODS II: COLUMN CHROMATOGRAPHY

where DP ¼ prep-scale column diameter (cm); V ¼ prep-scale column volume (mL); L ¼ column length (cm).

B. Flow Rate To achieve the same level of separation at the large scale as at the analytical scale, the linear velocity through the column should be maintained and the ﬂow rate adjusted. This can be done by the following calculations va ¼

Qa ðDa =2Þ2

where va ¼ velocity at analytical scale (cm/min); Qa ¼ ﬂow rate at analytical scale (mL/min); Da ¼ diameter of analytical column (cm). The prep-scale ﬂow rate is then determined:

DP Q P ¼ va 2

2

where QP ¼ ﬂow rate at prep-scale (mL/min); DP ¼ diameter of preparative column.

C. Particle Size The same particle-size resin as used for the loading studies should be used for scaleup. The choice of particle size is a trade-off between resolution, which decreases with an increase in particle size, and pressure drop, which also decreases with an increase in particle size. Often, bulk resins are not offered in the full range of particle sizes that analytical columns are. Additionally, a size should be selected so that the column pressure drop does not exceed the resin limitations. A summary of the effect of changing the operating conditions is shown in Table 2.

TABLE 2 Effects of Changes in Column Conditions Effects of change Change

Resolution

Run time

Pressure drop

Column length

"

"

"

"

Column diameter

"

$

$

$

Particle size

"

#

$

#

Linear velocity

"

# (case-dependent)

#

"

Column loading

"

#

$

$

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VIII. SUMMARY This chapter brieﬂy describes the various types of packing materials and equipment that can be utilized to isolate impurities from drug substances. It then goes into how to screen for a preparative HPLC method that could be used for impurity isolation and how to move from the screening phase of the project to develop an analytical-scale preparative method. The chapter concludes by giving rules on how to scale that preparative method up to any size column that will be used in the impurity isolation.

REFERENCES 1. Brinker, C. J. and Scherer, G. W. Sol-Gel Processing, Academic Press, London, 1990. 2. Layne, J. Characterization and comparison of the chromatographic performance of conventional, polar-embedded, and polar-endcapped reversed-phase liquid chromatography stationary phases. J. Chromatogr. A. 957:149–164, 2002. 3. Pirkle, W., H. Hyun, Myung Ho and Bank, B. A rational approach to the design of highly effective chiral stationary phases. J. Chromatogr. 316:585–604, 1984. 4. Snyder, L. R. et al. Practical HPLC Methods Development, John Wiley and Sons, NY, 1997. 5. Meyer, V. R. Practical High-Performance Liquid Chromatography, 3rd ed., John Wiley and Sons, NY, 1998. 6. Stahl, E. Thin Layer Chromatography: A Laboratory Handbook, 2nd ed., Academic Press, NY, 1969. 7. Dolan, J. W. Starting Out Right, Part VI—The Scouting Gradient Alternative LC-GC, 18:478–487, 2000. 8. Snyder, L. R. and Kirkland, J. J. Introduction to Modern Liquid Chromatography, John Wiley and Sons, NY, p. 49, 2000.

11 MASS SPECTRAL CHARACTERIZATION DAVID J. BURINSKYAND FENG WANG GlaxoSmithKline, ResearchTriangle Park, NC 27709

I. INTRODUCTION II. RELEVANCE OF IMPURITY CHARACTERIZATION III. THE COUPLING OF LIQUID-PHASE SEPARATIONS AND MASS SPECTROMETERS A. High-Performance Liquid Chromatography (HPLC) B. Capillary Electrophoresis (CE) IV. ION FORMATION V. ANALYZERS VI. ION STRUCTURE INTERROGATION VII. DATA ACQUISITION AND INTERPRETATION VIII. APPLICATIONS IX. CONCLUSIONS X. SUMMARY REFERENCES

I. INTRODUCTION The quality and safety of modern medicines are ensured by rigorous control of related substances that arise during and after the manufacturing process. Such substances are generally grouped into two categories: (1) compounds emanating from the synthetic scheme (i.e., residual reactants, intermediates, unintended side reaction products, and residual solvents)1 and (2) compounds resulting from the decomposition of the active pharmaceutical ingredient (API), its reaction with the formulation excipients, or the interaction of product with packaging materials.2–5 Characterization of these related substances in pharmaceutical products is an important facet of the regulatory approval process. Knowledge about trace-level compounds that either remain from the synthesis or form under recommended conditions of storage can be used to improve synthetic processes for the API as well as formulation processes for the ﬁnished product. The extent and rate of degradation often can be altered by appropriate selection of formulation ingredients, dosage form, or packaging material. The shelf life or expiry date for candidate pharmaceutical products is established through real-time monitoring of various experimental or probe formulations and packaging

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conﬁgurations maintained under the same conditions that a marketed product may encounter during its lifetime (i.e., from primary and secondary manufacture through pre- and post-sale storage and ultimately administration to or consumption by the patient). These stability studies are intended to demonstrate that the potency of the product will be sustained for the full extent of its shelf life and that no foreseeable mode of degradation will produce compounds having undesirable pharmacological or toxicological effects. Because much of the development work must be completed prior to the end of the real-time stability-monitoring program, potential formulations are routinely exposed to artiﬁcial conditions (commonly referred to as accelerated stress conditions) in order to predict the outcome of the real-time studies far in advance of their completion. These stress conditions are intended to produce the same outcome as those the product may encounter during its shelf life. The need to identify low-level compounds in the ﬁnished formulation is dictated by the relative levels at which they are present with respect to the API. Speciﬁc action levels are reﬂective of the proposed daily dose of the medicine under development (e.g., 1% for a maximum daily dose of < 1 mg to 0.1% for a maximum daily dose of > 2 g). Impurities present below the 0.1% level generally do not merit identiﬁcation (qualiﬁcation) unless the compound in question is expected to be unusually potent or toxic. The International Conference on Harmonisation (ICH) Guidelines6 set forth speciﬁc conditions of pH, temperature, oxidation, and light exposure for use in producing mixtures or proﬁles of representative decomposition products, in addition to the acceptable levels at which such compounds may be present. Degradants produced under accelerated stress conditions are presumed to be predictive of product stability over the course of its life. These mixtures of degradation compounds are useful in establishing and validating the speciﬁcity of analytical methods and for understanding the intrinsic chemistry of the molecules. Insight into the fundamental chemical reactions to which a molecule is prone can be fed back into the formulation process resulting in improved product stability. Identiﬁcation of related substances is ﬁrst undertaken during development of the drug substance synthetic route and continues throughout formulation optimization activities. By tracking these related substances in a systematic fashion, it is possible to rapidly identify new compounds resulting from modiﬁcations in either the synthetic scheme or the formulation strategy. Because of the high attrition rate for potential drug candidates, the rigor and level of detail required for structural characterization varies during the development life cycle. At early stages of the process, when candidate compounds tend to be short-lived, the demands are less stringent than at later stages when pivotal studies are conducted and information (e.g., synthetic routes, formulations, analytical methods, etc.) is being assembled in the regulatory dossier. Identiﬁcation at these early stages often consists of mass spectrometric analysis to determine molecular mass and relevant fragmentation pathways, and perhaps an NMR spectrum (1H, 13C, or both), with much or all of this information being acquired online (i.e., with direct coupling of the separation and spectroscopic techniques). This level of

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spectroscopic information, taken together with knowledge of the reaction scheme, is often sufﬁcient to adequately characterize compounds that are synthesis-related. The situation differs somewhat for drug product degradants. Unknown compounds that result from the decomposition of the API (e.g., hydrolysis, oxidation, thermolysis, etc.), reaction of the API with itself or another component of the formulation (i.e., an excipient or another API in the case of combination products), or the physical manipulation of the material (e.g., milling, spray-drying, compression, etc.) can require more extensive characterization, even at early stages. Unlike the body of knowledge that exists for the synthetic reactions and can aid in the identiﬁcation of reaction by-products, many of the compounds resulting from formulation activities may be unique, because of unknown and unconventional chemical processes. It may be necessary to isolate and characterize such compounds so that their toxicological properties can be evaluated. Synthetic or semisynthetic means can be employed to boost the amount of material available for isolation and characterization. In this context, ‘‘semisynthetic’’ refers to procedures or conditions used to convert the API to the related substance of interest in higher yields and shorter times than those unaided processes responsible for its formation in the product. The use of classical de novo syntheses is an available option; however, the time required to design and optimize a suitable synthesis often is not merited, given the limited amount of material (several milligrams) that will be required. Interest in the identiﬁcation of pharmaceutically related substances dates back to the early 1960s.7 Methods of separation were simple8,9 compared with those employed routinely today. The analytical techniques of choice included infrared (IR) and ultraviolet (UV) absorption spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry (MS). The primary difference between efforts then and now is that isolation and puriﬁcation of a compound generally was required prior to spectroscopic analysis. This paradigm was highly effective but required relatively large quantities of material and time. Despite such drawbacks, it remains the most comprehensive means for establishing deﬁnitive chemical structure because of the correlation between sample purity and spectral quality, which leads to greater interpretive certainty. With increasing demands for more information about a larger number of candidate compounds in ever-shorter time frames, came an evolution in analytical instrumentation. Beginning with the general acceptance and use of gas chromatography/mass spectrometry (GC/MS), the analysis of pharmaceutically relevant mixtures was undertaken in a dynamic or ‘‘online’’ fashion.10–13 The limitations of GC for the analysis of thermally labile compounds led to the development and widespread deployment of high-performance liquid chromatography (HPLC), and the development of the ﬁrst liquid chromatography/mass spectrometry (LC/ MS) interface devices in the 1970s. Over the course of the subsequent 30 years, technological advances in interface design, including the moving belt,14 direct liquid introduction,15 continuous-ﬂow fast atom bombardment (CF-FAB),16 and thermospray ionization,17 steadily improved the range and variety of analytes that could be introduced into mass spectrometers.

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The most recent products of this evolution are electrospray ionization (ESI)18 and atmospheric pressure chemical ionization (APCI).19 These techniques enable the routine on-line analysis of a wide variety of compounds using a variety of separation conditions (i.e., solvents, additives or buffers, ﬂow rates, etc.).

II. RELEVANCE OF IMPURITY CHARACTERIZATION The preclinical development process is comprised of a diverse collection of activities. The outcome of many of these activities is dependent on the qualitative analysis of a wide variety and large number of analytes. This characterization effort serves as the basis of understanding for chemical and physiological processes upon which successful development of a medicine depends. Traditionally, the product development process for pharmaceuticals has relied on separation techniques, such as HPLC, employing nonspeciﬁc detectors (e.g., refractive index, UV/visible absorption, electrochemical, ﬂuorescence, etc.). These detectors provide sufﬁcient information in many instances and are inexpensive, reproducible, rugged, and simple to operate. In the absence of authentic standards, they do not provide any information that might lead to the identiﬁcation of compounds. In contrast, the use of a speciﬁc mode of detection, such as mass spectrometry alleviates the dependency on standards because compounds can be identiﬁed directly, provided they respond under the conditions of the analysis and that the molecular mass can be associated with the identity of a particular compound. The most prevalent use of qualitative mass spectrometry during the course of preclinical pharmaceutical development is structure elucidation of related substances (metabolites, synthetic impurities, and degradants), often at trace levels. Insight into chemical structure is a key factor in reﬁning pharmaceutically desirable attributes of a molecule such as potency, safety, and bioavailability. Thus, elucidation of structure is critically important for accelerating the pace of the development process. For the purposes of this discussion, the scope of pharmaceutical development activities will be limited to those concerned with the synthesis of the API or drug substance and the formulation of the ﬁnished product. No discussion on the contributions of MS to the drug discovery process will be undertaken. Any mention of protein or peptide analysis will be in the context of their use as therapeutic agents, with the associated need for impurity characterization. Examples will describe the analyses of complex samples of chemical rather than biological origin. Despite obvious differences in the collection and handling of the samples, the tactics used to characterize trace compounds in pharmaceutical matrices have much in common with those employed for the qualitative analysis of metabolites in biological matrices. Knowledge about the identity or nature of related substances enables proactive decisions to be made with respect to synthetic routes and formulation activities. Characterization of bulk drug substance and its various intermediates and precursors provides information that synthetic chemists use to optimize chemical processes, resulting in fewer synthetic

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impurities and a lower cost-of-goods. In order to evaluate the durability of a molecule toward the conditions it is likely to face during secondary manufacture (formulation), packaging, shipment, and storage (either before or after sale of the ﬁnal product), the pharmaceutical development process incorporates intentional chemical and physical challenges to the wouldbe product and its components. In order to mimic the variety of known or anticipated conditions, the API may encounter, a series of standard conditions have been established to test potential drug candidates. Combinations of elevated temperature and humidity, high and low pH, oxidative environment, and exposure to speciﬁc wavelengths (visible and ultraviolet) of light constitute the conditions that candidate therapeutic molecules and their formulations must endure during stability evaluation. These conditions are intended to accelerate likely routes or mechanisms of degradation the ﬁnished pharmaceutical product may be expected to undergo while being stored before or after sale (shelf life). In addition to providing important information about the fundamental chemical behavior of pharmacologically active molecules under rather unique conditions, the samples that result from the intentional stressing of the drug substance and product are useful for developing and challenging the separation methods that will be registered with regulatory authorities for release testing of manufactured product. Information about the structure or nature of unanticipated synthetic by-products can be important not only in the rapid and efﬁcient optimization of cost-effective synthetic routes, but also in aiding the design of future therapeutic leads that will possess increased resistance toward degradation (either chemical or metabolic). In a similar way, complete or partial identiﬁcation of degradation products or other compounds that form from the interaction of the API with excipients in the formulation can direct future formulation development and provide a starting point for the toxicological evaluation of signiﬁcant degradants. Impurities that arise during the synthesis of would-be therapeutics have implications for the manufacture and safety proﬁle of the ﬁnal product. Common impurities include residual reactants, catalysts, side reaction products and solvents. The number of synthetic impurities, and their levels, are indicative of the overall process quality (e.g., reaction yield, effectiveness of recrystalization and washing steps, etc.). The use of MS in combination with LC provides for separation and characterization (ideally identiﬁcation) of trace components in complex mixtures, but it is also amenable to automation schemes that include sample handling and preparation. There are numerous examples of the important role that LC/MS has played in the characterization of drug substance impurities as a prelude to the registration of a new pharmaceutical product. The challenges of such work are many, including the signiﬁcant number of structurally similar compounds, the complexity of separation strategies required to purify a single desired compound, and the need for detailed structural analysis. The attributes of LC/MS techniques that make them methods of choice for such work are the speed with which analyses can be conducted, the wide range of chemical structures that generate a response (although not all compounds respond, and there can be differential responses between

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chemically disparate classes of molecules), and the achievable sensitivity of such analyses. The characterization of related substances20–29 generally requires the collaborative efforts of synthetic organic chemists, pharmaceutical scientists responsible for formulation development, and analytical scientists. The analytical chemists utilize the latest separation techniques (e.g., HLPC, GC, CE, etc..) and structure elucidation methods (e.g., NMR, IR and Raman spectroscopy, X-ray crystallography, MS, etc.) in combination with the insight provided by physical organic chemists versed in the degradation behavior of various classes of therapeutic compounds.30–32 The online capability of both MS, and more recently NMR spectroscopy, make them preeminent techniques in providing preliminary information about the related substances proﬁle of a drug substance or product obtained using HPLC separations.33–68 Subsequent changes in either synthetic route or composition of the formulation are tracked, using the initial proﬁle as a comparative baseline. Other separation techniques (such as GC,69,70 capillary electrophoresis (CE),71–78 ion chromatography (IC),79 thin-layer chromatography (TLC),80–84 etc. are used for classes of compounds where they offer signiﬁcant or unique advantage, but HPLC remains the most-often used separation technique in pharmaceutical research and development. In the context of mass spectral characterization, the term ‘‘identiﬁcation’’ can take on various meanings. Unequivocal identiﬁcation of a completely unknown compound using MS alone is rare. The technique does provide invaluable and unique information about compounds (molecular mass) that cannot be obtained by other techniques. Ascertaining the molecular mass of a compound that elutes in an HPLC method at a known or expected retention time can constitute ‘‘identiﬁcation’’ of a compound of interest (although ‘‘recognition’’ would be a more appropriate term) when taken in context with previous knowledge about its synthetic origin or other spectroscopic information from techniques such as NMR spectroscopy (see Chapter 12) or single-crystal X-ray crystallography (see Chapter 3). Structure elucidation by MS depends heavily on the availability of standard compounds or mass spectral data for analogs of the compound of interest. If authentic standards or analogs are unavailable, reasonable proposals of structure are put forth based on the available information (molecular mass, fragmentation spectra, and accurate mass measurements). Differences in mass-to-charge ratios of the intact molecular ion and speciﬁc fragment ions between spectra of known and unknown molecules often provide sufﬁcient information to surmise the location of structural modiﬁcation within the molecule, and in ideal cases, its cause. However, it is not uncommon for ions to fragment such that neither the identity nor the location of the chemical modiﬁcation can be deduced. These situations require a technique such as NMR spectroscopy, which can establish connectivity between atoms and groups of atoms within a molecule. This is particularly true when isomeric compounds are encountered, since mass spectral differentiation is unlikely. Although not the topic of this account, there have been reports describing the use of LC/NMR/MS instrumentation for the characterization and identiﬁcation of low-level related substances.85–95 However, despite the

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appeal of such a sophisticated, integrated, and comprehensive approach, the complexity and cost of such instrumentation has limited its availability and use. In most cases, compounds requiring qualiﬁcation and inclusion in regulatory submissions undergo comprehensive spectroscopic characterization after isolation by preparative chromatography (see Chapters 9 and 10) and/or synthesis by classical or semisynthetic means. Compounds prepared (and characterized) in this way serve as reference standards in chromatographic methods used to evaluate pivotal stability study samples, release ﬁnished product, and facilitate method validation activities. The appeal of MS as a potential solution to pharmaceutically relevant structure elucidation problems is based on the diversity of molecules that can be probed successfully. When used in combination with what have come to be known as ‘‘standard’’ or ‘‘generic’’ HPLC methods (i.e., steep gradient elution separations performed using short, narrow-bore columns and elevated ﬂow rates), information about a wide variety of compounds can be obtained quickly. While the modest peak capacity generally exhibited by such methods makes them less elegant than customized methods, they have been demonstrated to perform satisfactorily for a signiﬁcant portion of the problems with which they are challenged, while providing desirable consistency and time savings throughout the development process. This performance has been made possible, at least in part, by the speciﬁcity of the mass spectral detector, which compensates for some of the chromatographic resolution that is often sacriﬁced for reduced analysis time. MS requires very little material to yield information about the identity or nature of a particular chemical compound. Also, the technique can provide either nominal or exact molecular mass information, depending on the hardware conﬁguration. Determination of molecular mass information is facilitated by the use of ionization techniques such as electrospray ionization (ESI)18 and atmospheric pressure chemical ionization (APCI).19 Much like traditional chemical ionization (CI),96 fast-atom bombardment (FAB),97 and thermospray (TSP)17 ionization, these techniques minimize the internal energy deposited into the molecule upon ionization, and thus fragmentation. Unlike some of their predecessors, the APCI and ESI techniques are amenable to interfacing with commonly used separation techniques such as HPLC and CE. Since most of the ion current is concentrated in a single signal, determination of molecular weight is generally straightforward. Among currently available instruments, there are several conﬁgurations that yield nominal m/z measurements (quadrupole mass ﬁlters and quadrupole ion traps) and others capable of measuring mass-to-charge ratio values at high resolving powers and with a high degree of accuracy and reproducibility (double-focusing sector mass spectrometers, time-of-ﬂight analyzers, and ion cyclotron resonance spectrometers). The accuracy and precision of such measurements permit determination of potential elemental formulae. The likely identity of a degradant can be ascertained from nominal mass measurements in many cases because of the structural homology that such compounds often share with their precursors, coupled with knowledge about the conditions under which they form. However, the ability to generate a list of likely elemental formulas for ions that either bear molecular weight

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information or provide some insight into structural subunits (i.e., fragment ions) can improve the speed and efﬁciency of the structure elucidation process. But, even in light of potential elemental formulas, unequivocal determination of ion structure is often a daunting task. Fragmentation spectra of ions produced from conventional therapeutic molecules (i.e., heterocyclic organic compounds other than peptides) sometimes contain only a fraction of the information necessary for complete de novo interpretation, particularly as the size of analyte molecules increases (see Figure 1). Consequently, interpretation often takes the form of comparative analysis using spectra of related molecules, for which a deﬁnitive link has been established independently between chemical structure and mass spectral features, as guides or templates.98,99 Fragment ions (or the complementary neutral losses) that are common to both unknown and template spectra provide indications or conﬁrmation of changes in the structure of speciﬁc functional groups in the molecule that is due to chemical reactions induced by changes in the compound’s environment (i.e., storage or intentionally imposed degradation conditions). The identiﬁcation of trace-level organic compounds of various types in complicated matrices is a strength of MS. For example, the types and structures of molecules encountered in the analysis of an environmental soil sample will be quite different from those of interest to protein scientists probing cellular function. These kinds of structural differences may dictate the optimal choice of instrumental conﬁguration required to answer the relevant analytical questions. Ionization of high molecular weight proteins is best accomplished by electrospray ionization, following separation by microbore HPLC or CE. Conversely, small nonpolar halogenated hydrocarbons are ionized most effectively by electron ionization following gas chromatographic separation. Similarly, the choice of mass analyzer(s) is at times dictated by the size and character of the molecules being interrogated. For example, the identiﬁcation of small molecules can be aided substantially by accurate mass information.100–102 The relatively small number of possible elemental compositions that meet the acceptance criteria for low molecular weight molecules can lead to a single solution, in favorable cases. If there is little else known about an analyte, this high degree of certainty can mean the difference between success and failure. In contrast, as molecular weight increases, the value of accurate mass measurements diminishes rather quickly because of the large number of possible formulas for any accurate mass measurement (within the achievable uncertainty of the measurement). While the value of accurate mass measurements is low for large polypeptide or protein ions, so too is the need for such information. The nature of the amino acid constituents (i.e., a ﬁxed number of known and well-characterized structural subunits) makes characterization or identiﬁcation a more tractable task than the analogous small molecule scenario. The recent popularity of chromatographic systems with mass spectrometric detection is a result of the evolution and development of small, relatively inexpensive instruments that are capable of obtaining information about compounds in a stream of HPLC eluent using standard, rugged,

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FIGURE 1 Impact of the extent of ion fragmentation on spectral interpretation.Top panel Example product ion spectrum exhibiting relatively few fragment ions at higher m/z values. Bottom panelProduct ion spectrum showing a more uniform distribution of fragment ions throughout the spectrum.

and easily implemented experimental conditions. Undoubtedly, such instruments have made and are making contributions to a signiﬁcant number of analytical problem-solving efforts. In contrast, success in solving the most challenging problems of structure elucidation or compound characterization requires a multifaceted, customized approach. Expertise, experience, and creativity come into play in all phases of the process and are required to surmount the many challenges and difﬁculties often encountered. The origin and condition of the bulk sample often can dictate some of the analysis parameters. Factors such as the level of analytes present

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in the sample as well as their solubility, lypophillicity, or volatility with respect to the other mixture components, all play an important role in the design and execution of the appropriate experiments. Such characteristics may inﬂuence how the analyte is best sampled, prepared, or handled, what means of separation may be most appropriate (and which ones may be ruled out), and which modes of ionization may prove most fruitful. Experiments are often conducted in an iterative fashion. Results are obtained and quickly evaluated for their quality and information content. Despite careful planning, the results of preliminary experiments are sometimes insufﬁcient and a second (or third) round of experiments must be conducted. When compound isolation is warranted, two of the most important factors are sources from which the compound can be obtained and the purity of the ﬁnal isolates. The amount of time, effort, and expertise required to achieve successful isolation of a compound is inversely proportional to the amount present in the source material. Crude reaction mixtures and mother liquors are preferred sources from which synthetic impurity compounds can be isolated. Related substances in drug product can pose particular challenges. Compounds originating solely from the API (either degradants or condensation products) may be synthesized using a traditional approach. More likely, however, the drug substance will be subjected to speciﬁc conditions designed to accelerate or amplify the reaction(s) causing the formation of the degradant of interest. The intent of this approach is to mimic the products of the degradation chemistry but on a time scale that is more consistent with the demands of the development process. Additionally, these ‘‘semisynthetic’’ conditions generally produce enhanced levels of the degradants. The shortened time frame and elevated levels reduce the level of difﬁculty and time required for isolating and purifying the compound for spectroscopic analysis. Working with only the API simpliﬁes puriﬁcation considerably since the degradants are not formed in the presence of the various formulation excipients. Of course, in cases where formation of a degradant results from some interaction between the API and another component of the formulation (either an excipient or another API in the case of combination products), any and all means capable of boosting the levels must be brought to bear on the problem. Not only does this approach increase the complexity of isolation and puriﬁcation, but it also has the potential for complicating the desired outcome by preferentially generating unwanted and interfering compounds. The importance of the techniques and skills required to isolate and purify degradants successfully cannot be overemphasized. The quality of the characterization or identiﬁcation effort (including data acquisition and interpretation of results) hinges on the quality of the sample. The requisite separation, puriﬁcation, and sample handling techniques are often not straightforward, despite their simplicity of appearance. For this reason, highly skilled and experienced separation scientists are crucial to a successful and timely outcome. While there are a variety of techniques that can be used to isolate related substances, preparative or semipreparative HPLC are used widely and provide the separating power and capacity necessary for most applications.

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III. THE COUPLING OF LIQUID-PHASE SEPARATIONS AND MASS SPECTROMETERS A. High-Performance Liquid Chromatography (HPLC) The relative responses of HPLC detectors that employ or probe different physiochemical phenomena are often (and not surprisingly) dissimilar. Despite this fact, the use of multiple, complementary detectors (such as an UV absorption spectrophotometer either in parallel or series with a mass spectrometer) inevitably generates discussion on the comparability of the information or the degree of ‘‘universality’’ of one type of detector versus another. The proliferation of mass spectrometric detectors in the past several years has sparked considerable commentary regarding their ‘‘universality’’ as detectors for HPLC separations. Comparisons between the output of a mass spectrometric detector (in the form of a total ion chromatogram) and the very familiar single (or composite) wavelength trace from the ubiquitous UV-VIS detector are commonplace. For many organic compounds, empirical observations reveal some degree of correlation between these two detector responses (i.e., a compound generates a response from both detectors). What must not be overlooked or forgotten, however, is the highly selective nature of most analytical detectors. Monitoring the chromatographic eluent at a single wavelength is a widely accepted practice in HPLC analyses. There is an implicit assumption associated with this approach—all components or potential components of the sample will respond and do so uniformly at the selected wavelength. Since the nearly inﬁnite variety of organic molecules renders this assumption far from valid, quantitative methods compensate for dissimilarities in the absorption maxima (max) and molar absorptivities (") of various analytes by incorporating normalizing factors (response factors) into formulas used for calculating analyte quantity or concentration. The response for a molecule passing through the ﬂow cell of an HPLC/UV detector is related directly to its electronic structure. There may or may not be any correlation between these properties and the solution- or gas-phase basicity (or acidity) of a molecule, the primary attribute(s) responsible for ionization (protonation or deprotonation) in LC/MS applications. The manifestation of these distinct underlying causes of detector response can be two chromatograms that are quite similar in appearance, somewhat similar in appearance, or have little or no resemblance to one another. There are few analytical techniques with the versatility of MS. Various combinations of analyzers and ion sources have been used successfully to characterize or identify elements and compounds (inorganic and organic) of virtually any atomic or molecular weight. Sensitivity, dynamic range (particularly for quantitative applications), ability to deliver accurate mass measurements, data acquisition rate, compatibility with the most appropriate separation technique or ionization mode, accessible m/z range, and ability to carry out one or more stages of collision-induced dissociation are but some of the factors that ﬁgure into the selection of the most appropriate instrument for a given analytical problem. The combination of MS with chromatographic or other separation methods expanded the impact and utilization of the technique. However, the process of interfacing two analytical techniques

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imposes certain restrictions and limitations. These compromises limit the modes of ionization that can be utilized with particular inlet (separation) systems. Therefore, while MS by itself possesses great versatility, as a chromatographic detection system, its power and ﬂexibility are reduced making it far from ‘‘universal.’’ Atmospheric pressure ionization techniques favor ion formation for some types of molecules, but not all molecules. Even among compound classes that ionize successfully, the efﬁciency can vary widely. The upshot of this variable behavior is a technique that displays extraordinary sensitivity (attomoles or zeptomoles) for certain molecules103 while producing absolutely no response to others. Differences in proton afﬁnities between molecules can produce a mass spectrum that tells a very different story than another type of detector for the same sample. As an example, the ratio of relative abundances for two signals (both [MþH]þ) in a mass spectrum can be 1 : 10 or 10 : 1 because of respective solution- or gasphase basicities,104 despite the compounds’ being present in equimolar quantities. Of course, this kind of differential detectivity is observed for virtually all chromatographic detectors. The very popular UV-VIS absorption detector is prone to similar behavior for molecules that vary widely in their molar absorptivities or lack chromophores entirely. Similarly, the GC ﬂame ionization detector does not detect water and underestimates the amount of highly saturated compounds or those that contain large numbers of heteroatoms, when compared to the response for fully saturated hydrocarbon compounds. For these reasons, the mass spectrometer is most effective when used in combination with other detectors, so that the most comprehensive data set can be assembled. The choice of detector for a particular HPLC analysis is usually based on the type of information desired and the ‘‘compatibility’’ of the device with the analytes of interest. Electrochemical detection105 is appropriate for the detection of neurotransmitters such as catecholamine but is of little use for molecules that are not electroactive. Similarly, UV–VIS absorption detectors are the best choice for analytes that have high molar absorptivities for electromagnetic radiation in the wavelength range of 200–600 nm. However, while the primary interaction is between the detector ‘‘probe’’ (be it photons from an ultraviolet lamp or electrons from a working electrode surface) and the analyte molecules, the effect of the mobile-phase constituents on the fundamental process often cannot be ignored. The impact of solvent or mobile-phase composition is important because of the boundary conditions imposed by both concentration and physiochemical properties. Molar concentrations of common solvents used for HPLC mobile phases range from approximately 14 M for an organic modiﬁer such as tetrahydrofuran to 56 M for water. In contrast, the concentrations of minor mixture components can be on the order of 2 mM—a difference of 8 orders of magnitude. Even minor interactions between solvent molecules and the detector probe can easily overwhelm any response that analyte molecules may be generating. Thus, physiochemical properties of solvent molecules (or additives) such as high molar absorptivity or low redox potential can be detrimental to the detectivity of an analyte molecule by either preventing or obscuring its response. These same principles apply to the mass spectrometric HPLC

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detector. For example, the pH leveling effect of water hinders the solution phase protonation of weakly basic molecules. Thus, the types of organic molecules that can be ionized (protonated) successfully under electrospray ionization (ESI) conditions are limited, since solution phase processes play a substantial role. The gas-phase basicities of water, methanol, tetrahydrofuran, and acetonitrile are signiﬁcantly higher than reagents used in classical chemical ionization experiments (i.e., methane, butane, etc.). They are also very close to, if not higher than, the basicities of some classes of molecules for which HPLC is the separation technique of choice. This relationship between the gas-phase basicities of analytes and reagents has a tremendous impact on the response one observes for a compound under APCI conditions. Aside from the chemical implications of HPLC mobile-phase composition on the performance of the mass spectrometric detector, the use of particular compounds (e.g., sodium salts of phosphoric, citric, or boric acid) can have a deleterious effect on the physical integrity of the ion source or interface components. Unlike other HPLC detectors in which the mobile phase enters and exits the detector unchanged (save for a benign encounter with a beam of light), the mobile-phase entering the mass spectrometer interface undergoes a phase transition. The internal environment of a mass spectrometer is maintained at low pressure (high vacuum) in order to efﬁciently transmit and electrostatically manipulate ions as they move into and through the instrument. This pressure regime necessitates that everything entering the mass spectrometer be in the gas phase. The implication of this requirement for HPLC mobile-phase components (both solvents and any additives such as buffers) is that they must be sufﬁciently volatile to allow their removal from the system through the vacuum pumps. Any compounds that are not volatile will precipitate out of solution as the solvent evaporates and be deposited on the surfaces of the interface/ion source. These deposits impede the performance of the electrostatic elements within the instrument and can lead to dielectric breakdown, poor focusing of the ions, and irrevocable plugging or clogging of apertures or channels through which ions must pass in order to enter or traverse the mass spectrometer. The pH and ionic strength of mobile phase solutions can be adjusted and maintained using volatile low molecular weight organic acids such as formic, acetic, and triﬂuoroacetic acid, ammonium hydroxide or combinations thereof in order to avoid difﬁculties. The use of a UV-VIS absorption detector in series (or parallel) with the mass spectrometer is desirable, so the absorption characteristics of mobile-phase components should be considered so that the responses of low-level analytes are not obscured. Triﬂuoroacetic acid (TFA) offers some advantage over either formic or acetic acid because of its lower UV wavelength cutoff. The absence of signiﬁcant absorbance in the low-wavelength region (below 220 nm) of the TFA absorption spectrum facilitates the detection of low-level compounds with minimal masking. Additionally, being a very strong acid, TFA produces low pH mobile phases for reversed-phase HPLC separations at relatively low concentrations. Triﬂuoroacetic acid also displays ion-pairing characteristics106 that can be of beneﬁt in the separation of polar compounds, and it can provide varied selectivity. When additional ion-pairing capacity is required, homologues of TFA such as the perﬂuorinated C4 or C5 carboxylic acids have been

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demonstrated107 to be effective substitutes for classical reagents such as octane (or longer-chain hydrocarbon) sulfonic acid. Volatility is an important attribute for mobile-phase components, but it is not the only requirement. Compounds such as triethylamine (or its homologues) and sample diluents such as dimethylsulfoxide are sufﬁciently volatile but are best avoided because of interferences resulting from their persistence in the ion source. In the past several years there has been widespread acceptance of standard or so-called generic separation methods at the early stages of the pharmaceutical development process.108,109 Employing short, small-diameter columns, unsophisticated mobile phases, steep gradients, and high ﬂow rates, these methods serve as a foundation for a signiﬁcant portion of screening analyses conducted in chemical and formulation process research. The shift away from the previously accepted use of customized methods came with the realization that excessive effort spent on projects destined for termination was an ineffective use of analytical resources. This generic approach employs the same set of conditions for all samples and reduces the time required for method development, validation, and instrument conﬁguration. These generic or ‘‘fast’’ methods have lower peak capacities than individually optimized methods, thereby increasing the probability of chromatographic coelution. However, in many cases the separations are sufﬁcient to provide the desired information. The presence of a highly speciﬁc detector such as a mass spectrometer can compensate for some of the lost chromatographic resolution, but there are many cases when there is no substitute for adequate chromatographic separating power. Generic methods are popular because they can serve as a starting point for further reﬁnement of the HPLC separation, often through utilization of computer simulations. Major beneﬁts of adopting general methods early in a project’s lifetime include eliminating unnecessary iteration cycles for method development or validation,110 obtaining a signiﬁcant amount of information in a short period of time, and having a consistent data set, which often reduces the need for subsequent analyses. When coupled with MS, relative retention time (RRT) information is augmented with molecular mass and possibly some degree of structural information derived from fragmentation reactions.

B. Capillary Electrophoresis (CE) The preponderance of analytical (and preparative) separations that precede mass spectrometric analyses are carried out using HPLC. Despite its potential for extremely high efﬁciencies, capillary electrophoresis (CE) remains something of a specialized application, particularly with mass spectrometric detection. The technique is hampered by its reliance on small sample volumes (typically nL injections) in order to avoid loss of separation efﬁciency. The consequence of these small volumes is a concentration detection limit that is substantially higher than that of techniques such as HPLC. Despite the exquisite sensitivity of mass spectrometers, characterization of related substances present at low femtomole levels (assuming a hypothetical related substance compound with molecular weight 500 daltons present at 0.1% w/w in an equally hypothetical drug substance sample

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prepared for analysis at a concentration of 1 mg/mL) remains far from routine. Although the application of CE to problems of pharmaceutical impurity analysis has been less extensive than that of HPLC, the technique does merit some mention. There have been three primary approaches for the interfacing of CE to mass spectrometers — coaxial liquid sheath,111,112 liquid junction,113,114 and sheathless interfaces.115-117 One of the fundamental requirements for CE separations is a high electrical potential drop across the length of the fused silica capillary, which requires electrical continuity at both ends. Another characteristic of some CE separations is little or no ﬂow of supporting electrolyte. This absence of electrolyte mass transport poses a difﬁculty from the perspective of the atmospheric pressure ionization techniques, since they require a ﬂowing stream of solvent in order to form ions. While electrospray ionization is an optimal choice for mass spectral analysis of compounds that exhibit electrophoretic mobility, their solution-phase charge, the potential absence of a solvent stream from which the analyte ions can be sprayed may be problematic. The coaxial liquidsheath interface addresses both the issue of electrical continuity and lack of solvent by providing a makeup ﬂow. This makeup ﬂow both closes the electrical circuit enabling electrophoresis to occur and provides the necessary electrospray solvent, making the liquid sheath a very ﬂexible and versatile means for interfacing a CE capillary to a mass spectrometer. In addition to its applicability for electrospray ionization, the liquid-sheath interface has been demonstrated to be an effective means of transporting electrophoretically separated analytes to the tip of a continuous-ﬂow fastatom bombardment (FAB) probe. A second type of interface, the liquid junction, also has been successfully employed for both ESI and CF-FAB applications. The use of a transfer capillary for transporting analytes to the FAB probe tip or the ESI sprayer offers the advantage of optimizing ionization parameters independent of the CE experiment. While there are varying accounts with regard to issues such as peak broadening and relative sensitivity when using the liquid-junction interface, there is agreement that the device suffers from issues of dead volume and reduced sensitivity and is more challenging to assemble and optimize than the sheath-ﬂow interface. The sheathless CE interface was developed to minimize the limitations of the sheath-ﬂow and liquid-junction interfaces by eliminating the need for the makeup ﬂow. In contrast to the other two approaches, the sheathless interface employs the end of the analytical CE capillary as the point of electrical continuity and as the electrospray sprayer tip. Typically, the end of the capillary is either pulled or electropolished to form a nanospray ESI tip. Electrical conductivity is achieved through various means, including the use of microelectrodes or metal plating of the fused silica capillary. Sufﬁcient solvent ﬂow to sustain the electrospray process is achieved by operating the CE in a mode that produces electroosmotic ﬂow in the direction of the interface. However, excessive buffer ﬂow into the ion interface/source region can be detrimental when the solution contains non-volatile electrolytes or additives such as cyclodextrins. In this regard, CE shares the same limitations as HPLC with respect to the conﬂict (and compromise) between optimal separations and optimal conditions for mass spectrometer performance.

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IV. ION FORMATION The intrinsic basicity of most biologically relevant molecules makes the formation of solution-phase ions possible in low pH solutions. Conversely, carboxylic acids and molecules containing a limited number of other functional groups can be deprotonated to form anions in high pH solutions. When these solutions are sprayed as a stream of small droplets through a high-potential ﬁeld drop, evaporation of the solvent and concentration of residual charge eventually results in gas-phase ions that can be analyzed mass spectrometrically. Electrospray ionization (ESI) is amenable to small molecules and large molecules alike. Unlike other ionization modes, there is little thermal energy transferred to the analyte molecules during the ESI process. The amount of energy imparted to the molecules is so small that even very large noncovalent protein complexes survive the ionization process intact.118 For the most part, electrospray ionization mass spectra have a simple appearance, provided analytes are introduced into the source individually and not as a mixture. The probability of encountering complex spectra or uninterpretable signals is fairly low, but there are occasions when such complications arise. Some speciﬁc situations that result in complex ESI mass spectra include analyte molecules that contain more than one basic site, analyte molecules that are electroactive, and situations where the concentration of analyte in the ion source is very high or desolvation is incomplete. The presence of more than one basic site in a molecule makes multiple proton transfer reactions possible. Molecules that protonate simultaneously at multiple sites are multiply charged and generate additional signals in the mass spectrum. These signals are easily recognized so long as only a single analyte is ionized at any one time. Introducing large quantities of analyte into the ESI source can result in additional signals in the mass spectrum due to clustering reactions between analyte molecules. The resulting proton-bound dimers ([analyteþHþanalyte]þ) can be the source of interpretive uncertainty. Insufﬁcient or incomplete desolvation also can produce proton-bound dimers in ESI mass spectra. It is not uncommon to observe signals such as [MþHþCH3CN]þ or [MþHþCF3CO2H]þ for various classes of organic molecules. In addition, some ESI sources (due to either the geometry or composition of the interface components) produce signiﬁcant abundances of [MþNa]þ species for a wide variety of analytes. The electrospray ion source is generally accepted to be a rather passive device that desolvates preformed solution-phase ions. In reality, the ESI source is an electrochemical cell. This attribute seems to manifest itself rather infrequently, but holds the potential of transforming analytes into new chemical species.119 This prospect is undesirable from the analytical perspective since the creation of new compounds does nothing but complicate spectral interpretation. While the ESI technique has revolutionized the analysis of biomolecules such as peptides, proteins, polysaccharides, and nucleotides, the constraints imposed by its dependence on solution chemistry for ionization limit its applicability in the realm of small- to medium-sized nonpolar molecules (i.e., weakly basic or neutral compounds).120 Atmospheric pressure chemical ionization (APCI), on the other hand, takes advantage of gas-phase processes

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such as proton donation or abstraction to affect ionization of analyte molecules that have been volatilized by spraying them (entrained in a stream of solvent such as the HPLC eluent) through a heated region of the interface. The thermodynamics of the gas-phase reactions occurring in APCI promote the formation of ions from some molecules that are insufﬁciently basic (or acidic) to undergo ionization under ESI conditions. While broadening the spectrum of low molecular weight molecules that can be ionized, the solvents and additives commonly used as components of HPLC mobile phases also impose certain limits on the degree of success that is achievable. The common HPLC solvents such as water, methanol, acetonitrile, and tetrahydrofuran produce reagent ions ([RH]þ) upon vaporization and excitation in the corona discharge region of the APCI source. However, the acidity of these reagent ions is fairly modest, particularly in comparison to species formed under þ þ classical CI conditions (e.g., CHþ 5 ; C2 H5 ; or H3 ). Proton transfer (ionization) occurs if the basicity of the analyte molecule is greater than that of the conjugate base of the reagent ion (i.e., the neutral solvent molecule). There are two additional complications that can arise when APCI is selected as the ionization method. The ﬁrst is the potential for thermolysis of analyte molecules prior to gas-phase ionization. Unlike ESI, APCI imparts some thermal energy into molecules as they pass through the heated vaporizer region of the interface. The result can be unanticipated complexity in the mass spectrum. Instead of exhibiting a single signal such as [MþH]þ or [M-H], spectra contain or may be dominated by one or more additional signals representing thermal decomposition products.121–123 In extreme cases, there will be no intact analyte remaining. This can lead to a high probability of misinterpretation or misidentiﬁcation since the absence of the true [MþH]þ in the spectrum leads to erroneous assignment of a lower-mass thermal fragment as the protonated molecule. In addition to complications caused by thermally induced processes, chemical ionization conditions (whether APCI or convention CI) can produce ion/molecule reaction products that appear as spurious signals in the mass spectrum. Some of these ion/ molecule processes are easily recognized and understood. Most common among this type of artifact are solvation or association complexes. The formation and appearance of these ions is often dependent on the design and conﬁguration of the ion source/interface. Some instruments consistently exhibit one type or another of these adduct ions while others rarely produce that particular ion/molecule complex for the same analyte. Some examples of these solvation or ion/molecule complexes include [MþHþH2O]þ, [MþHþCH3CN]þ, [MþHþC4H8O]þ, [MþHþCH3OH]þ, [M-HþHCO2H], and [M-HþCF3CO2H]. These species are noncovalent in nature and revert to their constituents with the deposition of only a modest amount of energy into the complex. The appearance of such cluster ions in mass spectra can serve as conﬁrmatory evidence of molecular weight. However, there can be confusion or ambiguity if functional groups in the analyte can be eliminated as the same neutral molecule as the solvent. For example, if methanol is used as the organic modiﬁer in the HPLC mobile phase and a spectrum exhibits two signals separated by 32 m/z units, it can indicate one of the two possibilities: a protonated analyte molecule and a solvation complex,

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[MþH]þ/[MþHþCH3OH]þ or a protonated analyte molecule or a fragmentation product consistent with the presence of a methyl ester or methyl ether substituent, [MþH]þ/[MþH-CH3OH]þ. More troublesome, however, are ion/molecule reaction products that represent unpredictable processes of unknown origin. Such signals can lead to considerable confusion during spectral interpretation. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the most commonly used ionization modes for pharmaceutical analysis. Both methods operate primarily based on proton transfer reactions, making them suitable for the analysis of polar compounds. However, as the polarity and basicity of molecules decreases, so does the ionization efﬁciency of these two techniques. A new atmospheric pressure ionization technique has recently been introduced to address some of the limitations. Atmospheric pressure photoionization (APPI)124 utilizes 10 eV photons to affect ionization of analyte molecules. Background signals that are due to ionization of the common HPLC solvents are reduced because the ionization potentials (H2O ¼ 12.6 eV; CH3OH ¼ 10.8 eV; CH3CN ¼ 12.2 eV) are above those of many organic molecules (7–10 eV). In principle, a molecule will be converted to a radical cation ðMþ Þ by single-photon ionization, after which it may react with solvent molecules ultimately producing [MþH]þ ions. Ionization efﬁciency and the sensitivity of APPI can be improved by adding dopants such as acetone or toluene to the eluent stream prior to vaporization or directly to the vapor generated from the eluent. To date, successful experiments using APPI have been reported for the analysis of a number of compound classes including polyaromatic hydrocarbons (PAH)125 and ﬂavinoids.126 The ionization mechanism of APPI is not yet completely understood due to limited experimental data. The appearance of protonated molecules ([MþH]þ) for analytes with some polar character and radical cations ðMþ Þ for non-polar analytes in positive ion APPI mass spectra indicates a mixed ionization mechanism involving both charge transfer and proton transfer reactions. Figure 2 compares the relative responses observed for nonpolar aromatic compounds (similar to those found in pharmaceutical packaging container/closures or delivery device seals) using diode array UV detection, ESI, APCI, and APPI. The contrived mixture of seven aromatic compounds was analyzed using a reversed-phase HPLC method (gradient elution from a 50 3 mm C18 column with methanol as organic modiﬁer). While the analysis of large biomolecules is not the primary focus of this account, peptide mass spectra constitute a special case worthy of mention. Electrospray ionization of polypeptides and proteins generally produces more than a single signal in the mass spectrum. The multiple signals are due to the simultaneous protonation of the numerous basic sites (predominantly arginine, lysine, and some amide nitrogen atoms) distributed throughout these large molecules. The formation and stabilization of these multiply charged ions is due not only to the proton-acceptor nature of the basic functional groups of the amino acid residues but also to the intercharge distances accommodated by the size and conformational ﬂexibility of these molecules. The fact that these species are formed in solution (prior to the

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FIGURE 2 Comparison of LC/MS responses for different ionization modes. Top panel Chromatographic trace (HPLC/UV) recorded at 260 nm, showing all seven compounds. Second panelESI trace for the sample mixture, showing responses for four components of the sample.Vertical scale of the ESI trace is expanded by a factor of ten with respect to those of the APPI and APCI chromatograms.Third panelAPPI trace for the sample mixture, showing responses for four components of the sample. Bottom panelAPCI trace for the sample mixture, showing responses for four components of the sample.

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desolvation processes that ultimately result in gas-phase ions) helps to explain their presence in the mass spectra of these molecules. In the electrospray technique, ions are formed and exist in some solvated state long before the electrostatic ﬁelds within the ion source or interface region act upon them. This is in contrast to other ionization modes such as classical chemical ionization or atmospheric pressure chemical ionization. In these techniques, molecules become ions upon the addition or removal of a charged particle (e.g., protonation or deprotonation) and are acted upon immediately by the electrical potentials that extract them from the source region and transmit them into the analyzer. Once ions begin to move out of the source, the probability of their interacting with other charged species (protonating agents) is reduced, making acquisition of additional charge unlikely. The appearance of multiply charged ions is not unique to electrospray ionization. Matrixassisted laser desorption ionization (MALDI) generates doubly charged ions for biological molecules such as proteins, but none of the high charge states commonly encountered in ESI spectra. Prior to the advent of these two ionization modes, providing access to large thermally labile biomolecules, some of the only reported observations of doubly-charged ions had been for aromatic systems under conditions of kilovolt energy collisional activation.127 The observation of doubly charged ions in the electrospray mass spectra of conventional therapeutic molecules (i.e., synthetic non-peptide heterocycles) is not unexpected if the distance between basic sites is sufﬁcient to prevent coulombic repulsion from destabilizing the ion. As the complexity and size of these molecules continues to increase, so too does the probability of doubly charged ions. Recognition of spectral signals representing multiply charged ions is fairly simple when there are many charge states, as can be the case for proteins and oligonucleotides. The relative abundances of the signals generally reﬂect some distribution of ion stability (indicative of protein conformation),128 and they are spaced at irregular intervals over the m/z range of the spectrum. The molecular mass of a protein or peptide can be determined (either manually or using commercially available software applications) by multiplying the magnitude of the charge for one of the signals times the m/z value of that signal. The charge value of an ion can be calculated by substituting the m/z values of two adjacent multiply charged signals into the following expression and solving for the variable x: (high m=z value)x ¼ ðlow m=z valueÞðx þ 1Þ For example, two adjacent signals in the multiply charged signal envelope observed in the ESI mass spectrum of a peptide have m/z values of 824.7 and 660.0, respectively. According to the expression, the charge state (x) of the higher m/z signal is 4. Simple multiplication reveals the mass of the peptide to be 3295 daltons (see Figure 3). Doubly charged ions of conventional therapeutic molecules (with molecular weights less than 1000 u) can either help or hinder the interpretation process. Their positive contribution is the access they can provide to additional structural information about the molecule. Product-ion MS/MS spectra can be obtained for both the singly-charged ([MþH]þ) and

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FIGURE 3 ESI mass spectrum of a 3.2 kDa peptide showing the 2þ, 3þ, 4þ, 5þ and 6þ charge states. InsetMagnification of the high m/z region of the spectrum showing the low abundance of the [MþH]þ ion.

doubly charged ([Mþ2H]2þ ions, which can provide complementary information about distinct and structurally diagnostic dissociation channels. This approach of employing doubly charged ions as the precursor ions for CID experiments is commonplace in the arena of peptide sequencing by MS. Most of the peptides sequenced by MS result from the tryptic digestion of some larger protein. The speciﬁc nature of the enzyme trypsin to cleave amide linkages adjacent to basic residues such as lysine and arginine results in peptides having these basic residues at each terminus. Under electrospray ionization conditions, these two basic residues are readily protonated to yield intense signals for the doubly charged peptide ion. In nonpeptide molecules, the ability to fragment a doubly charged ion also can be useful. The majority of fragmentation processes encountered in MS are accepted to be chargeinduced.129 Therefore, regions of ions that are far removed from the charged site tend to be less susceptible to fragmentation reactions that provide insight into ionic structure (with the exception of speciﬁc examples of charge-remote fragmentation130). The introduction of a second charged site into the ion, which is by necessity reasonably far removed from the ﬁrst, can alleviate the problem by providing access to alternative dissociation channels (see Figure 4). While the formation of doubly charged ions can be advantageous, their appearance in mass spectra can also pose some interpretive difﬁculty if they are not recognized, since they may be confused with structurally indicative fragment ions. Recognizing doubly charged ions depends on factors that include the type of analyzer, the nominal numerical value of the mass-tocharge ratio for the protonated molecule, and the possible presence of cluster ions in the spectrum. The spacing of isotope signals on the m/z scale (x-axis) of a mass spectrum is a function of charge state, with isotope ions of increasing charge being spaced increasingly closer together. The separation is

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FIGURE 4 Utility of doubly charged ions in the structure elucidation of nonpeptide mole-

cules.Top panelProduct-ion spectrum for the [MþH]þ ion (m/z 529) of a substituted nucleoside showing several fragmentation products. Bottom panelProduct-ion spectrum for the [Mþ2H]2þ ion (m/z 265) for the same compound. The presence and location of the second charge site induces a number of fragmentation processes (resulting in product ions at m/z 140, 198, 332, 374, and 459) not observed for the singly charged ion upon collisional activation.

1 m/z unit between adjacent singly charged signals, 0.5 m/z units for doubly charged ions, and so on as the charge state increases. Analyzers such as double focusing sector conﬁgurations, ion cyclotron resonance spectrometers, and time-of-ﬂight instruments are capable of achieving the mass resolving power (resolving power ¼ mass-to-charge ratio/ mass-to-charge ratio, whereas resolution is the inverse, or mass-to-charge ratio/mass-to-charge ratio) necessary to separate adjacent isotope ions having multiple charges. Quadrupole mass ﬁlters and quadrupole ion traps are typically operated at unit mass resolving power, with each adjacent singly charged ion signal being nominally resolved from the next. Adjacent doubly charged signals

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(or higher charge states) are not completely resolved under these conditions (see Figure 5). The implication of unresolved signals is manifested when data are displayed in the traditional histogram representation. If the computer algorithm that calculates the centroid m/z values fails to recognize unresolved peaks (because of signals spaced at 0.5 m/z intervals), the resulting mass spectrum will exhibit inaccurate isotopic patterns that are due to peak merging in the ﬁnal display. Without proper recognition and representation of the multiply charged ions, interpretations can be erroneous. Quadrupole mass ﬁlters are capable of operating at higher resolving powers, but until recently131 doing so resulted in unacceptable losses in ion transmission (sensitivity). Similarly, quadrupole ion traps can be operated in a mode that provides mass resolving power sufﬁcient to distinguish isotopes of ions in the 5þ charge state. However, these specialized scan modes132 are operable only over a limited portion of the scan range, and acquisition times for these scans are signiﬁcantly longer than for the routine mass analysis modes. Other analyzers are more easily operated at the mass resolving powers necessary to resolve the isotopes of multiply charged ions and with less impact on sensitivity and acquisition or scan times. The nominal numerical value (odd number versus even number as a function of the number of nitrogen atoms in the molecule—the nitrogen rule) and magnitude of the m/z values of the singly and doubly charged ion signals also play roles in their recognition and ultimate impact on spectral interpretation. The cumulative contribution of fractional mass from the various atoms that make up organic molecules results in mass spectral signals having noninteger m/z values. As the number of atoms in a molecule increases, the m/z value for the 12C isotope signal of the molecular ion shifts from an integer (or near integer) value at low mass to fractional values at higher masses, as illustrated for hypothetical ions in Table 1. Also shown in the table are the effects of the odd/even nature of the m/z value for the singly charged ion with respect to a doubly charged ion of the same compound. Doubly protonated molecules of low to medium molecular mass containing an odd number of nitrogen atoms yield mass spectral signals that fall at fractional intervals (e.g., m/z 66.56, m/z 235.70, etc.) making them recognizable. However, as the molecular mass increases, the trend reverses (i.e., doubly charged ions of even-number-nitrogen compounds fall at recognizable fractional values). It is clear from the information in the table that in cases where adjacent isotopes are not adequately resolved from one another, determining whether a signal in a mass spectrum reﬂects the presence of doubly charged ions (versus singly charged ions arising from other sources such as coeluting compounds or unimolecular fragmentation reactions) can be nontrivial. The recognition of doubly charged signals in a mass spectrum is further complicated by the tendency of some compounds to form noncovalent selfassociation complexes under electrospray ionization conditions. Much like doubly charged signals, these cluster ion signals appear in the mass spectrum with those representing singly charged ions. Distinguishing these signal pairs ([MþH]þ/[Mþ2H]2þ versus [MþH]þ/[MþHþM]þ) requires close examination of the data, to ascertain the charge state of both ions in question (vide supra), and can be subject to misinterpretation.

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FIGURE 5 Electrospray ionization (ESI) mass spectrum of a 27-residue therapeutic peptide, showing the signal at m/z 824 ([Mþ4H]4þ).Top panelData acquisition conducted at nominal or ‘‘unit’’ resolution using a triple quadrupole or quadrupole ion trap instrument operated in typical fashion. Middle panelData acquisition conducted using the charge state recognition feature (slow scan at higher resolving power) of a commercially available quadrupole ion trap instrument. Bottom panelData acquisition conducted using a time-of-flight mass spectrometer.

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TABLE 1 Implication of the Nitrogen Rule and Molecular Mass on Accurate m/z Values for Singly and Doubly Charged Ions Elemental formula (hypothetical compounds)

[MþH]Q (m/z)*

[MQ2H]2Q (m/z)*

C6H13NO2

132.1

66.6

C6H14N2O

131.1

66.0

C24H46N4O5

471.4

236.2

C24H47N5O4

470.4

235.7

C48H90N8O9

923.7

462.4

C48H91N9O8

922.7

461.9

C72H134N12O13

1376.1

688.5

C72H135N13O12

1375.1

688.0

*m/z of the most abundant isotopic signal (12C) for the protonated molecule. Doubly protonated molecules of low to medium molecular mass that contain an odd number of nitrogen atoms yield mass spectral signals that fall at fractional intervals (e.g., m/z 66.6, m/z 235.7, etc.) making them recognizable. However, as the molecular mass increases, the trend reverses (i.e., doubly charged ions of even-number-nitrogen compounds fall at recognizable fractional values). These examples illustrate the potential difficulty in recognizing mass spectral signals attributable to doubly charged ions.

V. ANALYZERS The selection of a particular instrumental platform can provide distinct advantages for ion structure characterization of unknown compounds, regardless of their origin. One way that mass spectrometric analyzers can be differentiated is according to the manner in which ions traverse them. In beam-type instruments, ions are extracted from a source (or interface) region and accelerated or steered through one or more analyzer components by electrical or magnetic ﬁelds with the aid of various electrostatic focusing elements, ultimately arriving at a detector. The translational (kinetic) energy of the ions can be either high (kilovolts) or low (volts) and is dictated by the type(s) of analyzers being employed. Magnetic sectors or electrostatic analyzers perform momentum-to-charge or kinetic energy-to-charge analysis of ions possessing high kinetic energies (e.g., 2–10 KeV). Similarly, time-ofﬂight (TOF) analyzers separate ions possessing high kinetic energies (e.g., 10–40 KeV) as a function of their velocity. This is in contrast to quadrupole mass ﬁlters, which measure m/z values of ions having kinetic energies on the order of a few to a few tens of electron volts. Operationally, all beam-type instruments share certain attributes, regardless of the energy regime. Ions make a single pass through the instrument (from source to analyzer, and on to detector), and the source produces and transmits ions to the analyzer in a continual fashion (note: time-of-ﬂight instruments produce a modulated stream of ions and the presence of a reﬂectron ion mirror causes ions to make multiple passes within the device, albeit in a linear path). Thus, all of the various processes and events that ions undergo (formation, analysis,

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activation, and detection) are separated from one another spatially. This approach is fairly simple to implement and offers great experimental ﬂexibility, but requires redundancy or duplication of components and considerable physical space in order to accomplish multiple processes such as tandem (MS/MS or higher order MSn, where n ¼ 3 or more) mass spectrometric experiments. Since ion selection or analysis is required both before and after any excitation event, multiple analyzers must be joined in series to carry out the desired experiment. In trapping instruments, ions are also produced in a source region (external to the analyzer) and transmitted to a detector after traversing an analyzer. However, there are several notable differences in the operating principles of these devices compared to their beam-instrument counterparts. The most obvious characteristic of a trap (as implied by its name) is that the ions spend some ‘‘extended’’ period of time within the analyzer during the analysis process. It is not uncommon for ions to reside within a trapping device for times ranging from tens or hundreds of milliseconds to seconds. These residence times are quite long when compared to the typical transit times of ions through quadrupole mass ﬁlters (hundreds of microseconds) or time-of-ﬂight analyzers (tens of microseconds). This ability to accumulate and repetitively analyze ions within the trap is the basis of the excellent detection limits and signal-to-noise ratios that can be achieved by such devices. Multiple stages of mass analysis and ion excitation all occur within the same spatial region of the instrument and are separated from one another in time rather than space, as in the beam-type instruments. The advantages of temporally-based instruments are compact size, ability to perform multiple experiments with relatively small losses of signal, and no need for many expensive analyzer elements. Disadvantages of the time-based (trapping) analyzers include inherently lower dynamic range (because of the ﬁnite capacity of the trap) than beam instruments and the complexity of executing the timing events necessary for mass analysis and other ion manipulation events. Tandem mass spectrometry experiments (product ion, precursor ion, and neutral loss scans) were ﬁrst performed using double-focusing sector mass spectrometers (having either forward or reverse geometry).133–137 Introduction of the triple quadrupole conﬁguration in the early 1980s138 resulted in a rapid uptake of the MS/MS technique for a wide variety of analytical problems. Some of the reasons for the shift from sector instruments to quadrupole instruments had to do with size, complexity, and price of the hardware, as well as scan speed and simplicity of operation (either perceived or real). One factor that is generally overlooked is the signiﬁcant difference in the ability to fragment certain types and classes of ions when changing from the high-energy (kilovolt) regime accessible in sector instruments to the lowenergy (volt) regime of quadrupoles. While the fragmentation behavior for some compounds (e.g., peptides) was investigated and shown to be similar,139 small, stable ions (e.g., heterocyclic aromatic species) tend to be much less likely to yield complete structural information under low-energy conditions. Triple quadrupole instruments were also the ﬁrst to be used for ‘‘intelligent’’ or ‘‘data-dependent’’ acquisitions.140 Data-dependent acquisition takes

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advantage of rapid scanning and preset thresholds (or other parameters) to obtain various types of data without real-time intervention by the instrument operator. Operating on a predetermined set of parameters, the instrument switches between various acquisition modes based on the presence of particular signals that meet speciﬁed criteria. If an ion of interest is detected, the mass spectrometer switches in real time from full-scan mode to another scan mode. Thus, more extensive information can be obtained in fewer analyses. Introduction of the Paul quadrupole ion trap141 delivered new and different capabilities from those offered by triple quadrupole instruments. Excellent sensitivity (stemming from the accumulation of ions), faster scan speeds, the ability to directly establish the generational relationship between various fragmentation products through use of the so-called MSn scan mode(s), and full implementation of data-dependent acquisitions were some of the advantages offered by the device. This ability to perform more scans per unit time made ion traps the ﬁrst instruments capable of fully utilizing ‘‘intelligent’’ data acquisition. Modern separation techniques are producing shorter analysis times with proportionally narrower peaks. Not long ago, a typical HPLC peak was on the order of 15 or 20 seconds wide at its base meaning that the analyte eluting into the mass spectrometer source was present for about 20 seconds. Typical quadrupole instruments could scan a range of 1800 m/z units in approximately 1–2 sec. Therefore, the ‘‘data yield’’ for an average experiment was 10–15 mass spectra. Allowing for adding or averaging of spectra in order to improve the S/N, only 2–3 different experiments (e.g., normal mass spectrum in positive ion mode followed by normal mass spectrum in negative ion mode, or normal mass spectrum followed by one of the available MS/MS scans for one or two precursor ions of interest, etc.) could be performed on any given chromatographic peak for a single injection. If additional experiments were desired or required, another chromatographic analysis (a second injection followed by the waiting time until elution of the compound of interest) would need to be conducted in order to expand or augment the data set. The improved cycle times achieved by ion trap instruments increases the number of scans that can be accumulated during chromatographic elution, but not without limits. In fact, because additional generational scans (i.e., MSn) were required to acquire important structural information (vide supra) obtained in a single scan on a triple quadrupole instrument, gains in data accumulation rates because of lower cycle times (faster scans) were offset to some degree. The analytical utility and appeal of the quadrupole mass ﬁlters and ion traps is undeniable. However, one of the major attributes these devices have lacked (when compared to their predecessors, the double-focusing sector mass spectrometers) is the ability to resolve isobaric species and perform measurements of mass-to-charge ratio with high accuracy and precision. Aside from double, focusing magnetic/electric sectors instruments, the ion cyclotron resonance (ICR) spectrometer or Fourier transform (FT) mass spectrometer142,143 had been the only instrument capable of high mass resolving power and m/z measurements having sufﬁcient accuracy and precision to permit the determination of empirical formulas. These instruments have long been of interest in academic environments but have

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lagged commercially because of complexity and cost issues. Despite these drawbacks, the capability of the ICR spectrometer to carry out accurate m/z measurements for virtually any ion at very high resolving power is of tremendous utility in the characterization of unknown pharmaceutical compounds and has been demonstrated recently.144 The most recent addition to the list of mass analyzers capable of accurate m/z determinations (with their attendant correlation to the elemental composition of ions) is the TOF analyzer. The popularity of instruments based on TOF mass analysis145 stems from their ability to provide extremely rapid rates of data acquisition, excellent sensitivity (due to the lack of a scan function as well as the absence of physical constraints to the ion beam such as slits or apertures), and the ability to measure m/z values with sufﬁcient accuracy and precision for empirical formula determinations (i.e., accurate or exact mass measurements). The TOF analyzer is not a new instrument, but the reincarnation of technology introduced in the 1960s.146 Hindered at the time of its original introduction by a limited mass range (strictly speaking, m/z range) and modest mass resolving power, the instrument has reappeared because the advent of sophisticated high-speed digital electronics and the development of the reﬂectron ion mirror.147 Together, these two improvements have yielded an instrument with impressive capabilities. It should be pointed out that the description of modern TOF analyzers as being capable of high resolving power and accurate m/z measurements is relative. These instruments have demonstrated mass resolving powers ranging from 5000 to 20,000 and accuracies in the measurement of m/z values on the order of 3– 5 ppm. By comparison, double-focusing sector instruments (past and present) have been capable of similar or slightly better accuracies (perhaps 1– 3 ppm), but signiﬁcantly higher mass resolving powers (50,000 to > 100,000 in some cases). Yet even these impressive statistics pale in comparison to the capabilities of FT mass spectrometers, some of which can deliver sub-ppm mass accuracies with mass resolving powers on the order of 1,000,000 or higher. Together, high mass accuracy (with precision in the low ppm range) and the ability to uniquely measure a single ionic species (i.e., purity of the analyte ion population of interest is guaranteed by the resolving power) combine to provide a limited list of potential elemental formulas. This information can be extremely valuable in the characterization of unknown ion structures,148 which explains the reemergence of accurate mass determinations in the recent mass spectrometry literature. That being said, the utility of accurate m/z measurements diminishes with increasing mass of the analyte because the number of possible formulas having m/z values that differ by less than the uncertainty of the measurement increases geometrically. It is also important to mention that the execution of such experiments is far from trivial if reliable data are desired. Calibration procedures, internal standardization, and the requirement of appropriate signal levels (necessary to avoid peak skewing due to pulse pileup) all contribute to the complexity of the experiment. While the reemergence of TOF technology has resulted in widespread interest in accurate mass measurements, to date only the m/z values of precursor ions and ﬁrst generation fragment ions (i.e., MS and MS/MS, or MS2) have been measured using these devices. In this respect, the

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TOF instruments are similar to sector instruments because of the need to have n þ 1 analyzers for every n stage of structural interrogation (i.e., precursor selection, excitation, and product analysis).

VI. ION STRUCTURE INTERROGATION Beginning in the 1950s with chemical ionization and continuing to the present day (a progression that includes ﬁeld ionization,149 plasma desorption,150 fast-atom bombardment, thermospray, matrix-assisted laser desorption,151 electrospray, and atmospheric pressure chemical ionization), ‘‘soft’’ ionization methods have continually expanded the range, type, and size of molecules that can be analyzed by MS. The ability to ascertain the molecular weight of very large and very fragile molecules (presumably having structures that closely resembled their neutral precursors) has come as a consequence of the minimal amount of energy these techniques impart to newly formed ions. Mass spectra obtained using the techniques mentioned above generally exhibit few signals, which greatly limits their structural information content. In order to recover information about ion structure that traditional electron ionization had always provided, these ‘‘soft’’ ionization techniques were coupled with an activation event designed to increase the internal energy of the analyte ion. The ﬁrst, and still most prevalent, means of achieving this activation or deposition of internal energy is through the conversion of some portion of the analyte ion’s translational or kinetic energy. The simplest way to accomplish this desired translational/internal energy conversion is through glancing collisions with gaseous target atoms or molecules. Described initially over 30 years ago,152 collision-induced dissociation (CID) or collisionally activated decomposition (CAD) continues to be an important means for probing ion structures. Other techniques for energizing ions have been described, including the use of solid surfaces in lieu of gaseous targets (surface-induced dissociation, SID),153 the use of photon beams (photodissociation, PD)154 and the use of electron beams (electroninduced excitation in organics, EIEIO).155 However, none have gained the commercial acceptance of CID for conducting tandem mass spectrometric experiments. Ideally, an ion activation method would impart a desired (or controllable) quantity of energy in a desired or controllable time frame and the distribution or spread of energies imparted would be narrow. None of the methods available possess all of these characteristics. The precision with which the various methods deposit energy into ions generally is not high. Most of the methods produce an ion population that is best described as possessing a distribution of internal energies. This range of internal energies results in a diversity of fragmentation reactions, which is generally viewed as a beneﬁcial attribute when ion structure elucidation is the goal. Unfortunately, having a range of internal energies also allows for the possibility of isomerization (rearrangements) within the ion during the time between excitation and dissociation. These rearrangement processes and their resulting products are no different from those observed in EI spectra and are no less likely to confuse or complicate the interpretation of fragmentation

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spectra. The degree of fragmentation achievable in a CID MS/MS experiment is controllable (to the extent allowed by the intrinsic properties of the analyte molecule) in some instrumental conﬁgurations through the selection of the kinetic energy of the ions (i.e., collision energy), and the number (collision gas pressure) or type of targets that an ion encounters in the excitation region. The maximum achievable degree of fragmentation will be observed for ions that interact with a large number (high pressure) of massive target particles at relatively high kinetic energy. Due to drastic differences in operating pressures, experimental time scales, and the translational energies of ions as they traverse analyzers, the parameters of the CID experiment can be quite diverse and the terminology somewhat ambiguous. CID experiments intended to probe ion structure for the purpose of compound characterization or identiﬁcation should maximize fragmentation in terms of both the variety and abundance of ions produced. From the earliest days of MS, it was recognized that the yield and variety of dissociation processes could be enhanced by raising the internal pressure of the instrument.156 Until the popularization of the triple quadrupole mass spectrometer in the 1980s, most of the fundamental investigations into CID processes had been conducted at kilovolt energies using sector instruments. The use of reverse geometry instruments (magnetic sector preceding the electric sector) and the importance of collision gas identity and pressure were the contributions of early research efforts.157 The most common conditions for high-energy CID experiments included the use of helium as collision gas at a localized pressure sufﬁcient to diminish the intensity of the mass-selected precursor ion beam by approximately 30–70%. This degree of beam suppression correlated with multiple collision conditions and a maximum in the signal intensity versus collision gas pressure function for fragment ions.158 Multiple collision conditions are important for enabling access to higher energy processes, particularly when the precursor ions are formed by ionization events that are ‘‘soft’’ and impart very little internal energy. They result in consecutive dissociation reactions for one or more subsequent generations of fragment ions. The use of helium was determined to be optimal because energy deposition is reasonably efﬁcient and losses due to scattering or neutralization of fragment ions are minimal. Increasing the mass of the collision gas target by using nitrogen, argon, or some other gas was found to improve the energy deposition process only marginally while degrading transmission (i.e., increasing scattering or neutralization) unacceptably. A special case where using a collision gas other than helium was advantageous was the investigation of charge permutation reactions, which beneﬁted substantially from the use of oxygen.159 The sector instruments were not amenable to signiﬁcant adjustments in the translational energy of the ions. Even in cases where this was possible, little if any difference was observed in the appearance of fragmentation spectra as a function of collision energy. The situation is quite different for low-energy CID experiments. The effect of changing either translational energy, collision gas, or collision gas pressure can be quite dramatic. Trapping instruments have greater limitations than quadrupole mass ﬁlters with respect to the maximum kinetic energy they can impart to ions, the pressure of collision gas used, or which gases can be used.

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These shortcomings are overcome by the ability of these devices to subject the ions multiple times to the lower energy collisions occurring at lower pressures. Thus, restrictions posed by the need to maintain ions within the trap under stringent pressure requirements (either low pressures in the case of the ICR cell or a constant pressure of helium bath gas for the quadrupole ion trap) are offset by the long residence times of the ions within these analyzers. The linear beam-type instruments, such as the recently popularized quadrupole time-of-ﬂight (QTOF) or the triple quadrupole instrument, are more ﬂexible in terms of the upper limits of translational energy that can be used in CID experiments. The collision gas pressure can be varied in both of the quadrupole-based instruments for the purpose of achieving multiple collision conditions. However, much in the same way that too high a pressure was found to be detrimental in the high-energy regime, so too is excess pressure a liability in the low-energy regime of CID. Diminished intensity of fragment ion signals due to scattering losses is equally undesirable in these experiments. The merits of choosing a more massive gas target over a less massive target for CID experiments are manifested in the degree or extent of fragmentation that can be achieved. The upper limit for the amount of internal energy that can be imparted in a single collision event in the lowenergy regime (i.e., approximately 1–100 eV) is deﬁned by the following expression, Ecom ¼ Elab ðmt =mt þ mp Þ where Ecom is the energy of the ion in the center-of-mass frame of reference (related to the internal energy of the ion), Elab is the kinetic energy of the selected projectile ion in the laboratory frame of reference (i.e., the offset potential of the collision quadrupole in a triple quadrupole instrument), mt is the mass of the target atom or molecule and mp is the mass of the projectile (analyte) ion. The importance and impact of target mass are illustrated by the examples for singly charged ions shown in Table 2. Increasing the mass of the collision target causes the conversion of a larger portion of the projectile ion’s translational energy into internal energy, thus producing more structurally diagnostic fragment ions of higher abundance. The most likely amount of energy imparted by any single collision with a gaseous target is only some fraction of the theoretical limit. Nevertheless, the increase of internal energy as a function of target mass has clear utility in the CID of larger species (having many degrees of freedom among which the collision-induced excitation energy can be dissipated) or small ions having very stable structures. Figure 6 illustrates the effect of varying collision energy and the impact of target mass. The interpretive utility of a fragmentation spectrum is based on (a) the quantity of information available, which is reﬂected in the number and intensity of the signals in the spectrum and (b) establishing precursor/product (familial) relationships between ions represented by the signals in the spectrum. The derivation of an ion structure requires a fragmentation scheme of most probable structures for the various fragment ions (and neutral products that are eliminated in the fragmentation processes), as well as their relationships to one another. The dissociation processes responsible for the

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TABLE 2 Illustration of the Relationship Between Target Gas Mass and Energy Deposition in Collision-Induced Dissociation Reactions Elab (eV)

m*t

10

28 (N2)

100

2.2

10

28

500

0.5

10

28

900

0.3

40

28

100

8.8

40

28

500

2.1

40

28

900

1.2

10

131 (Xe)

100

5.7

10

131

500

2.1

10

131

900

1.3

40

131

100

22.7

40

131

500

8.3

40

131

900

5.1

mp

Ecom (eV)

*Increasing the mass of the collision target causes the conversion of a larger portion of the projectile ion’s translational energy into internal energy, thus producing more structurally diagnostic fragment ions of higher abundance. (see Figure 6).

SCHEME 1

formation of product ions in CID experiments are of one of two types, either consecutive or competitive, as illustrated in Scheme 1. Product-ion MS/MS spectra recorded using triple quadrupole or QTOF instruments are composite pictures of all fragmentation reactions (consecutive and competitive) captured in a single experiment. While information rich, these spectra can pose an interpretive challenge because of the ambiguous origins of various fragment ions. Additional information about some of the relationships that exist between ions can be obtained from subsequent experiments. One approach employs a sequential CID experiment in which a ﬁrst stage of CID is undertaken in the source region of the instrument (so-called in-source or skimmer/cone CID) and followed by the standard product-ion MS/MS experiment. Using this procedure, the fragmentation spectrum for each fragment ion observed in the original product-ion MS/MS spectrum of the analyte ion can be recorded. The precursor/product relationships between various fragment ions can be deduced upon compilation of such data sets. The success of this experiment depends on two critical factors: ﬁrst, that the fragment ions generated in the source CID

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FIGURE 6 Product-ion spectrum for the [MþH]þ ion (m/z 529) of a therapeutic azasteroid illustrating the dependence of structural information on both collision energy (translational energy of the analyte ion) and the mass of the target (identity of the collision gas). Top panelTranslational energy of the precursor ion was 40 eV (Elab, laboratory frame of reference) with xenon as collision gas. Middle panelTranslational energy of the precursor ion was 20 eV (Elab) with xenon as collision gas. Bottom panelTranslational energy of the precursor ion was 40 eV (Elab) with air as collision gas.

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stage of the experiment are identical in both mass and structure to the fragment ions observed in the product-ion MS/MS spectrum of the analyte ion; and second, that the intensities of the in-source CID product-ion signals are sufﬁcient to yield second-generation fragmentation spectra of adequate quality (S/N) to interpret. Another approach amenable to triple quadrupole but not QTOF instruments, is to record precursor-ion spectra for each fragment ion observed in the original product-ion spectrum of the analyte ion. Each of these spectra will display all of the precursor ions responsible for the fragments’ formation through either single or multiple step processes. Both of these approaches are time-consuming and labor-intensive. Product-ion MS/MS spectra for many compound classes obtained using trapping instruments tend to have a simpler appearance (i.e., fewer fragment signals) than comparable triple quadrupole or QTOF spectra. The minimal number of signals can be a hindrance to interpretation since only minimal insight can be gained into the ion structure being examined. Fortunately, this shortcoming can be overcome by utilizing a series of sequential CID experiments. These sequential experiments are made possible by the ion trap’s capacity to isolate and energize subsequent generations of precursor ions, each a fragment of a previously mass-selected precursor ion. However, this cascade of ionic dissociation products is not unending. Despite their very high efﬁciencies for trapping and fragmenting ions, the trapping instruments are constrained by certain practical limits. In general practice, there are few situations that require more than 4 or 5 stages of MS/MS (sometimes denoted as MS4 or MS5), and even these situations are rare. Ion traps generally scan more rapidly than quadrupole instruments, although specialized functions such as those required for increased mass resolving power160 or high mass analysis161 take considerably more time than a conventional analytical scan.

VII. DATA ACQUISITION AND INTERPRETATION The popularity of HPLC in pharmaceutical analysis is due not only to the wide variety of compounds to which it is amenable but also to the variety of detectors that can be used to obtain information about compounds once separated from one another. Several detectors can be employed simultaneously in order to boost both the amount of information and the rate at which it can be obtained. Each detector adds a new dimension of information to the resulting data set. Simple instruments use refractive index or single wavelength UV detectors to produce characteristic two-dimensional response traces for eluting compounds that plot intensity (absorption or refraction of light) versus time. Replacing the single wavelength detector with a diode array adds a third dimension to the data set, associating a complete absorption spectrum with each time/intensity data point. Introduction of a mass spectrometer, either in series or parallel, adds one or more additional dimensions of data, depending on the type of instrument chosen. Single analyzer instruments, be they quadrupole mass ﬁlters or TOF analyzers, add

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the fourth dimension to the data set—molecular mass. If tandem instruments such as triple quadrupole or QTOF instruments are used, a ﬁfth (and possibly a sixth) dimension are added to the data set—m/z values derived from a precursor m/z value. Finally, with the use of ion traps (either quadrupole ion traps or ion cyclotron resonance spectrometers), a sixth, seventh, eighth, or higher (in theory up to thirteenth, with currently available instruments capable of 10 stages of analysis) dimension of data is accessible for a single compound eluting from an HPLC column. However, visualization of ndimensional information (where n 4 or 5) poses challenges. Commercially available data-processing software deals effectively with three-dimensional data (either time/intensity at a speciﬁc UV wavelength coupled with diode array spectra or time/intensity for all masses coupled with complete mass spectra). The most common representation of chromatographic data when a mass spectrometer is incorporated into the detection scheme commonly is referred to as a total ion chromatogram or TIC. This trace correlates time (x-axis) with a summation of the abundances (y-axis) of all ions detected in the speciﬁed scan range (or acquisition range for nonscanning instruments). Because each data point in the TIC is composed of a complete mass spectrum, it is a trivial operation to produce the second most common and possibly the most useful data representation, the mass chromatogram or selected ion trace (also referred to as an extracted ion trace). This trace highlights one or more speciﬁc compounds by virtue of their unique molecular masses. The utility and value of mass chromatograms is realized in situations where the molecular weights of related substances can be predicted or inferred from other information. Any and all compounds having the requisite molecular mass (assuming that they ionize to produce an ion with the expected m/z value) will be differentiated from all other responses in the total ion chromatogram. Once the information is obtained, processed, and appropriately displayed in a meaningful fashion, the process of spectral interpretation can begin. Considerable information regarding compound class or the presence of structural analogs can be obtained from the UV absorption spectrum of HPLC peaks if diode array detection (DAD) is employed. Although the UV absorption spectrum of any particular compound may not provide speciﬁc information upon which a deﬁnitive identiﬁcation can be based, these spectra often make it possible to differentiate drug-related compounds from excipient compounds. This differentiation can be an important ﬁrst step in the characterization process because it can eliminate compounds of little or no importance. The molecular mass of an analyte compound is generally reﬂected in the m/z value of the most abundant ion in the mass spectrum (e.g., [MþH]þ for positive ESI or APCI and [MH] for negative ionization modes). This conclusion can be substantiated by the appearance of other ions such as adducts ([MþNa]þ, [MþK]þ, [MþHþCH3CN]þ, [MHþ HCO2H], [M-HþCF3CO2H], etc.) and dimer species ([MþHþM]þ, [Mþ NaþM]þ, [MHþM], etc.). Aside from the immediate information offered by the signals in a mass spectrum, that is m/z values for ions that represent the intact molecule or its fragments, there are also a number of more subtle details about the analyte that can be extracted upon closer examination.

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The observation of naturally occurring isotopes was one of the ﬁrst important accomplishments achieved using early mass spectrometers. Although isotopic patterns are often ignored, they can provide useful information. For compounds containing elements that have a distinct and recognizable distribution of isotopes, such as some halogens and most transition metals (among others), identiﬁcation or recognition of a compound can be immediate. The majority of molecules encountered in pharmaceutical analysis lack atoms that possess distinctive isotope patterns. However, at the early stages of some synthetic schemes it is not uncommon to encounter synthons that contain atoms such as chlorine, bromine, sulfur, and boron. The appearance of characteristic isotopic signals in a mass spectrum can also aid in the interpretation of fragmentation pathways. When tandem MS experiments are used to obtain structural information, the isotopic information is no less valuable, provided the necessary experimental conditions are used. One of the hallmarks of product-ion MS/MS spectra is the speciﬁcity of the information that comes from the selection of ions having a speciﬁc m/z value. This monoisotopic (at either unit or high resolving power) selection of precursor ions was initially an important element of the MS/MS experiment because there was no separation technique prior to ionization. The impact of monoisotopic precursor ion selection is a loss of the informative isotopic pattern. The lost information can be recovered through multiple experiments in which each isotopic signal is successively interrogated. The multiple fragmentation spectra are then compared in order to identify the mass shifts that indicate the presence of the isotope in a particular ionic fragment or its elimination in a dissociation process. Alternatively, when a separation technique such as HPLC is used to introduce a compound into the mass spectrometer, guaranteeing the homogeneity of the ion source contents, the importance of monoisotopic selection of precursor ions is reduced. Consequently, the resolving power of the ﬁrst analyzer (in a beam instrument) or ﬁrst stage of analysis (in a trapping instrument) can be relaxed so that ions of several m/z values are passed through to the activation stage. The integrity of the isotopic abundances that are ultimately reﬂected in the fragmentation spectrum may be less than accurate because of imperfections in the ﬁelds and functions responsible for discrimination between adjacent masses in the mass-selection stage. Despite less than perfect abundance ratios, the appearance and location of recognizable isotopic signals in a single product-ion MS/MS spectrum can provide valuable insight into fragmentation processes and ion structure. Direct interpretation of a mass spectral fragmentation pattern in the absence of other information about a molecule is challenging. This sometimes laborious process is carried out according to various interpretive rules based on fundamental thermodynamic and kinetic relationships (e.g., quasi-equilibrium162 or RRKM163 theory). The inherent complexity of the processes in combination with the large number of degrees of freedom for average-sized organic molecules makes interpretation or rationalization of anything but the major signals in a spectrum difﬁcult at best. As the population of unique chemical structures continually increases, the number of exceptions to fundamental rules becomes larger and larger making

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interpretation all the more difﬁcult. There has been some recent progress in the development of interpretive software applications based on fundamental rules and empirical observations.164 However, these computerized algorithms cannot interpret mass spectra completely and accurately. This means that many, if not most, fragmentation spectra are interpreted in a manual fashion. The large number of interpretive rules, the signiﬁcant number of possible exceptions to those rules and the increasing complexity of pharmaceutically relevant molecules has made pattern- or template-based interpretation a widely practiced approach. The tactic employs the parent drug molecule as a template upon which interpretation of unknown structures is based. The identiﬁcation of compounds from molecular weight information and the fragmentation products observed in the product-ion MS/MS spectrum depends on signiﬁcant (and characteristic) portions of the parent molecular structure being retained in the impurities, degradants, or metabolites.165–167 Differences in molecular weights of the intact molecules or the m/z values of the fragment ions reﬂect changes in the substructures leading to a proposed structure for the unknown compound. This paradigm has proven to be highly successful for the identiﬁcation of drug substance and drug product related substances for both conventional therapeutics168 and biotechnology products (proteins).169-171 From an interpretive perspective, maximum (or complete) information is obtained when the spectrum displays a large number of signals that are distributed at regular intervals across the m/z range. Unfortunately, in many cases the information provided by the mass spectral data is less than complete because of a general lack of interpretable signals in regions of the spectrum that would be indicative of important structural elements. Although the identity or meaning of a particular fragment ion can be quite speciﬁc, the information in many ways is similar to that obtained from vibrational spectroscopy in that it indicates the presence of a structural subunit or functional group, but not its speciﬁc location within the molecule or its spatial relationship to other components (atoms or other functional groups). For example, a difference in mass of 18 u between the protonated molecule ([MþH]þ) and an adjacent signal in a fragmentation spectrum indicates the presence of an –OH functional group in the molecule. However, the precise nature (e.g., carboxylic acid, alcohol, phenol, etc.) and location of the functional group is not evident from this spectral information. Similarly, signals indicating the loss of 42 u (ketene, CH2CO) from a protonated molecule or the appearance of a complementary ion with m/z 43 are clear indications of an acyl functional group, although the position of the substituent cannot be deduced from this information. The information present in mass spectra takes many forms. Because the unequivocal determination of structure by MS alone is an elusive and sometimes unattainable goal, in many circumstances it can be the more subtle information that contributes to the understanding of a molecule’s structure or identity. In situations where identiﬁcation is not possible, the mass spectral information can still contribute substantially to the characterization process by providing insight into the points of similarity (or dissimilarity) that the analyte shares with other known compounds or constituents of a given sample. This ability to immediately eliminate a compound (represented by a

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particular peak in a chromatogram) from further consideration is a critical contributory factor to optimum operational efﬁciency. Some of the subtler and less quantiﬁable kernels of information sometimes evident in mass spectra include characteristic isotopic abundances, stable solvation complexes, and multiply charged ions. For example, the presence of chlorine, bromine, sulfur, or other less common elements produces unique and recognizable isotope patterns in a mass spectrum because of abundant naturally occurring isotopes. While the more common elements comprising biologically relevant molecules contain unremarkable isotopic abundances, the relative abundance of the 13C signal (normalized to that of the 12C signal) for the parent ion of a compound provides an approximation of the number of carbon atoms in the molecule. This information can be used to constrain the list of possible elemental formulas generated from an accurate mass measurement or can be correlated with 13C NMR spectroscopic data and knowledge of the molecule’s chemistry to ascertain a structure. In fact, any one of these seemingly benign spectral features could augment other information about the sample (e.g., details about the synthetic scheme, features of the compound’s UV–VIS absorption, IR, Raman, or NMR spectrum, etc.) and result in the determination of compound identity.

VIII. APPLICATIONS The goal of chemical and pharmaceutical development efforts is to establish ﬁnal synthetic routes and effective formulations as rapidly as possible. These activities provide the materials necessary for safety evaluation (i.e., screening for adverse toxicological effects) and human clinical trials, as well as information leading to large-scale production of commercial medicines. All of the various processes must be optimized by the time the manufacturing batch sizes (typically many kilograms) for the clinical trial supplies are reached. This ensures that the analytical methods used for release testing of manufactured drug substance and drug product are capable of detecting and quantifying known or expected related substances that may be present (i.e., according to approved regulatory speciﬁcations). There are numerous examples detailing the contributions of MS to the various processes associated with the development of new pharmaceutical products. Qualitative information about related substances172–183 (see Figure 7) provides a useful baseline against which any future changes in the impurity proﬁle of a product can be compared. The existence of a knowledge base can facilitate the recognition of compounds that may have been encountered under various conditions during synthetic scale-up reactions. In cases where the speciﬁc compound has not been encountered, information about structural analogs generally results in rapid identiﬁcation of the new compound, along with recommendations for the prevention of its formation in the future. The structure elucidation of metabolites in many ways parallels the qualitative analysis efforts that are undertaken for the chemical and pharmaceutical aspects of the drug development process. The determination

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FIGURE 7 Representative data set for the characterization and structure elucidation of low-level related substances in an active pharmaceutical ingredient (API). Top panelMass chromatogram (ESI) representing the 1200 mass spectra (one per second) acquired throughout the HPLC separation. Mass spectra are acquired to determine nominal and/or exact molecular masses183 for all observed analyte ions. Subsequent CID experiments yield fragmentation (product-ion MS/MS) spectra that contain structural information about the ions of interest. Second panelCID product-ion spectrum of related substance A (m/z 132 for [MQH] Q). Third panelCID product-ion spectrum of related substance B (m/z 156 for [MQH]Q). Bottom panelCID product-ion spectrum of the API, peak C (m/z 315 for [MQH]Q).

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of structure enables a more complete understanding of metabolic pathways, which in turn contributes to knowledge about the potential pharmacological effects of the putative medicine. Realization that many biochemical transformations leave the core structure of the pharmacological agent largely intact has led to widespread use of a rational interpretive philosophy. Once the fragmentation spectra of the parent molecule and the metabolite have been compared, deduction of the site of structural modiﬁcation can be straightforward.184,185 As with any tandem mass spectrometric analysis, success requires analytes of interest to yield abundant signals (indicative of molecular weight) upon ionization and that these ions can be efﬁciently (and extensively) dissociated. Spectral interpretation reveals relationships between sites of metabolic modiﬁcation and systematic fragmentations (producing characteristic ions or neutral losses). Information about metabolic pathways is fed back into the ‘‘design stage’’ therapeutic molecules so that synthetic modiﬁcations can be made that will either increase efﬁcacy, decrease toxicity, or prolong the duration of action (by reducing the clearance rate). The characterization of compounds arising from biochemical processes is fraught with many of the same difﬁculties encountered during the characterization of compounds of chemical or pharmaceutical origin (i.e., related substances emanating from synthetic or formulation activities). However, the relatively limited set of potential biochemical reactions is an advantage in that it restricts the total number of potential structures that might reasonably be encountered. That being said, mass spectral characterization alone is incapable of complete and unequivocal structure determination. Conjugates frequently dissociate by eliminating the endogenous molecule, providing little or no indication as to its site of attachment to the drug molecule. Similarly, primary oxidation products rarely fragment sufﬁciently to shed any light on the identity of the speciﬁc substructure or atom that has undergone the biotransformation. The use of interpretive guides or templates (i.e., the product-ion CID spectra of the parent molecule and other structural analogs) can lead to conclusions regarding the portion of the molecule (e.g., oxidation of an aromatic ring to produce a phenol versus production of an N-oxide or sulfoxide). However, certain metabolic transformations will produce structures that fragment very differently from any of the ‘‘standards.’’ Complementary information from other spectroscopic techniques (primarily NMR spectroscopy) becomes critical for success in such cases.

IX. CONCLUSIONS The characterization (identiﬁcation or structure elucidation) of chemical compounds at trace levels in complex matrices is a crucial activity during many stages of the pharmaceutical development process. Complete and unequivocal determination of chemical structure is the product of a comprehensive and coordinated process that involves a variety of instrumental techniques and information from various sources (e.g., synthetic scheme, storage conditions, nature, and composition of excipients, published scientiﬁc literature, personal experience, etc.). The source, purity, and

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available quantity of sample material are important factors since they inﬂuence both data quality and interpretive certainty. Great strides have been made in the past several decades in the development of hybrid techniques that combine chromatography (or other separation techniques) with spectroscopic methods capable of providing insight into molecular structure. These approaches yield valuable information about compounds related to the API, some of which are present in the drug substance or ﬁnished product at very low levels. One of the primary tools that has emerged and become a mainstay in this work is HPLC coupled with tandem mass spectrometry (LC/MS/MS). This dynamically linked combination of two powerful analytical techniques was the logical successor to GC/MS and was ﬁrst highlighted in the 1980s. Since that time it has been adopted in many areas of chemical and biological research as an important means of analyzing (both quantitatively and qualitatively) a wide variety of complex samples not amenable to other techniques. However, hybridization of techniques always comes with compromise. For techniques such as LC/MS or LC/NMR the compromise is acquisition time or less than ideal matrices that degrade (if only slightly) spectral quality. Despite the utility and convenience of these online techniques, they do not eliminate the need for compound isolation. Expertise in separation science (on the preparative, semipreparative, and analytical scales), puriﬁcation, and sample handling is still a critical component of a successful structural characterization effort. Mass spectral characterization constitutes only a single facet of the pharmaceutical analysis paradigm. The ultimate importance and value of mass spectrometric characterization comes when it is integrated with other information, whether from complementary chemical and physical measurements or from knowledge of and experience with the chemistry at hand. Final conﬁrmation of identity comes only with independent synthesis of the compound suggested by the spectroscopic conclusions. The process of structure elucidation for low-level components of complex mixtures, such as those encountered in the pharmaceutical development process, has been aided tremendously by the availability and utilization of sophisticated instrumentation. However, the endeavor remains largely empirical in nature, requiring experience, dedication, perseverance, and attention to detail.

X. SUMMARY Mass spectrometry has had an increasingly signiﬁcant impact on the pharmaceutical development process over the past several decades. Advances in the design and efﬁciency of the interfaces that directly connect separation techniques with mass spectrometers have afforded new opportunities for monitoring, characterizing, and quantifying drug-related substances in APIs and pharmaceutical formulations. Possessing exceptional analytical speciﬁcity and sensitivity, MS dramatically reduces the cycle time of chromatographic method development, validation, and sample analysis. The popularity of LC/ MS/MS systems for complex mixture analysis of thermally labile, biologically relevant molecules is largely attributed to the ‘‘soft’’ nature of atmospheric

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pressure ionization techniques such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). This chapter describes the fundamentals of LC/MSbased techniques for ion structure analysis, including aspects of ion formation in API sources, attributes of various mass analyzers and scan modes used for collision-induced dissociation experiments, and issues surrounding the interpretation of the resulting mass spectra. Although the determination of chemical identity or molecular structure for related substances in pharmaceutical products has continuously beneﬁted from the availability and evolution of modern instrumentation, fundamental knowledge about solution phase chemistry, ionization, and gas-phase processes is still vitally important for achieving success in this endeavor.

ACKNOWLEDGMENTS The authors wish to acknowledge Dr. Kevin Facchine for his encouragement and support of this work.

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110. Olah, T. V., McLoughlin, D. A. and Gilbert, J. D. The simultaneous determination of mixtures of drug candidates by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry as an in vivo drug screening procedure. Rapid Commun. Mass Spectrom. 11:17–23, 1997. 111. Smith, R. D., Barinaga, C. J. and Udseth, H. R. Improved electrospray ionization interface for capillary zone electrophoresis-mass spectrometry Anal. Chem. 60, 1948–1952, 1988. 112. Moseley, M. A., Deterding, L. J., Tomer, K. B. and Jorgenson, J. W. Coupling of capillary zone electrophoresis and capillary liquid chromatography with coaxial continuous-flow fast atom bombardment tandem sector mass spectrometry. J. Chromatogr. 480, 197–209, 1989. 113. Lee, E. D., Mueck, W., Henion, J. D. and Covey, T. R. On-line capillary zone electrophoresis-ion spray mass spectrometry for the determination of dynorphins J. Chromatogr. 458:313–321, 1988. 114. Reinhoud, N. J., Niessen, W. M. A., Tjaden, U. R., Gramberg, L. G., Verheij, E. R. and van der Greef, J. Performance of a liquid-junction interface for capillary electrophoresis mass spectrometry using continuous-flow fast-atom bombardment Rapid Commun. Mass Spectrom. 3:348–351, 1989. 115. Gale, D. C. and Smith, R. D. Small volume and low flow-rate electrospray ionization mass spectrometry of aqueous samples. Rapid Commun. Mass Spectrom. 7, 1017–1021, 1993. 116. Chowdhury, S. K. and Chait, B. T. Method for the electrospray ionization of highly conductive aqueous solutions. Anal. Chem. 63:1660–1664, 1991. 117. Wahl, J. H. and Smith, R. D., Comparison of buffer systems and interface designs for capillary electrophoresis-mass spectrometry. J. Cap. Elec. 1:62–71, 1994. 118. Pramanik, B. N., Bartner, P. L., Mirza, U. A., Liu, Y.-H. and Ganguly, A. K. Electrospray ionization mass spectrometry for the study of non-covalent complexes: an emerging technology. J. Mass Spectrom. 33:911–920, 1998. 119. De la Mora, J. F., Van Berkel, G. J., Enke, C. G., Cole, R. B., Martinez-Sanchez, M. and Fenn, J. B. Electrochemical processes in electrospray ionization mass spectrometry. J. Mass Spectrom. 35:939–952, 2000. 120. Thurman, E. M., Ferrer, I. and Barcelo, D. Choosing between atmospheric pressure chemical ionization and electrospray ionization for the HPLC/MS analysis of pesticides. Anal. Chem. 73:5441–5449, 2001. 121. Mochida, Y., Yokoyama, Y. and Nakamura, S. Effect of ammonia addition to the mobile phase on atmospheric pressure chemical ionization mass spectrometry of methyl hydroperoxyoleates. J. Mass Spectrom. Soc. Japan 46:246–249, 1998. 122. Tong, W., Chowdhury, S. K., Chen, J. C., Zhong, R., Alton, K. B. and Patrick, J. E. Fragmentation of N-oxides (deoxygenation) in atmospheric pressure ionization: investigation of the activation process. Rapid Comm. Mass Spectrom. 15:2085–2090, 2001. 123. Burinsky, D. J., Williams, J. D., Thornquest, A. D. Jr. and Sides, S. L. Mass spectral fragmentation reactions of a therapeutic 4-azasteroid and related compounds. J. Am. Soc. Mass Spectrom. 12:385–398, 2001. 124. Robb, D. B., Covey, T. R. and Bruins, A. P. Atmospheric pressure photoionization: an ionization method for liquid chromatography-mass spectrometry. Anal. Chem. 72:3653– 3659, 2000. 125. Hanold, K. A., Evans, M. D., Fischer, S. M., Cormia, P. H. and Syage, J. A. Photoionization interface for LC/MS. Abstr. Pap.—Am. Chem. Soc. 221st ANYL-108, 2001. 126. Rauha, J.-P., Vuorela, H. and Kostiainen, R. Effect of eluent on the ionization efficiency of flavinoids by ion spray, atmospheric pressure chemical ionization, and atmospheric pressure photoionization mass spectrometry. J. Mass Spectrom. 36:1269–1280, 2001. 127. Kemp, D. L., Beynon, J. H. and Cooks, R. G. Charge stripping and E/2 mass spectra. Org. Mass Spectrom. 11:857–866, 1976. 128. Loo, J. A., Loo, R. R. O., Udseth, H. R., Edmonds, C. G. and Smith, R. D. Solvent-induced conformational changes of polypeptides probed by electrospray-ionization mass spectrometry. Rapid Commun. Mass Spectrom. 5:101–105, 1991. 129. McLafferty, F. W. (1980). Interpretation of Mass Spectra, 3rd ed., Chapter 4. University Science Books, Mill Valley.

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130. Deterding, L. J. and Gross, M. L. Tandem mass spectrometry for identifying fatty acid derivatives that undergo charge-remote fragmentations. Org. Mass Spectrom. 23:169–177, 1988. 131. Thomson, B. A., Douglas, D. J., Corr, J. J., Hager, J. W. and Jolliffe, C. L. Improved Collisionally Activated Dissociation Efficiency and Mass Resolution on a Triple Quadrupole Mass Spectrometer System. Anal. Chem. 67:1696–1704, 1995. 132. Williams, J. D., Cox, K. A., Cooks, R. G., Kaiser, R. E. Jr. and Schwartz, J. C. High massresolution using a quadrupole ion-trap mass spectrometer. Rapid Commun. Mass Spectrom. 5:327–329, 1991. 133. Jennings, K. R. Collision-induced decompositions of aromatic molecular ions. Int. J. Mass Spectrom. Ion Phys. 1:227–235, 1968. 134. Beynon, J. H., Morgan, R. P. and Brenton, A. G. New methods of identifying organic compounds. Philos. Trans. R. Soc. London, Ser. A 293:157–160, 1979. 135. Boyd, R. K., Porter, C. J. and Beynon, J. H. Linked-scan laws to detect fragmentations in the second field-free region of a double-focussing mass spectrometer. Int. J. Mass Spectrom. Ion Phys. 44:199–214, 1982. 136. Boyd, R. K., Porter, C. J. and Beynon, J. H. A new linked scan for reversed geometry mass spectrometers. Org. Mass Spectrom. 16:490–494, 1981. 137. Kemp, D. L., Cooks, R. G. and Beynon, J. H. Simulated mass-analyzed ion kinetic energy (MIKE) spectra from a conventional double focusing mass spectrometer. Int. J. Mass Spectrom. Ion Phys. 21:93–101, 1976. 138. Yost, R. A. and Enke, C. G. Selected ion fragmentation with a tandem quadrupole mass spectrometer. J. Am. Chem. Soc. 100:2274–2275, 1978. 139. Bean, M. F., Carr, S. A., Thorne, G. C., Reilly, M. H. and Gaskell, S. J. Tandem mass spectrometry of peptides using hybrid and four-sector instruments: a comparative study. Anal. Chem. 63:1473–1481, 1991. 140. Josephs, J. L. Detection and characterization of fumonisin mycotoxins by liquid chromatography/electrospray-ionization using ion trap and triple quadrupole mass spectrometry. Rapid Commun. Mass Spectrom. 10:1333–1344, 1996. 141. Stafford, G. C., Jr., Kelley, P. E., Syka, J. E. P., Reynolds, W. E. and Todd, J. F. J. Recent improvements in and analytical applications of advanced ion trap technology. Int. J. Mass Spectrom. Ion Processes. 60:85–98, 1984. 142. Baldeschwieler, J. D. Ion cyclotron resonance spectroscopy. Science. 159:263–273, 1968. 143. Comisarow, M. B. and Marshall, A. G. Fourier transform ion cyclotron resonance spectroscopy. Chem. Phys. Lett. 25:282–283, 1974. 144. Winger, B. E. and Kemp, C. A. J. Characterization of pharmaceutical compounds and related substances by using HPLC FTICR-MS and tandem mass spectrometry. Am. Pharm. Rev. 4:55–63, 2001. 145. Price, D. and Milnes, G. J. The renaissance of time-of-flight mass spectrometry. Int. J. Mass Spectrom. Ion Processes 99:1–39, 1990. 146. Gohlke, R. S. Time-of-flight mass spectrometry and gas-liquid partition chromatography. Anal. Chem. 31:535–541, 1959. 147. Mamyrin, B. A., Karataev, V. I., Shmikk, D. V. and Zagulin, V. A. Mass reflectron. New nonmagnetic time-of-flight high-resolution mass spectrometer. Zh. Eksp. Teor. Fiz. 64:82–89, 1973. 148. Beynon, J. H. Qualitative analysis of organic compounds by mass spectrometry. Nature 174:735–737, 1954. 149. Beckey, H. D. Field desorption mass spectrometry: a technique for the study of thermally unstable substances of low volatility. Int. J. Mass Spectrom. Ion Phys. 2:500–503, 1969. 150. Macfarlane, R. D. and Torgerson, D. F. Californium-252-plasma desorption time-of-flight mass spectrometry. Int. J. Mass Spectrom. Ion Phys. 21:81–92, 1976. 151. Karas, M., Bachmann, D., Bahr, U. and Hillenkamp, F. Matrix-assisted ultraviolet laser desorption of non-volatile compounds. Int. J. Mass Spectrom. Ion Processes 78:53–68, 1987. 152. McLafferty, F. W., Bente, P. F., III, Kornfeld, R., Tsai, S.-C. and Howe, I. Metastable ion characteristics XXII: Collisional activation spectra of organic ions. J. Amer. Chem. Soc. 95:2120–2129, 1973.

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153. Mabud, M. A., Dekrey, M. J. and Cooks, R. G. Surface-induced dissociation of molecular ions. Int. J. Mass Spectrom. Ion Processes 67:285–294, 1985. 154. Harris, F. M., Mukhtar, E. S., Griffiths, I. W. and Beynon, J. H. Design of a high-resolution mass spectrometer for studying the photodissociation of organic ions in the gas phase. Proc. R. Soc. London, Ser. A 374:461–473, 1981. 155. Cody, R. B. and Freiser, B. S. Electron impact excitation of ions from organics: an alternative to collision induced dissociation. Anal. Chem. 51:547–551, 1979. 156. Aston, F. W. A positive-ray spectrograph. Phil. Mag. 38:707–715, 1919. 157. Hemberger, P. H., Laramee, J. A., Hubik, A. R. and Cooks, R. G. Angle-resolved mass spectrometry. Target effects upon energy deposition. J. Phys. Chem. 85:2335–2340, 1981. 158. Glish, G. L. and Todd, P. J. Collision region for mass spectrometry/mass spectrometry. Anal. Chem. 54:842–843, 1982. 159. Ast, T., Porter, C. J., Proctor, C. J. and Beynon, J. H. Charge stripping reactions in mass spectrometry: ionization energies of some mono- and disubstituted benzene ions. Glas. Hem. Drus. Beograd 46:135–151, 1981. 160. Schwartz, J. C., Syka, J. E. P. and Jardine, I. High resolution on a quadrupole ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 2:198–204, 1991. 161. Kaiser, R. E., Jr., Cooks, R. G., Moss, J. and Hemberger, P. H. Mass range extension in a quadrupole ion-trap mass spectrometer. Rapid Commun. Mass Spectrom. 3:50–53, 1989. 162. Rosenstock, H. M., Wallenstein, M. B., Wahrhaftig, A. L. and Eyring, H. Absolute rate theory for isolated systems and the mass spectra of polyatomic molecules. Proc. Natl. Acad. Sci. U.S. 38:667–678, 1952. 163. Lin, S. H., Lau, K. H., Richardson, W., Volk, L. and Eyring, H. Stochastic model of unimolecular reactions and the RRKM [Rice-Ramsperger-Kassel-Marcus] theory. Proc. Nat. Acad. Sci. U.S. 69:2778–2782, 1972. 164. Hart, K. J., Palmer, P. T., Diedrich, D. L. and Enke, C. G. Generation of substructure identification rules using feature-combinations from tandem mass spectra. J. Am. Soc. Mass Spectrom. 3:159–168, 1992. 165. Kerns, E. H., Volk, K. J., Hill, S. E. and Lee, M. S. Profiling new taxanes using LC/MS and LC/MS/MS substructural analysis techniques. Rapid Commun. Mass Spectrom. 9:1539–1545, 1995. 166. Volk, K. J., Klohr, S. E., Rourick, R. A., Kerns, E. H. and Lee M. S. Profiling impurities and degradants of butorphanol tartrate using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry substructural techniques. J. Pharm. Biomed. Anal. 14:1663–1674, 1996. 167. Straub, K. M., Rudewicz, P. and Garvie, C. ‘Metabolic mapping’ of drugs: rapid screening techniques for xenobiotic metabolites with MS/MS techniques. Xenobiotica 17:413–422, 1987. 168. Tyczkowska, K. L., Voyksner, R. D. and Aronson, A. L. Solvent degradation of cloxacillin in vitro. Tentative identification of degradation products using thermospray liquid chromatography-mass spectrometry. J. Chromatogr. 594:195–201, 1992. 169. Pflumm, M. N., Gruber, S. C., Tsarbopoulos, A., Wylie, D., Pramanik, B., Bausch, J. N. and Patel, S. T. Isolation and characterization of an acetylated impurity in Escherichia coliderived recombinant human interleukin-10 (IL-10) drug substance. Pharm. Res. 14: 833–836, 1997. 170. DiPaolo, B., Pennetti, A., Nugent, L. and Venkat, K. Monitoring impurities in biopharmaceuticals produced by recombinant technology. Pharm. Sci. Technol. Today 2:70–82, 1999. 171. Oliva, A., Farina, J. and Llabres, M. Development of two high-performance liquid chromatographic methods for the analysis and characterization of insulin and its degradation products in pharmaceutical preparations. J. Chromatogr., B: Biomed. Sci. Appl. 749:25–34, 2000. 172. Eckers, C., Hutton, K. A., de Biasi, V., East, P. B., Haskins, N. J. and Jacewicz, V. W. Determination of clavam-2-carboxylate in clavulanate potassium and tablet material by liquid chromatography-tandem mass spectrometry. J. Chromatogr., A 686:213–218, 1994.

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12 NMR CHARACTERIZATION OF IMPURITIES LINDA L. LOHR, ANDREW J. JENSEN, AND THOMAS R. SHARP Pfizer, Inc.,Groton,CT 06340

I. II. III. IV. V.

VI.

VII. VIII.

IX. X.

INTRODUCTION TO NUCLEAR MAGNETIC RESONANCE (NMR) INFORMATION GATHERING SAMPLE PREPARATION FOR NMR SAMPLE PREPARATION FOR LC-NMR NMR INSTRUMENTATION A. General Hardware Considerations B. Probe Specifications NMR EXPERIMENTS A. Getting Started B. One-Dimensional Experiments C. Two-Dimensional Experiments CHOOSING AN EXPERIMENT SET DATA INTERPRETATION A. Primary Interpretation B. Secondary Interpretation C. Tertiary Interpretation: Spectral Simulations FINAL STEPS SUMMARY REFERENCES

I. INTRODUCTION TO NUCLEAR MAGNETIC RESONANCE (NMR) The ability of NMR to provide information regarding the speciﬁc bonding structure and stereochemistry within a molecule has created broad applicability across physics, chemistry, biology, and medicine.1–5 NMR provides a powerful analytical tool for structural elucidations. Unfortunately, NMR has traditionally been sensitivity-limited compared with other analytical techniques. Conventional sample requirements for NMR are on the order of 10 mg, as compared with mass spectroscopy, for example, which requires < 1 mg. Therefore, NMR spectroscopy historically has not been the ﬁrst approach for an analytical chemist when identifying an unknown compound. Technological advancements in the ﬁeld of magnetic resonance have made signiﬁcant strides in improving sensitivity levels.6–8 This is particularly important in the structural characterization of drug impurities and

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degradants, which often are available only in extremely limited quantities.9 The nondestructive, noninvasive nature of NMR spectroscopy makes it a valuable tool for the characterization of low-level impurities and degradants. In addition, NMR can be considered close to a ‘‘universal detector’’ for hydrogen and carbon, as well as for other magnetically active nuclei. This may or may not be considered advantageous, since all signals are detected — those arising from the compound of interest as well as all other components in the sample, such as solvent and starting materials. Quantitation is accurate over a dynamic range of nominally four orders of magnitude, although not as precise as other analytical tools particularly at low levels. This poses a challenge for studying mixtures containing low-level components. It is therefore preferred, if possible, to isolate a given impurity prior to study by NMR rather than to analyze a mixture. This also will dramatically simplify spectral interpretation. The following discussion focuses exclusively on what is termed ‘‘small molecules’’ in the industry, namely compounds with a molecular mass on the order of 1000 Da or less. The study of proteins, polymers, and other such ‘‘macromolecules’’ by NMR warrants an entirely different approach that is beyond the scope of this book. Similarly, we will restrict our discussion to liquid-state NMR spectroscopy, since solid-state NMR techniques are discussed elsewhere in this book (see Chapter 3). The general characterization strategy we will take for NMR is shown schematically in Figure 1. The ﬁrst step in the NMR characterization process is to determine sample purity, solubility, and quantity. This will enable one to assess the feasibility of an NMR evaluation, as well as estimate time frames for data acquisition. If any of these three criteria is low, then data acquisition times will be long, since only a limited amount of target sample will be in the detection region of the NMR spectrometer. Low sample purity will additionally complicate interpretation of the data. Depending on the importance of the structural elucidation, a practical upper limit for acquisition of mass limited NMR data is typically a weekend run. Longer acquisitions on unknown entities may bring sample stability issues into question. Acquisition times should be discussed with the project team to appropriately organize time allocations. If the degradant or impurity is proposed to be a previously characterized species, then simply matching onedimensional proton and carbon spectra to that of the known structure provides sufﬁcient conﬁrmation of structure. Otherwise, a full NMR analysis must be conducted. In this case, one must ﬁrst decide whether to run tubebased, nonﬂow NMR on an isolated sample or alternatively run LC-NMR (HPLC-NMR) on a sample mixture. NMR analysis of an isolated sample is typically more straightforward than that of a mixture. Most obviously, there are not competing signals from multiple components that may be difﬁcult to distinguish. In addition, there is probably not an overwhelming solvent signal, which commonly dominates an LC-NMR spectrum, even if solvent suppression techniques are employed. However, LC-NMR of a sample mixture may be the preferred route if the sample is not readily isolatable (see Chapter 13). This may also be the preferred approach if only a quick analysis is required. One may ﬁnd it valuable, for example, to ﬁrst perform an

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FIGURE 1 Flowchart for structural elucidation of impurities and degradants by NMR.

LC-NMR run and collect a 1-D proton spectrum, and then decide whether to isolate additional sample, based on the results. The next step is to identify an appropriate hardware conﬁguration. For tube-based NMR, one must select the appropriate NMR probe to match the coil size and frequency to the quantity and concentration of sample to be investigated. An appropriate set of experiments must also be determined. For LC-NMR, on the other hand, in addition to selecting an appropriate set of experiments, one must establish optimized chromatographic conditions for maximizing the chromatographic separation and the quantity of sample in the active NMR coil region. Once the data have been collected, one should compare the results to NMR spectra of any relevant standard samples, such as that of the parent or precursors. This will aid in the structural elucidation process, as will other supporting information such as mass spectral results and possible sources of the impurity or degradant. If appropriately selected, the collection of NMR spectra thus obtained should lead to one or at most just a few plausible structures. These then can be presented to the project team to determine synthetic plausibility and the next steps.

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This outlines a logical approach for making key decisions to move effectively and efﬁciently through the characterization process. Each of these steps will be discussed at greater length in the sections that follow. Alternative schemes may be employed, such as the order of procession for interpreting the NMR data. However, the overall logic should be the same as that outlined here, particularly regarding instrumentation and experiment selections.

II. INFORMATION GATHERING Arguably the single most important piece of non-NMR data used by the NMR spectroscopist is the total molecular mass. Although not required for structural elucidations, this information greatly facilitates the characterization process by reducing the number of possible structures. Certain mass changes are indicative of speciﬁc structural fragments. Furthermore, in many cases fragmentation patterns can identify which portion of the molecule differs from the parent species, thus pinpointing the degradation site. One should be careful, however, to remember the capabilities and limitations of each technique when identifying possible structures. For example, if LC-MS was performed, one should consider the possibility of co-elution of multiple components, especially stereoisomers. All of this information must be balanced with the NMR data when considering plausible structures. Another fruitful source for identifying possible structures comes from knowing the sample’s origin. Was the sample extracted from a mother liquor, isolated from a bulk lot, or synthesized from precursors? It is useful to know both the original synthetic route as well as the isolation pathway followed to prepare the sample. Not only will this provide clues of the possible structure based on predicted chemistry, it will also reveal any potential contaminants in the NMR sample. This will be important if the sample purity is low. Some examples of common contaminant sources include parent compound, precursors, catalysts, stereoisomers, excipients, extractables, and reaction vessel. Often little is known about the inherent properties of a degradant or impurity before it has been fully characterized. If the information is available, there are several properties, that are useful to know for proper handling of the NMR sample. First, it is essential to have an approximate idea regarding the quantity and purity of the sample, since this will determine which NMR hardware is most appropriate. This is commonly estimated using liquid chromatography. We will discuss speciﬁc quantity requirements in the ‘‘NMR Instrumentation’’ section, but for now, let’s say that approximately 1 mg of targeted compound is required. Of course, this value is directly related to the sample purity, since 1 mg of sample with a purity level of 50% would contain only 500 mg of targeted compound. For tube-based NMR, one would prefer a purity level of at least 80%, and 90% or greater should be the target for isolation. Sample mixtures containing components in approximately equal quantities are particularly challenging to investigate, since it is usually difﬁcult to discern which peaks correspond to which component. A 40:60 mixture, for instance, is actually preferred over a 50:50 mixture, since it is more straightforward to distinguish spectral contributions from each

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component. The quantity of sample observed by NMR also relates to the solubility in the selected solvent. Preliminary solubility tests therefore are important to identify an appropriate NMR solvent and hence maximize sensitivity. Choosing an amenable solvent system may also improve spectral resolution, since proton chemical shifts are solvent-dependent. Solubility in methanol and acetonitrile are often known beforehand based on chromatographic methods used for isolation. Related to purity is salt content. A large amount of salt in the sample, that is several times the amount of degradant, signiﬁcantly changes the magnetic susceptibility of the sample. In unfavorable cases, this may be beyond the tuning range of the probe and will therefore make it challenging or impossible to tune and match the probe. The effect on the resulting data is lower resolution, reduced sensitivity, and poor coherence selection, which is essential for indirect detection experiments. If information is available regarding stability, it is useful for appropriate sample handling. Possible sources of instability include light, heat, air, and stability in solution. Unfortunately, this information is usually not known a priori. Therefore, one should minimize risk by taking suitable precautions, as described in the sample preparation section. Since this is an unknown research sample, safety hazards are also unknown, so one should proceed with at least the same level of caution that applies for the parent species. The parent is often a useful starting point to assist in the interpretation of the NMR spectra of the unknown degradant or impurity. One-dimensional proton and carbon NMR spectra of the parent species, along with those of any high-level sample contaminants, are valuable references to expedite the data interpretation process. Ideally, because of the solvent dependency of proton chemical shifts, one should acquire reference proton spectra using the same solvent as for the target degradant or impurity. Unfortunately, this may not be possible because of solubility or stability limitations. Two-dimensional proton–carbon correlation spectra may also be necessary to make all resonance assignments. Additional information may come from a variety of sources. Certainly a proposed structure based on plausible chemistry and the total molecular mass is helpful. However, one must be careful to keep in mind that proposed structures are based on preliminary data only and thus may not be consistent with subsequently collected data. If LC-NMR is to be performed, then it is essential to obtain details of the chromatographic method to be used. Other information may include color to suggest conjugation, IR absorption to detect carbonyl stretches, and relative chromatographic retention time to evaluate polarity compared to the parent and other known compounds.

III. SAMPLE PREPARATION FOR NMR Deuterated solvents are used in NMR for two reasons. First, deuterium provides a signal on which to lock the spectrometer frequency, thus enhancing long-term stability. Second, it dramatically reduces the protonsignal that arises from the solvent, which would otherwise overwhelm the signal of the target compound. Solvents should be 99.9þ% deuterated. It is

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best to use 0.5–1.0 ml ampoules rather than larger solvent bottles. This ensures the integrity of the solvent and avoids contamination during sample preparation. It also prevents absorption of water and deuterium exchange with hydrogen in air over time. Some common deuterated solvents used for NMR studies include dimethyl sulfoxide (DMSO), chloroform, methanol, water, acetone, and acetonitrile. DMSO is considered by many to be a ‘‘universal solvent,’’ since it provides excellent solubility for a wide variety of compounds. In addition, its high boiling point makes it ideal for variable temperature experiments, which are useful for dynamics studies including rotamer investigations. However, because of this, it is nontrivial to remove DMSO from the sample, so it may be difﬁcult to retrieve the sample for subsequent non-NMR tests. It is also extremely viscous, so sample preparation and retrieval using small diameter tubes, including capillaries and submicro tubes, is not easy. One additional point to note is that DMSO absorbs water over time, so careful storage conditions are suggested. Protic solvents such as water and methanol provide proton sources for rapid exchange with labile protons. This makes NMR detection of species such as N–H and O–H protons impossible in these solvents. Also, some compounds, such as peroxides, may dissociate in protic solvents. When using water as the solvent, the pH may need to be adjusted to maximize solubility. Where possible, use deuterated compounds to adjust the pH to minimize undesirable background proton signals. In general, it is recommended to use the same solvent as used for the parent or other proposed impurity source to facilitate subsequent comparison of spectra. As we will see in our discussion of NMR instrumentation, the volume of solvent depends on the speciﬁc probe and tube size chosen. In general, one should minimize the total amount of solvent in order to concentrate the sample in the detection region and hence increase the observed signal. Concurrently, adequate solvent must be used so that the sample height is above the top of the coil to ensure proper ﬁeld homogeneity across the sample. Ideally the sample should be symmetric about the coil region for maximum ﬁeld homogeneity. A 1:2:1 ratio is a good rule of thumb for volume of sample below, in, and above the active coil region respectively. To minimize the degree of shimming required to achieve good resolution, ideally the sample height should be roughly equal to that of the standard NMR sample used to create the shim ﬁle. Otherwise, there may be a signiﬁcant difference in the shim values necessary to optimize the line shape. A standard proton sample used to measure resolution is 1% chloroform in deuterated acetone. To properly reference the spectra, tetramethylsilane (TMS) is often added as an internal reference standard. The chemical shift for the TMS resonance is set equal to 0.0 ppm for both protons and carbons. One must be extremely careful to add only a minuscule amount of TMS to the sample to avoid overwhelming the target signals. Typically, not more than vapor is added. Alternatively, one can use the solvent signal itself as an internal reference for chemical shift values. Clean glassware is essential for characterization of low-level impurities and degradants. Remember that one is often working with the ‘‘world’s supply’’ of the sample, so it is imperative to avoid contamination. For this

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reason, we recommend not reusing 5-mm NMR tubes for this purpose, but rather use a new tube for each impurity or degradant characterization. The justiﬁcation is that the price of a 5-mm NMR tube is much less than the value of the sample. Similarly, we recommend not reusing capillary or 1.7-mm tubes for the same reason. In addition, these ultra small diameter tubes are challenging to clean thoroughly. The cost of most 2.5–3.0 mm NMR tubes, however, is more substantial, so often these tubes are reused. For any of these sizes, a high quality tube should be selected. This ensures a high degree of tube colinearity and concentricity, which affects observed line shape. Tubes speciﬁcally designated by the vendor as disposable usually do not meet these quality standards. Special NMR tubes with plugs is also available that are susceptibility-matched to particular solvents.10 This can increase sensitivity, although the cost of these tubes and plugs is signiﬁcantly higher than for traditional glass tubes. These tubes are reused. Rinsing tubes with solvent prior to use washes away any residual contaminants that may be present. Similarly, an LC-NMR system, including the probe and any sample storage loops to be used, should be washed for several minutes prior to use. Disposable syringes are available for LC-NMR injections as an alternative to glass syringes, although one must be certain that the sample is inert to the plastic material of the syringe. Unless otherwise known, proceed with a level of safety precaution at least as rigorous as for the parent species. Samples should be provided in the smallest vessel possible, preferably with a conical bottom, to reduce loss. Once the sample has been transferred to the NMR tube or syringe, inspect the tube for any residual solids. Solids decrease both the sensitivity and the resolution achievable and may produce a broad hump in the spectral baseline. Finally, before placing the sample tube into the magnet or injecting into an LC-NMR system, be sure to eliminate any air bubbles, since bubbles will make shimming more challenging and will cause adverse effects in the LC-NMR system. Based on available stability information, keep the sample out of light by covering it completely with aluminum foil when not in the magnet. To avoid thermal instability, do not heat the sample above ambient conditions. Keep the sample capped or sealed to minimize exposure to air. Dissolve the sample in solvent only immediately prior to running NMR experiments to evaluate its stability in solvent. Run a standard proton spectrum immediately after making up the sample as a stability reference. Reacquire the proton spectrum following the ﬁnal NMR experiment to verify that sample integrity was maintained. If the sample needs to be stored for possible future use, it is recommended that it be stored under refrigerated conditions and protected from exposure to light. Reacquire the proton spectrum if any additional experiments are subsequently required.

IV. SAMPLE PREPARATION FOR LC-NMR As a technique requiring compatibility with both chromatographic and spectroscopic instrumentation, LC-NMR samples require special considerations beyond those described so far. For example, for the

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FIGURE 2

Calculation to determine the quantity of sample in the active volume of an LC-NMR flow cell. The conditions in this example result in less than the desired amount of sample in the coil, i.e., nominally one microgram. Note that some sample loss throughout the system may be observed. In this case, a smaller amount of sample will be present in the active region of the coil.

applications described here, NMR is not used quantitatively. However, in order to forecast feasibility of an LC-NMR approach, it is beneﬁcial to estimate sample quantity. Depending on the source of the sample and its stability, the sample can be a solid or liquid. A solid sample is convenient since an accurate weight can be obtained. If the sample is a liquid, it can be compared with a known solution to estimate an approximate concentration. Given the sample concentration, the amount of sample in the active volume of the probe can be calculated and then used to determine if LC-NMR will be a viable technique (see Figure 2). One microgram or more is desirable for a reasonable acquisition time. Sample handling requirements and safety considerations are analogous to nonﬂow NMR. The minimum amount of sample to target is 1 mg in the active volume of the coil. The concentration and conditions normally used for optimization of LC detection are not relevant for LC-NMR analysis since the sensitivity of NMR is much less in comparison. The optimization of LC conditions for NMR compatibility is extremely important. Normally a sample concentration of 0.6 – 2.0 mg/mL is used for LC detection. For LC-NMR analysis, this concentration needs to be as high as possible, potentially as high as 50 mg/mL depending on percent impurity level. Parallel to sample concentration is amount of sample injected. Attempt to inject the maximum amount possible. Optimize resolution and peak shape to maximize the amount of sample in the active volume of the probe. The impurity concentration occasionally can be increased either by using a mother liquor or other fraction of sample that may be enriched. Using other concentrating techniques, such as column trapping11 or solid-phase extraction (SPE),12 can also enrich the amount of impurity introduced to the NMR system. Finally, peak width is optimized to produce a peak that is as narrow as possible yet ﬁlls the active volume of the ﬂow cell. This may involve the use of different columns or changing

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mobile-phase conditions such that the peak of interest elutes as soon as possible, since peak width is normally narrower for early eluting peaks. A variety of solvents and buffers or modiﬁers can be used for LC-NMR. However, one must bear in mind that protonated species used in the chromatographic separation will be observed in the NMR spectrum. The exception is when using sample concentrating techniques such as solid-phase extraction. If LC-NMR-MS13 is used, then any required buffers must be compatible with all three techniques. Examples include formate and acetate buffers. If possible, use deuterated solvents and buffers or modiﬁers to reduce unwanted proton resonances in the NMR spectrum. Deuterium oxide and protonated acetonitrile have historically been used as mobile phase constituents since deuterated acetonitrile is usually much more expensive than LC-NMR–grade protonated acetonitrile. Alternatively, deuterated methanol may be used, but it is even more expensive than deuterated acetonitrile. In addition, methanol is not recommended for LC-NMR applications since it is retained in the tubing and leaches out over an extended period of time. This causes very noticeable peaks in the proton spectrum. Additionally, non-salt buffers are preferred since organic solvents may evaporate over time during analysis, resulting in the buffer’s potentially salting out and clogging the ﬂow probe. Since protonated acetonitrile has a proton resonance that saturates the dynamic range of the NMR receiver, the signal must be reduced by solvent suppression techniques. Examples include presaturation,14 WET,15 and other shaped pulse sequences. Since there is often residual protonated HOD in D2O, this resonance is also reduced by solvent suppression. The primary disadvantage of solvent suppression is that it dramatically distorts the spectrum at the solvent chemical shift, thereby obscuring any target signals in these regions. When setting solvent suppression parameters, it is best to reduce the residual acetonitrile and water resonances to an intensity below the 13C satellite signals of acetonitrile. Another option is to use all deuterated solvents, thereby eliminating the need for solvent suppression. By using deuterated rather than protonated acetonitrile, the capacity of the receiver is effectively increased by a factor of 10. The primary disadvantage of using fully deuterated solvents is cost. One should also be aware of potential retention time shifts because of deuterated solvents. Comparison of retention time and peak area should provide conﬁdence that the peak of interest is tracked properly. If the peak of interest cannot be easily identiﬁed using LC-NMR conditions, then LC-NMR-MS can alternatively be used to track peaks.

V. NMR INSTRUMENTATION A. General Hardware Considerations The three main sample factors that determine the appropriate selection of NMR equipment are quantity, solubility, and stability. The quantity of sample determines the detection limits required. Because we are usually

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sample-limited, it is important to identify the NMR probe that can accommodate the maximum quantity of sample without unnecessarily diluting the sample in excessive solvent. This then relates to the sample’s solubility in the selected solvent. Obviously one should choose a solvent that yields high solubility to maximize the amount of sample in solution. Stability is important since we wish to ensure that the sample does not change over the course of acquiring the data. For unstable compounds, it may be necessary to separate additional degradants from the target species. For this purpose, chromatographic techniques can be combined with traditional NMR techniques.13,16–18 Regardless of individual sample characteristics, there are several general instrumentation guidelines to perform the experiments described here. Mass and solubility limitations, combined with complex mixtures and the need for rapid problem-solving, demand high-end instrumentation, including a highﬁeld spectrometer with a fast CPU, lots of computer memory, and an assortment of probes or a cryoprobe. Antivibration legs reduce spectral noise that arises from ﬂoor vibrations. This is essential at high ﬁeld strengths, 400 MHz and above. Similarly, temperature control minimizes thermal instabilities, which may produce unwanted ridges in 2-D data sets, particularly during long acquisitions. System temperature control is also useful for certain experiments such as the investigation of rotamers. A minimum magnetic ﬁeld strength of 400 MHz is recommended to achieve the resolution and sensitivity necessary for reliable low-level impurity characterizations. The experiments described here are for protons and carbons, so two radio frequency (RF) channels optimized for these frequencies are required in addition to the deuterium lock channel. One channel is dedicated to protons, while the other (sometimes designated ‘‘X’’) channel covers a broad range of lower frequencies that include carbon. A single Z-axis gradient channel is recommended both for solvent suppression and to signiﬁcantly improve coherence selection and hence the overall spectral quality of 2-D experiments. A deuterium switch for deuterium gradient shimming is also desirable for fast, reliable shimming. Another desirable capability is LC-NMR, which combines the mixture separation capabilities of liquid chromatography with the structural elucidation capabilities of NMR.13,16,18 In order to perform LC-NMR experiments, one must have both the LC accessories for the NMR console and an LC-NMR probe. The LC accessory typically consists of an LC system with either a variable wavelength UV detector or a diode array detector. It therefore requires a UV active chromophore for the targeted compound to be detected. An optional loop collector allows individual fractions to be stored for future NMR experiments. Individual fractions are sent to the LC-NMR probe, and NMR experiments that employ solvent suppression are performed. Currently, only LC-NMR probes optimized for proton detection are available since the sensitivity prohibits reasonable carbon detection. If using protonated solvents, single or double solvent suppression techniques must be incorporated into the experiments. Gradient capabilities are required for some solvent suppression techniques. Sample sizes range from hundreds of micrograms down to hundreds of nanograms. LC-NMR is suitable for

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compounds that are difﬁcult to isolate or are unstable for prolonged periods when isolated. LC-NMR is also useful for initial NMR screening to determine if subsequent sample isolation is necessary. When preparing to purchase an LC-NMR accessory, one must make a number of choices. They include the following questions: Is mixture analysis required, or can samples be isolated for use with microprobes? What sample quantities will be routinely studied? If isolated samples will be available in greater than 10-mg quantities, then tube-based NMR will probably be a more efﬁcient route for structural elucidations. Will fully deuterated solvents be used for the chromatographic separations? Deuterated acetonitrile can be quite costly. Often deuterated water and protonated acetonitrile are thus used to create the mobile phase for LC-NMR. However, this approach produces a sizeable acetonitrile signal in the proton NMR spectrum, so solvent suppression techniques must be employed. Unfortunately, detection of signals in this spectral region is obscured. It is therefore desirable to use deuterated solvents for the structural elucidation of impurities and degradants. Should mass spectrometry (MS) or solid-phase extraction (SPE) capabilities be added? Combining MS with an LC-NMR system provides mass information and facilitates chromatographic peak tracking. An SPE module is used to concentrate the chromatographic peak of interest in the active region of the NMR coil. Alternatively, column trapping techniques can be employed for a similar effect. Are single or triple axis gradients required? Triple axis gradients occasionally provide improved solvent suppression. However, a triple axis gradient probe typically has reduced sensitivity performance compared with an equivalent single axis gradient probe. The HPLC system contains the following components: degasser, binary or quaternary pump, ultraviolet detector, auto or manual injector, column oven, and loop collection (see Figure 3). A degasser is required to purge the system so gases do not cause bubbles in the tubing that can collect on the pump head or at the detector. A pump should be selected appropriate for the HPLC methods used. Signals of protonated solvents, buffers, and modiﬁers will be observed in the NMR spectrum. The exception to this is when SPE or column trapping is employed. An SPE unit contains cartridges used to concentrate individual sample fractions, then evaporate solvents, and ﬁnally elute the fraction off the cartridge with the desired deuterated solvent. This approach provides nontrivial operating cost savings since protonated solvents can be used for the primary chromatographic separation, and then a much smaller amount of deuterated solvent can be used after sample concentration prior to introduction into the NMR probe. The UV detector must satisfy any chromatographic method requirements. Variable wavelength (VW) or diode array detectors (DAD) are commonly employed for this application. There are three main modes of operation for LC-NMR: on-ﬂow, peak collection, and stopped ﬂow. On-ﬂow LC-NMR involves acquisition of realtime NMR scans during the chromatographic run, but requires high sample concentration since only a small number of scans can be averaged for each NMR spectrum. Stopped-ﬂow LC-NMR is normally used when a single peak of interest is to be analyzed. The peak of interest is selected either manually, by region of time, or by intensity threshold. In stopped-ﬂow mode, the LC

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FIGURE 3

LC-NMR hardware configuration.

pump is physically stopped when the component is in the active volume of the probe. The selected sample fraction is then analyzed by NMR for a duration of time. Peak collection involves an uninterrupted LC run, where the peaks of interest are sent to individual sample storage loops. Then, at the operator’s convenience, each fraction is sent to the NMR probe to be analyzed separately.

B. Probe Specifications Regarding probe speciﬁcations, if the console is equipped with gradient, capabilities then the NMR probe requires a corresponding gradient coil to accommodate it. A single z-axis gradient should sufﬁce for these applications. Triple-axis gradients may be used for solvent suppression, although typically at a slight sensitivity loss compared with equivalent single-axis gradient conﬁgurations. The strength of the gradient is nominally 60 Gauss/cm. The probe must also have at least two RF channels, one for protons and one for carbons or X nuclei, in addition to the deuterium channel used to lock the signal. Probes are often designed such that one coil is concentrically inside the other. The inner coil yields the highest sensitivity since it is closest to the sample. Therefore, for proton detection experiments, one should choose a probe with the proton coil as the inner coil. This is called a broadband inverse probe, or an H–X probe. Alternatively, for optimal carbon detection, one should designate the X coil to be the inner coil, which is called a

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broadband observe probe. If the X channel is limited to carbon detection only, the probe is called a dual probe. When dealing with mass limited samples, carbon detection is often not feasible in a reasonable amount of time. In this case, one should use an inverse probe. If sample quantity and solubility are not limited, then one may decide to spend the extra acquisition time necessary to collect a carbon spectrum. This may be desirable to conﬁrm the number and type of carbons present, especially for quaternary carbons. For these experiments, a broadband or dual probe should be used. The ﬁeld strength of the probe must match that of the magnet. At least 400 MHz is recommended. Several probe sizes are commercially available, and if one intends to do impurity and degradant characterizations routinely, it is important to have a representative range of sizes. The probe size is expressed in terms of the diameter of the sample tube for which it is designed. A common size is a 5-mm probe. This is an appropriate choice if one is not limited by sample quantity. The sample size is ideally 25–50 mg in 0.5–0.75 mL of solvent, although as little as 1 mg of material can be used. Obviously, increasing the sample concentration will reduce the experiment time required to obtain adequate signal-to-noise ratios. If the amount of sample is limited, a good alternative is the family of ‘‘microprobes.’’ Depending on the vendor, microprobes are available in either 2.5- or 3.0-mm sizes. The sample volume is approximately 200 mL. This is suitable for samples on the order of one milligram down to hundreds of micrograms. For smaller sample quantities, on the order of tens to hundreds of micrograms, a ‘‘submicro-probe’’ is available.6–8 This probe, also known as a ‘‘SMIDGE’’ probe, is an inverse detection probe — in other words, optimized for proton detection. The tube diameter is 1.7 mm and has an active volume of approximately 30 mL. Similarly, 1.0-mm diameter capillary tube probes have been used for even smaller sample volumes.19 It is important to note that decreasing the sample tube size effectively concentrates the sample by reducing the amount of solvent required. Concentrating the sample in the active coil region increases the number of spins detected and thus increases observed signal-to-noise ratios in the NMR spectra. Going to smaller tube sizes, therefore, will yield no signiﬁcant advantage if there are solubility limitations preventing an increase in concentration. In the case of very poor solubility, the standard 5-mm probe affords the greatest ease of use. LC-NMR requires use of a special probe that contains a ﬂow cell rather than using a conventional tube. Samples ﬂow into the ﬂow cell, halt for extended acquisitions, and then ﬂow out of the probe to either waste or a fraction collector. The LC-NMR probe is interfaced to the HPLC, using standard chromatographic tubing. LC-NMR ﬂow cells are available in various sizes covering the range of 30–240 ml. Cryogen-cooled tube and ﬂow NMR probes offer a signiﬁcant sensitivity enhancement. In the NMR industry, these are called ‘‘cryoprobes’’12 or ‘‘chiliprobes,’’20,21 depending on the vendor. While the sample remains at ambient conditions, the probe electronics are cooled to cryogenic temperatures to reduce therma noise and, hence, boost observed signal-to-noise

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ratios. In favorable cases, sensitivity enhancements of up to fourfold have been reported.12, 20 Clearly this is extremely advantageous for the characterization of low-level impurities and degradants. As with conventional NMR probes, cryogenic probes come in several possible conﬁgurations. When selecting a probe of this type, one needs to decide on sample size, inverse or dual detection, and tube or ﬂow conﬁgurations. The same considerations apply here as for conventional probes. One additional limitation of a cryogenic probe is the potential difﬁculty in removal from the magnet bore. This is because of the time required for warming up the probe as well as to the sheer weight of the probe. Similarly, when installing the probe, one must also allow sufﬁcient time to install and cool down the probe and establish thermal equilibrium. Additional supporting hardware and software may also be required for cryogenic probe use. For these reasons, it is common to dedicate an NMR spectrometer to cryogenic probe use and not to switch probes as one would do for a conventional conﬁguration.

VI. NMR EXPERIMENTS A. Getting Started Once the sample is loaded into the magnet, set the temperature control and wait for thermal equilibrium to be established. Depending on sample size, approximately 15 minutes should sufﬁce. If the sample does not adequately tune or match, there may be an excessive amount of residual salt present, in which case the sample must be further puriﬁed. This may also be exempliﬁed by an unexpected broadening of all observed proton resonances. Spinning the sample improves resolution by reducing ﬁeld inhomogeneities in the x–y plane. However, instabilities are introduced by the spinning itself, so one should never spin the sample for any long-term experiments, such as nOe measurements or 2-D experiments. The resolution improvement is trivial compared to the line width of carbon signals, so there is no advantage in spinning the sample when acquiring carbon-detected data. The single case, therefore, when one may want to spin the sample is for the simple 1-D proton experiment. However, it is important to remember that spinning sideband signals arise in the proton spectrum at multiples of the spinning speed on either side of each observed resonance.22 The intensity of sideband signals depends on the quality of shimming. Poor shimming gives rise to more intense sideband signals. This may confuse the interpretation of the data, particularly when deciphering low-level contributions to the spectrum. We therefore recommend running all experiments described here without spinning the sample for ease of data interpretation and for optimal stability. Deuterium gradient shimming, or proton gradient shimming if running LC-NMR with protonated solvents, yields reasonable shim values for optimal line shape and resolution. Depending on the amount of available sample, one should allow between 8 and 48 hours to acquire and process a standard set of experiments.

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B. One-Dimensional Experiments The most basic NMR experiment is the one-pulse proton experiment.23–25 Proton chemical shifts typically range from 0 to 10 ppm, so the spectral width should be set at least this large. A good approach is to set the spectral width to a larger value, such as 15 ppm, to identify the actual limits of the resonances observed for a given sample. Then the spectral width can be reset to a smaller value speciﬁc to the sample. Acquisition parameter values determined for the 1-D proton spectrum can be used as a guideline for other proton-detected experiments, including the proton dimension of twodimensional experiments. Integrating each of the observed proton resonances is useful for subsequent data interpretation. When the proton spectrum is integrated, one can compare integral values of the various resonances to estimate sample purity and determine if further puriﬁcation is necessary. Ideally, there should be no more than roughly 20% contaminants in the sample. Feasibility of 2-D acquisitions and ‘‘carbon observe’’ experiments can also be assessed, based on the sensitivity and resolution obtained from the proton spectrum. It is good practice to acquire a proton spectrum at the start and end of data collection. By comparing the two resulting spectra, one can conﬁrm if sample integrity was maintained over the course of the experiments. The standard carbon experiment decouples protons from carbons to enhance sensitivity.26 In the absence of proton decoupling, each carbon resonance is split by one more than the number of protons attached to that carbon. Since, unlike for protons, the natural abundance of carbon-13 is only 1.1%, it is critical to maximize sensitivity in order to detect carbon in a reasonable amount of time.27,28 Usually protons are the only nuclei that are decoupled in a standard carbon experiment. Other spin-1/2 nuclei, such as ﬂuorine, are not decoupled and thus split each carbon resonance by one more than the number of attached ﬂuorines (for example, see Figure 4). This can be particularly advantageous for identifying ﬂuorinated carbon entities. In order to simultaneously decouple both protons and ﬂuorines, an additional RF channel is necessary. Since carbon signals are often too weak to observe in a small number of scans, one needs to make a reasonable approximation for the spectral width. A standard carbon spectrum covers the range of 0 to 200 ppm. Because of the relatively long T1 relaxation time of carbons, common practice is to use less than a full 90 pulse, such as a 30 pulse, and a recycle time of 0.2 s. A line broadening of one Hz improves sensitivity without signiﬁcantly reducing resolution. Acquisition parameter values determined for the 1-D carbon spectrum can be used as a guideline for other carbon experiments as well, including the carbon dimension of 2-D experiments. Because of the inherently poor sensitivity, the acquisition of a carbon spectrum should be considered optional. Deciding whether to collect carbon data depends on the expected sensitivity based on the proton spectrum, the anticipated sample stability over the duration of the experiment, the required degree of carbon resolution necessary, and the importance of directly detecting quaternary carbons. Alternatively, the carbon resonances can be

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FIGURE 4

One-dimensional carbon spectrum showing splitting due to a tri-fluoro group. The effected resonances appear as quartets due to splitting by three equivalent spin-1/2 nuclei. The size of the coupling constant increases dramatically with proximity to the fluorines.This greatly facilitates the assignment of carbon resonances.

obtained in a fraction of the time indirectly from projections of 2-D heteronuclear spectra. However, 2-D projections exhibit poorer signal-tonoise ratio because of fewer scans, and poor resolution than for direct detection. In addition, some quaternary carbons may not appear in projections because of the presence of neighboring heteroatoms. The DEPT experiment, or distortionless enhanced polarization transfer, is a carbon selectivity experiment.29–35 Based on the pulse length selected, one

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can selectively observe different types of carbon entities depending on the number of attached protons. We recommend setting the DEPT proton pulse length to 135 degrees. In this case, quaternary carbons are suppressed, methylenes are inverted, and methine and methyl carbons appear upright in the phased spectrum. Methines and methyls are then distinguished based on chemical shift differences and 2-D proton correlations. Methines usually appear downﬁeld of methyls. If desired, the entire series of DEPT experiments can be performed to conclusively distinguish methine from methyl resonances. Similar experiments to the DEPT include APT36 and PENDANT,37 among others. APT affords the advantage over DEPT of detecting all carbon entities including quaternaries. However, it has lower sensitivity. PENDANT, on the other hand, incorporates both the observation of all carbon entities and an nOe enhancement. However, there is no phase cycle associated with this experiment, and hence phasing can occasionally be problematic. The same considerations apply as for the standard carbon experiment when deciding whether to run one of these optional carbon observe experiments. This information generally can be obtained by close analysis of the HSQC (or equivalent HMQC) spectrum, as we will see in the next section. However, overlapped proton signals may make conclusive assignment of carbon type challenging for some carbon resonances. Alternatively, as discussed in the following section, one can indirectly obtain carbon multiplicity information from a DEPT-HSQC experiment. Although lower in carbon spectral resolution, this approach affords a signiﬁcant saving in acquisition time, compared with direct carbon detection.

C. Two-Dimensional Experiments 1. Introduction While much information can be gleaned from simple 1-D experiments, a wealth of additional chemical structure information can be obtained from 2-D experiments. The general purpose of expanding to higher dimensions is to establish correlations either between the same nuclear type, known as ‘‘homonuclear’’ experiments, or between different types of nuclei, known as ‘‘heteronuclear’’ experiments. In either case, either short- or long-range interactions can be investigated. In addition to through-bond connectivities, spatial relationships can also be studied. Two-dimensional spectra are commonly displayed as contour plots with either the spectral projections or the corresponding 1-D spectra shown along the axes. For the 2-D experiments described here, the spectral width of the proton dimension should be equal to that determined for the 1-D proton spectrum, and the spectral width of the carbon dimension should be equal to that determined for the 1-D carbon spectrum. If the 1-D carbon spectral width has not yet been determined, then 0 – 200 ppm is a reasonable default range. A good compromise of data set size versus adequate resolution is 2048 points in the t2 dimension and 512 points in the t1 dimension. This data set size is appropriate for all the 2-D experiments described in this section. Linear prediction can be used to enhance the apparent spectral resolution of truncated data.

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There are several general considerations for identifying appropriate 2-D experiments to use for structural elucidation. First, most of the experiments described here are available both in gradient and nongradient versions. Gradients are used in these experiments to improve coherence selection that is otherwise performed using elaborate phase cycle schemes.38,39 It is therefore possible to achieve improved signal selectivity and reduced spectral artifacts with fewer acquisitions using gradient selection. The primary disadvantage of gradient coherence selection is reduced sensitivity. Therefore, if the low quantity of sample available requires a large number of scans for sensitivity reasons, one may prefer to use nongradient versions of the experiments described and instead rely on phase cycling for signal selectivity. On the topic of sensitivity, the 2-D experiments described here are all proton-detected experiments. The reason is that protons are naturally abundant and have a high gyromagnetic ratio — and hence are easily detected — while 13C nuclei have low natural abundance and gyromagnetic ratio, and thus are more challenging to detect. Experiments that traditionally were based on carbon detection, thus often have modern equivalent versions that transfer the carbon magnetization to protons and detect the corresponding proton signal. This is known as ‘‘inverse detection’’ or ‘‘indirect detection,’’ since the carbon signal is indirectly detected via its effect on the observed proton signal.40–43 Inverse detection experiments are particularly valuable for the characterization of impurities and degradants, since sample quantity is often quite limited. 2. HomonuclearTwo-Dimensional Experiments The experiment traditionally used to detect proton–proton scalar couplings is known as a COSY38,44 for correlation spectroscopy. The 1-D proton spectrum is shown along each of the two axes, and off-diagonal peaks show through-bond correlations between neighboring proton groups. Each unique proton group also exhibits a correlation to itself, which appears along the diagonal axis of the spectrum. These self-correlation peaks do not provide any additional information, and depending on resolution limitations, may obscure cross peaks near the diagonal. It may therefore be advantageous to add a ‘‘double quantum ﬁlter’’ to the COSY experiment to reduce contributions along the diagonal. The double quantum ﬁltered COSY, or DQFCOSY,45–47 provides the same information as a standard COSY with improved resolution near the diagonal. COSY-type experiments are useful for showing through-bond connectivity within individual spin systems. However, they cannot provide information to link spin systems separated by nonprotonated groups. COSY-type experiments target geminal or vicinal spin coupling between protons. For longer range proton correlations, the TOCSY is often beneﬁcial.48,49 This twist on the traditional COSY experiment shows through-bond correlations across an entire protonated chain, hence the name ‘‘total correlation spectroscopy.’’ When long spin-lock times are used, spatial correlation artifacts due to ROESY transfers (see ‘‘Additional Experiments’’ section of this chapter) may appear. However, these are easily distinguished, since they are anti-phase to TOCSY correlation peaks. Because of

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the additional observed resonances, TOCSY spectra are more complex to interpret than COSY spectra. As we will see in the next section, TOCSY spectra provide information analogous to heteronuclear HMBC spectra. TOCSY has a signiﬁcant sensitivity advantage over HMBC, since it is a proton–proton rather than a proton–carbon correlation experiment. Therefore, while the HMBC may be easier to interpret, the TOCSY may be the preferred choice for extremely mass-limited samples. This usually would be the case, for example, for LC-NMR applications. 3. Heteronuclear Two-Dimensional Experiments Regarding heteronuclear correlations, HETCOR historically was the standard experiment used to detect proton–carbon scalar couplings.50–52 The disadvantage of HETCOR is that it is a carbon-detection experiment. It is therefore limited in sensitivity as previously discussed. Recalling that the signal-to-noise ratio is proportional only to the square root of the number of acquisitions, it can readily be seen that it becomes unrealistically timeconsuming to attain desired sensitivity levels. For example, a sensitivity loss of a factor of four requires a 16-fold increase in the number of scans to compensate. There are therefore two modern, equivalent experiments commonly used for the indirect detection of short-range proton–carbon scalar couplings. They are heteronuclear single quantum coherence or HSQC,53–55 and heteronuclear multiple quantum coherence or HMQC.56–58 Both experiments show the 1-D proton spectrum along one axis and the 1-D carbon spectrum or carbon projection along the other axis. Cross peaks correlate protons directly bonded to carbons. Two-dimensional correlations can be used to distinguish poorly resolved resonances in either the proton or carbon dimension. This may be the case, for instance, in a complicated aromatic system or for a macrolide. A proton resonance showing no correlations thus must be bonded to a noncarbon entity, e.g., an O–H or N–H group. Conversely, a carbon resonance showing no correlations must be a quaternary carbon. Since quaternary carbons are not observed in this experiment, the carbon spectral width can be set to cover only the protonated carbon region, nominally 0–180 ppm, to improve resolution in this indirect dimension. As previously mentioned, this information is complementary to that obtained from the DEPT spectrum, since one can also match proton integrals to corresponding carbons to distinguish methine, methylene, and methyl carbons. This interpretation may be complicated by the presence of overlapping proton resonances. A version of this experiment, called the DEPT-HSQC (for example, see Figure 5), combines these two experiments into a single proton-detected experiment.59 The cross peaks of the resulting HSQC type experiment are phase-sensitive depending on carbon multiplicity. Methylene-derived cross peaks are negatively phased, while methine- and methyl-derived peaks are phased positively. By incorporating a standard carbon observe DEPT experiment into the proton observe HSQC experiment, a dramatic time saving over running both experiments separately can be achieved without foregoing pertinent structural information. Comparing HSQC and HMQC experiments, the HSQC tends to yield higher resolution,

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FIGURE 5

DEPT-HSQC spectrum. The four methylene resonances appear as negative correlation peaks, while the methyl resonance in the top right of the spectrum appears as a positive correlation peak.

while the HMQC tends to yield higher sensitivity.60 A 1J(C, H) coupling constant of approximately 145 Hz is recommended for this type of experiment. Long-range correlations can be investigated using the heteronuclear multiple-bond correlation experiment, or HMBC. It detects scalar couplings between protons and carbons that are separated by multiple bonds.43,61–63 We recommend setting the long-range coupling parameter, nJ(C, H), to approximately 10 Hz and the short-range coupling parameter, 1J(C, H) to 145 Hz. The pulse sequence ﬁlters out contributions from short-range scalar

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couplings. Given these parameter values, for aliphatic systems, peaks are typically observed for protons and carbons separated by two bonds. In aromatic systems, peaks are observed for protons and carbons separated by three bonds. Low-intensity 2J(C, H) and possibly 4J(C, H) correlations may also be observed in highly conjugated systems (see, for example, Figure 6). Because the HMBC experiment detects weak couplings, it is an inherently

FIGURE 6

Comparison of strong and weak long-range scalar correlations in an HMBC spectrum. A strong intensity correlation is observed between carbon 2 and protons 4 and 6, since the parameters are optimized for three-bond separations on an aromatic ring. Only a weak intensity correlation is observed between carbon 2 and protons 1 and 3, since this corresponds to a two-bond separation.

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insensitive technique compared to the other 2-D experiments described thus far. It is therefore wise to incorporate more scans and allow additional time for acquisition. Occasionally, expected long-range peaks are not observed in the HMBC spectrum. This may be because the chosen coupling values are not optimized for the chemical entity being studied. Unfortunately, for an unknown impurity or degradant being characterized, one usually does not know a priori what the long-range scalar couplings are. To circumvent this obstacle, a class of experiments was created to cover a range of coupling values. The original experiment of this type, called ACCORD-HMBC, splits each HMBC cross peak into its corresponding proton multiplicity.64 Thus for a proton quartet, for example, correlation peaks will arise in sets of four. The advantage of this approach is its ability to cover a range of coupling constants rather than necessitating a single value to be used. Additionally, the more deﬁned peak groupings potentially facilitate spectral interpretation by distinguishing more readily between resonances. The primary disadvantage of this HMBC derivative is its reduced sensitivity compared to the traditional HMBC due to T2 signal loss related to a lengthier acquisition. Apparent signal-to-noise ratio is also diminished, since observed correlation peaks are split into multiplets. As we saw earlier, COSY and TOCSY spectra are used to link proton resonances within a given spin system. However, they are unable to link neighboring spin systems. In this case, the HMBC spectrum is used to observe long-range couplings, particularly across nonprotonated carbons and heteroatoms. HMBC couplings can also be used to conﬁrm correlations within a given spin system. The carbon projection of the HMBC spectrum gives essentially an equivalent to the full carbon spectrum. Clearly this affords a signiﬁcant sensitivity advantage and time savings to acquiring the standard 1-D carbon spectrum. Limitations include reduced resolution, and the possibility of not observing all carbon resonances. This limitation would arise for neighboring quaternary carbons or other nonprotonated entities since longrange coupling to the nearest proton would be too weak to observe. By interpreting these data interactively with other data such as COSY and HSQC spectra, one can correlate carbons to their attached protons, establish individual spin systems, and then link them together to piece together a plausible chemical structure. 4. Additional Experiments If more information is required, one may need to perform additional experiments. One common example is when information regarding stereochemistry is necessary. Examples include investigation of chiral centers, E versus Z isomers, and overall structural conformation. The 2-D experiments previously described all relate to through-bond scalar correlations and not spatial relationships. One experiment that speciﬁcally addresses through-space interactions is known as the nuclear Overhauser effect, or ‘‘nOe,’’ experiment.65–68 The nOe experiment shows correlations between nuclei that are in close proximity to each other. A target signal is irradiated, and this alters the intensity of signals from neighboring nuclei. The resulting

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alteration of signal intensity is known as the nuclear Overhauser effect. Taking the difference between the irradiated spectrum and a standard spectrum leaves only the target signal, which is inverted, and the resulting nOe’s. Observations are independent of the number of bonds separating the protons of interest. This experiment requires very high instrument stability, so it is useful to employ temperature control to ensure thermal stability. The nOe interactions are typically very weak, approximately 1 – 3% of the original signal intensity. Therefore, this experiment is commonly limited to proton detection based on sensitivity considerations. For this reason, it is extremely challenging on mass-limited samples and is not considered realistically feasible for LC-NMR. As an added complication, depending on the molecular tumbling rate of the molecule, which relates generally to size, the nOe may be positive, negative, or even zero. Hence the inability to observe an expected nOe does not necessarily mean that two protons are not within close proximity. For small molecules, observed nOe signals are generally positive, while for macromolecules, they are generally negative. To avoid any potential for being in the zero nOe regime, one can use the alternative rotating frame equivalent experiment. In this case, the observed effect is always positive, regardless of molecular tumbling rate. Two-dimensional, homonuclear versions of these experiments are known as NOESY69 and ROESY experiments.70 Other possible 1-D experiments include detection of nitrogen, ﬂuorine, oxygen, and other nuclei of pharmaceutical interest. However, with the exception of ﬂuorine, these active nuclei have very low natural abundance which makes data acquisition prohibitively time-consuming.28 In most cases, impurity and degradant samples are too mass-limited to consider detection of these low abundance nuclei. If pairs of peaks are observed in the 1-D spectra, particularly for protons, this is often indicative of rotamers. Rotamers occur when the rotation about a bond, usually a nitrogen–carbon bond, is slow compared to the NMR time scale. In this case, NMR observes two distinct yet equivalent entities. Elevating the temperature increases the rate of rotation such that, at the coalescence temperature, pairs of peaks coalesce to one, and an average conformation is observed.71 While performing these experiments is not generally limited by sample quantity, sample stability at elevated temperatures should be strongly considered, particularly when dealing with an unknown chemical entity. The solvent’s boiling point should also be taken into consideration. A good choice for elevated temperature studies is DMSO, since its boiling point is quite high. Another useful experiment addresses labile protons, such as N–H and O–H groups. The experiments described thus far detect proton–carbon and proton–proton interactions and do not speciﬁcally address protons bonded to other nuclei. As mentioned when discussing NMR solvents, the presence of protic solvents causes labile protons to exchange rapidly, rendering them unobservable by NMR. We can use this to our advantage. A proton spectrum is ﬁrst acquired using an aprotic solvent. A few drops of deuterium oxide or deuterium chloride are then added to the sample, and the proton spectrum is reacquired. Labile protons will exchange with the D2O and hence no longer be observed in the spectrum.

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For any of the experiments described thus far, modiﬁcations can be made to the individual experiments to adapt to the requirements of a given sample. For example, if a signiﬁcant amount of residual protonated solvent is present, then solvent suppression techniques can be employed to reduce or eliminate this contribution to the spectrum. Also, shaped pulses can be used to select a desired frequency band within the spectral range.72,73 This enables one to observe only the region of interest and null all other regions. This may be advantageous for LC-NMR, for example, to eliminate the solvent region, or for improved resolution of a target region in the indirect dimension of a 2-D experiment.

VII. CHOOSING AN EXPERIMENT SET With this arsenal of techniques, we are now prepared to obtain sufﬁcient data to begin elucidating a structure. At this point, one must identify an appropriate set of experiments for the sample and issues at hand. The following should be taken simply as guidelines to experiment selection. Alternative approaches may be equally valid. A 1-D proton spectrum should be acquired at the start and end of any investigation. This is used to evaluate sample stability over the course of the experiments. As we have seen, carbondetected experiments should be reserved for those rare instances when sample quantity is not limited and time is not critical. If one is fortunate enough to be in this situation, a suggested set of experiments may include the following: 1-D proton, APT or equivalent for detection of carbon resonances and their corresponding multiplicity, DQFCOSY for establishing spin systems, ACCORD-HMBC for linking spin systems, and HSQC for correlating carbons to their attached protons. Supplemental experiments described in the previous section may also be relevant, depending on the questions to be addressed. This strategy is relevant for sample quantities in excess of approximately 10 mg. If, on the other hand, one has an isolated sample of limited mass and/or a limited amount of time, an alternative approach should be taken. Carbon resonances and their corresponding multiplicity should be detected indirectly to maximize sensitivity and minimize acquisition times. Therefore, instead of acquiring an APT plus an HSQC spectrum, the more time-efﬁcient DEPTHSQC can be used. COSY affords improved sensitivity over DQFCOSY at the cost of resolution along the diagonal. Similarly, ACCORD-HMBC can be replaced with the more sensitive HMBC. This approach is recommended for time-limited situations or for the nominal mass range of 100 mg–10 mg. For yet smaller sample sizes or time frames, a different tactic can be taken. The weak couplings of the HMBC experiment will probably be unattainable in this scenario, so long-range interactions can alternatively be probed with TOCSY. While this provides a much higher level of sensitivity, it will be more challenging to link neighboring spin systems. Therefore, it is possible to acquire a 1-D equivalent to the HMBC spectrum for individually designated resonances. Similarly, if heteronuclear 2-D experiments in general are not

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feasible given the amount of sample, then a 1-D version of the HMQC experiment can be employed on targeted resonances. A COSY spectrum will also facilitate the structural elucidation process. Unlike for the previous two cases, nOe measurements will most likely not be within these detection limits. This case applies to samples on the order of 10–100 mg. If one is truly pushing the limits of NMR detection or has only an extremely time-sensitive sample, yet another strategy is preferred. Here a 1-D proton spectrum may be all that is realistically attainable. Possible additional experiments include a 1-D version of TOCSY and a 1-D version of COSY, also known as a homonuclear decoupling experiment. This applies to sample quantities down to hundreds of nanograms, which is currently the practical limit of NMR detection using commercially available equipment. LC-NMR is typically limited to the last two options because of limited sample quantity. Keep in mind that LC-NMR may require incorporating single or double solvent-suppression techniques into these experiments if protonated solvent systems are used. Having selected and acquired a standard set of experiments, the next step is data interpretation as a ﬁrst pass for structural elucidation.

VIII. DATA INTERPRETATION A. Primary Interpretation Which set of NMR experiments employed will dictate the strategy for interpreting the data? The following discussion is therefore meant as a general guideline. In practice, not all of the spectra mentioned may be available for a given sample. Use the 1-D proton spectrum and either a 1-D carbon spectrum, including any multiplicity-edited version, or the carbon projection of a 2-D heteronuclear experiment as reference spectra for making proton and carbon assignments. Keep in mind that some multiplicity-edited experiments, including DEPT, do not show quaternary carbon resonances. When selecting a carbon projection, it is preferable to take it from a long-range rather than a short-range experiment, since one will be able to detect most, if not all, quaternary carbon signals. If available, begin by identifying the number of protons attached to each carbon. This information can be obtained from several potential sources including DEPT or equivalent, DEPT-HSQC, or by matching proton integrals to carbon resonances in an HSQC or HMQC spectrum. It is useful when interpreting data to label each carbon resonance as quaternary, methine, methylene, or methyl for easy reference. Next, total the number of each type of carbon and compare these numbers to those for the parent or other relevant related structure. Remember that NMR detects only magnetically inequivalent species, so when counting carbons in the parent structure, be sure to count all magnetically equivalent carbons as only one unique carbon entity. The differences in carbon counts between the impurity and the parent will point out any obvious changes from the parent. These numbers may also be compared with

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the corresponding numbers for any proposed structures, which may rule out some possibilities immediately. A comparison of parent and impurity chemical shifts will also identify any obvious differences. This is true for both proton and carbon resonances. This comparison is especially useful, for example, when determining the oxidation site of an N-oxide or a sulfoxide, since resonances neighboring the oxidation site will be shifted downﬁeld compared to the corresponding peaks in the parent (see Figure 7 for

FIGURE 7

Comparison of carbon spectra for a parent compound and its associated N-oxide.Notice the carbons immediately neighboring the oxidation site are shifted downfield, and carbon 5 is significantly broadened.The upfield shift of carbons 2 and 3 reflects this shift in electron density.

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example). When comparing spectra, recall that minor differences may arise if different solvents are used. This is usually insigniﬁcant for carbon spectra but is more noticeable for proton spectra. At this point, we can continue by identifying an appropriate starting point for speciﬁc assignments. There is inevitably at least one proton or carbon resonance, and usually several, that one can assign immediately. This may be based on chemical shift. For example, a carbonyl should appear as a quaternary signiﬁcantly downﬁeld in the carbon spectrum or projection, while aliphatic quaternaries appear signiﬁcantly upﬁeld. Carbon multiplicities that are due to ﬂuorine coupling are also excellent starting identiﬁers (see Figure 4). Recall that the size of the coupling constant will determine the actual number of bonds separating the carbon and ﬂuorine. In the proton spectrum, a unique peak may be assigned based on chemical shift, multiplicity, and proton integration. If no peaks can be identiﬁed using this direct approach, it is necessary to go to the next step by looking at correlations as a unique identiﬁer. For example, there may be two methyl triplets in the proton spectrum, but only one that is expected to neighbor two methines. This can be readily identiﬁed using the COSY or DQFCOSY spectrum (see Figure 8). Ideally, a starting point should be identiﬁed for each spin system in the molecular structure. It is therefore useful to identify several starting points to facilitate assignments. Once starting points have been identiﬁed, one can use 2-D correlations to map out the rest of the structure. First, if starting with a protonated carbon group, use the HSQC or equivalent to identify the carbon resonance directly bonded to the proton group or vice versa. Proton line shape will identify the number of neighboring protons since the observed multiplicity is equal to one more than the number of neighboring protons.74 Labile protons may show only weak couplings, so bear this in mind when evaluating proton multiplicities. Next use a COSY or TOCSY spectrum to identify any immediate protonated neighbors. Recall that TOCSY shows correlations between all protons within a given spin system, while COSY shows only immediate neighbors. Comparing COSY and TOCSY data then distinguishes immediate versus distant neighbors. Alternatively, HMBC-type spectra can be used to identify long-range correlations. This information is invaluable for linking spin systems together, particularly when heteroatoms or quaternary carbons are present. However, it may be challenging to link together molecular fragments separated by more than one heteroatom or quaternary carbon. In this instance, such long-range couplings between carbons and protons are typically too weak to observe. Remember that for two correlated protonated carbons in an HMBC spectrum, a cross peak will exist from the proton of the ﬁrst to the carbon of the second. An additional cross peak will exist from the carbon of the ﬁrst to the proton of the second (see Figure 9). This built-in redundancy is particularly useful when limited spectral resolution obscures a correlation peak. Of course, this redundancy is not possible for correlations to quaternary carbons. On the issue of redundancy, once all of the proton and carbon resonances have been assigned, it is a good idea to conﬁrm that all of the observed 2-D correlations are consistent with the proposed assignments. Throughout the initial assignment process, one

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FIGURE 8

DQFCOSY spectrum of an extended spin system. Methyl 1 correlates to methylene 2, which neighbors methine 3. Methine 3 correlates to methylene 4, which in turn neighbors hydroxyl proton 5.

should refer to an HSQC or HMQC spectrum if available to correlate carbon resonances to their corresponding attached proton resonances. Assignments based on 2-D correlations can often be conﬁrmed by information found in the 1-D spectra. For instance, if two protons neighbor each other as evidenced in the COSY spectrum, their proton resonances should have the same coupling constant. Aromatic ring protons often show an additional hyperﬁne splitting, which helps to distinguish ring protons from other electron-rich proton resonances. Labile protons found in N–H and

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FIGURE 9

This HMBC spectrum shows that for two protonated carbons correlated to each other, a cross peak will exist from the proton of the first to the carbon of the second, and an additional cross peak will exist from the carbon of the first to the proton of the second. Here we see a correlation between carbon 1 and proton 3, and an equivalent correlation between carbon 3 and proton 1.

O–H groups reduce the measured proton integration of these peaks. In addition, these proton resonances are often broader than other resonances in the spectrum (see Figure 10). This may make 2-D correlations to these groups too weak to observe. Summation of proton integrals should be consistent with any proposed structures. When sensitivity-limited, 1-D equivalents to the common 2-D experiments can be employed to selectively probe key correlations. In extreme cases, one may need to rely entirely on

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FIGURE 10

Labile protons found in N^H and O^H groups reduce the measured proton integration of these peaks. Here we see the integration of the hydroxyl proton is less than the expected value of1.00. Labile protons are also broadened, as seen in the line width of resonance 5 compared to that of 3 and 4.

1-D data for structural elucidation. Any required solvent suppression may further complicate spectral interpretation by dramatically distorting selected proton regions. When all possible proton and carbon assignments have been made, one should have a reasonable picture of the actual structure. Recall that any nuclei other than protons and carbons have not been directly investigated, so keep in mind other possible heteroatoms that may be present. Certainly proton and carbon chemical shifts and correlations (or lack thereof) will provide clues to the presence of other nuclei. Complementary data from other techniques, such as MS, may provide additional insights into the presence of heteroatoms. Also, as previously discussed, NMR is limited to identifying

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magnetically inequivalent nuclei.75 Therefore, NMR spectra for a particular molecule often will be indistinguishable from its corresponding dimer, trimer, etc. Possible structures will therefore include these equivalent entities as well. We need to rely on molecular mass and other complementary information to make this distinction. This brings us to the secondary interpretation.

B. Secondary Interpretation As mentioned, for the structural elucidation of impurities and degradants, it is useful to compare the proton and carbon assignments to those of the parent compound. It will be immediately obvious which peaks have changed, disappeared, or are new. This identiﬁes the site of degradation or reactivity. This is a valuable strategy for the determination of oxidation sites for N-oxides and sulfoxides (for instance, see Figure 7). In either case, resonances immediately neighboring the oxidation site should shift signiﬁcantly downﬁeld to reﬂect the enrichment of the electronic environment by the addition of oxygen. Also, any protons observed in the parent to be attached to the nitrogen will no longer be observed for the N-oxide. If there is no clear resemblance between the parent and impurity spectra at all, then the impurity may have derived from an alternative source. In this case, one should refer to the list of possible sources previously described to identify a likely candidate. One can then proceed in a similar fashion by comparing the proton and carbon assignments for the impurity to the corresponding chemical shifts for the excipient, precursor, or other identiﬁed source. Also bear in mind whether the parent or other source was run as a salt form or a free base, and similarly for the impurity or degradant, since resonances may be observed from the salt. These peaks should be easily identiﬁable by comparison to proton and carbon chemical shifts for known salts. Any supplemental data such as nOe results or variable temperature studies should also be evaluated at this time. Observation of an nOe between two protons conﬁrms spatial proximity. This information can prove invaluable in distinguishing cis versus trans conformations or other stereoisomers. Coalescence of proton resonances at elevated temperatures is consistent with chemical exchange or the presence of rotamers. Therefore, by acquiring a set of experiments at an elevated temperature, it greatly simpliﬁes the interpretation process for a rotamer system. As we will see subsequently, one may also use spectral simulations to lend additional credibility to a proposed structure.76 However, simulations should not be taken as proof of structure but rather be used to suggest plausible structures.

C. Tertiary Interpretation: Spectral Simulations In certain cases, the experimental NMR data may differ signiﬁcantly from expected chemical shifts of a proposed structure. When this occurs, one may need to use simulated spectra to provide conﬁdence in the assignments of the experimental chemical shifts. Using the previously obtained MS data, one can propose structures that satisfy the molecular weight requirement and are consistent with the experimental NMR data. These structures are then

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used to simulate proton and/or carbon spectra. Comparison of the simulated spectrum to the experimental spectrum will hopefully provide a match. If a match is not obtained, then at least the relative chemical environment can be assessed, reported back to the project team, and then used to propose plausible structures related to the original synthesis conditions. These simulations are especially important to assist in the assignment of heteroatom connectivity in the molecule. The simulated spectra may also be used to provide conﬁdence in unexpected chemical shifts. They also prove useful as a comparison with experimental chemical shifts of moieties that have not been previously analyzed. There are two primary methods to calculate simulated spectra, theoretically and empirically. Theoretical simulations establish a molecular model based on quantum mechanical calculations, while empirical simulations rely on a database of experimentally measured chemical shifts and coupling constants for known structures. There are a number of software programs available for either simulation method. Some software packages simulate only 1-D proton and carbon spectra, while others include additional analytical simulations, such as 2-D NMR, HPLC retention times, log P, log D, etc. 1. Empirical Simulations This approach uses a large database of experimental NMR data to simulate 1-D proton and carbon spectra. Databases commonly contain over a million chemical shifts and more than 250,000 coupling constants.77 Some databases can be tailored to speciﬁc needs by incorporating the user’s experimental data into the database.77 There are some cautionary notes to these simulation software packages that need to be addressed. One important consideration is that they normally do not accurately simulate tautomers, zwitterions, or charged species. Quadrupolar coupling effects are also difﬁcult to simulate. These are representative instances where one may need to check the references and structures that the software used to simulate the spectra. These cases are a result of situations where too few related examples exist in the database of chemical shifts. Remember that when entering new data into the database, the simulated spectra are only as good as the data in the database. 2. Theoretical Simulations The following discussion focuses on ab initio predictions of 13C chemical shifts.78 Calculations for predictions of proton resonances are possible as well, but are not presented here. In the comparison plots for all compounds, both the x- and y-axes have a range of 200 ppm. The diagonal line drawn from the lower left to upper right of the graph represents a theoretically perfect correlation. The observed experimental shifts, plotted along the x-axis, are the chemical shifts assigned by interpretation of a full NMR spectral data set. Assignments are based on the expected chemical shift ranges for a given type of carbon, experimentally determined proton multiplicities, both homonuclear and heteronuclear correlation experiments, and whatever additional experiments were necessary to assign a spectral line to a particular atom. Calculated chemical shifts are plotted along the y-axis. The line drawn

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through the data points is the best linear ﬁt of the data. The units generated by the DFT DGAUSS program have been converted to conventional ppm units by comparison with computations for TMS, used as a reference compound, computed using the same DFT program parameters. A theoretical approach to spectral simulations can, in many cases, provide an extremely accurate match to experimental values. Deviations may arise in the case of charged species, differing stereoisomers or conformations, and potentially the presence of atypical heteroatoms. Bearing these precautions in mind, simulations can provide valuable insights into plausibility of proposed structures. The 13C NMR chemical shift predictions for sertraline (Structure 1) are plotted in Figure 11. The correlation is quite high. The greatest deviations between observed and predicted chemical shifts are for the N-methyl carbon and for the two aromatic ring carbons to which chlorine atoms are attached. Computations for ziprasidone (Structure 2) are shown in Figure 12. A large variety of chemical shifts are present in this molecule. Nonetheless, the predictions are generally good as seen by the r2 value of 0.9899.

STRUCTURE 1 Sertraline

FIGURE 11 Observed versus predicted 13C chemical shifts for sertraline.

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STRUCTURE 2 Ziprasidone

FIGURE 12

Observed versus expected shift for ziprasidone.

One notable exception is for a quaternary carbon in the ﬁve-membered portion of the benzisothiazole ring. These computations took approximately 5 hours to complete. The trovaﬂoxacin (Structure 3) computed chemical shifts are plotted in Figure 13. The three aberrant chemical shifts at approximately 148 ppm are the ﬂuorinated carbons. Computing time for trovaﬂoxacin was 31.8 hours. Figure 14 is a composite comparison of observed versus expected shift for 12 compounds.

IX. FINAL STEPS After the required levels of NMR data interpretation have been completed, it is advantageous to reconvene with the characterization team to discuss the structural information obtained thus far. Speciﬁcally, the group should identify areas of consistency or inconsistency of information gathered from other sources compared with the NMR conclusions. For example, are the mass spectral fragment weights consistent with the molecular fragments determined by NMR? Is the degradation site or other deviation from known compounds identiﬁed by NMR consistent with the location identiﬁed by mass spectral fragmentation patterns? Is the total molecular mass consistent with

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STRUCTURE 3 Trovafloxacin

FIGURE 13

FIGURE 14

Observed versus expected shift for trovafloxacin.

Composite comparison of observed versus expected shift for 12 compounds, including data for sertraline, ziprasidone, and trovafloxacin.The three aberrant carbon shifts from trovafloxacin, previously mentioned, are identifiable in this composite plot.

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structures based on the NMR results? The characterization strategy outlined here does not explicitly detect nuclei other than carbon and hydrogen, and as previously mentioned, NMR cannot distinguish magnetically equivalent nuclei. Consistency between NMR and MS data is thus especially important for distinguishing monomers from dimers, trimers, etc. If a carbonyl was observed in the IR spectrum, was it also observed either directly or indirectly in the NMR data? Is the expected polarity of a proposed structure consistent with the observed chromatographic retention time? If any such inconsistencies are identiﬁed, focus on these areas to propose alternative structures. Even if a structure is identiﬁed to be consistent with the data, it is important to consider if any other structures may be consistent as well. Clearly this approach requires a high level of interaction among the members of the characterization team. If a structure has passed all the requirements thus far, one needs to determine if it can be derived from a plausible synthetic route. In other words, is it consistent with the reaction pathway and chemical entities present in the reaction vessel? If additional structural information is required, it is necessary at this point to identify appropriate follow-up experiments. For example, if the stereochemistry at a chiral center is critical to the understanding of the system, nOe experiments may be useful to investigate spatial relationships. To improve the conﬁdence level in a proposed structure, it may be necessary to synthesize the proposed structure using a wellestablished synthetic route and then repeat the NMR characterization on the synthesized compound. Comparing the proton and carbon spectra of the unknown with the known compound should conﬁrm consistency of structure. Finally, the team should draw conclusions based on the identiﬁed structure, plausible synthetic routes, and ways to prevent its formation.

X. SUMMARY As one can see from this discussion, the NMR characterization process plays a valuable role in the structural elucidation of impurities and degradants. A rigorous decision process enables a logical selection from the many choices of instrumentation and experiments available. The approach outlined in this chapter minimizes data-acquisition time while maximizing relevant information gathered. This becomes critical for rapid characterizations, either because of aggressive project time lines or sample instability. Mass limited samples, as well as low-solubility or low-purity samples provide additional challenges that can be appropriately addressed through prudent hardware and experiment selection. Interpretation of the NMR data requires careful diligence, usually involving an iterative process of studying several spectra simultaneously. This sometimes time-consuming effort is well worth the investment. NMR spectroscopy yields critical structural information that cannot be obtained readily, and in many cases not at all, by any other means. The results of the NMR analysis must be carefully weighed with observations from other analyses, such as MS and HPLC, to ensure a thorough understanding of the

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chemical system. NMR spectroscopy is often applied last in the characterization process, and because of the level of structural detail achievable by NMR, it often provides the missing information that ties all other experimental observations together. It is this philosophy of interactively collecting and interpreting complementary information that enables the successful characterization of the most challenging impurities and degradants, a requirement that continues to grow ever more stringent with evolving FDA guidelines.

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24. Friebolin, H. P. Basic One- and Two-Dimensional NMR Spectroscopy, 2nd ed. VCH, Weinheim, 1993. 25. Braun, S., Kalinowski, H.-O. and Berger, S. 150 and More Basic NMR Experiments. Wiley, VCH, Weinheim, 45–49, 1998. 26. Kalinowski, H.-O., Berger, S. and Braun, S. Carbon-13 NMR Spectroscopy, Wiley, Chichester, 1988. 27. Braun, S., Kalinowski H.-O. and Berger, S. 150 and More Basic NMR Experiments, Wiley, VCH, Weinheim, 50–54, 1998. 28. Harris, R. K. Nuclear Magnetic Resonance Spectroscopy, Longman Scientific & Technical, NY, 236, 1990. 29. Bendall, M. R., Pegg, D. T., Doddrell, D. M. and Field, J. J. Mag. Reson. 520–526, 1983. 30. Bendall, M. R. and Pegg, D. T. J. Mag. Reson. 53:40–48, 1983. 31. Bendall, M. R. and Pegg, D. T. J. Mag. Reson. 53:272–296, 1983. 32. Derome, A. E. Modern NMR Techniques for Chemistry Research, Pergamon Press, Oxford, UK, 143–151, 1987. 33. Schenker, K. V. and Philipsborn, W. V. J. Mag. Reson. 66:219–229, 1986. 34. Doddrell, D. M., Pegg, D. T. and Bendall, M. R. J. Mag. Reson. 48:323–327, 1982. 35. Braun, S., Kalinowski, H.-O. and Berger, S. 150 and More Basic NMR Experiments. Weinheim: Wiley, VCH, 180–182, 1998. 36. Patt, S. and Shoolery, J. J. Magn. Reson. 46:535–539, 1982. 37. Homer, J. and Perry, M. J. Chem. Soc. Chem. Commun. 373–374, 1994. 38. Hurd, R. E. J. Mag. Reson. 87:422–428, 1990. 39. Tolman, J. R., Chung, J. and Prestegard, J. H. J. Mag. Reson. 98:462–467, 1992. 40. Tyburn, J. M., Brereton, I. M. and Doddrell, D. M. J. Mag. Reson. 97:305, 1991. 41. Vuister, G. W., Boelens, R., Kaptean, R., Hurd, R. E., John, B. K. and van Zijl, P. C. M. J. Am. Chem. Soc. 113:9688, 1991. 42. Hurd, R. E. and John, B. K. J. Mag. Reson. 91:648, 1991. 43. Willker, W., Leibfritz, D., Kerschebaum, R. and Bermel, W. J. Mag. Reson. 31:287–292, 1993. 44. Jeener, J. Ampere International Summer School, Proposal. Basko Polje, 1971. 45. Piantini, U., Sorensen, O. W. and Ernst, R. R. J. Am. Chem. Soc. 104:6800–6801, 1982. 46. Brereton, I. M., Crozier, S., Field, J. and Doddrell, D. M. J. Magn. Reson. 93:54–62, 1991. 47. Braun, S., Kalinowski, H.-O. and Berger, S. 150 and More Basic NMR Experiments. Wiley, VCH, Weinheim, 168–371, 1998. 48. Braunschweiler, L. and Ernst, R. J. Magn. Reson. 53:521–528, 1983. 49. Kover, K., Uhrin, D. and Hruby, V. J. Magn. Reson. 130:162–168, 1998. 50. Martin, G. E. and Zektzer, A. S. Two-dimensional NMR Methods for Establishing Molecular Connectivity, VCH, Weinheim, C. 3, 1988. 51. Bax, A. and Morris, G. A. J. Mag. Reson. 42:501–505, 1981. 52. Freeman, R. and Morris, G. A. J. Chem. Soc. Chem. Commun. 684–686, 1978. 53. Bodenhausen, G. and Ruben, D. J. Chem. Phys. Lett. 69:185–188, 1980. 54. Braun, S., Kalinowski, H.-O. and Berger, S. 150 and More Basic NMR Experiments. Wiley, VCH, Weinheim, 397–400, 1998. 55. Kay, L. E., Keifer, P. and Saarinen, T. J. Am. Chem. Soc. 114:10663–10665, 1992. 56. Muller, L. J. Am. Chem. Soc. 101:4481–4484, 1979. 57. Braun, S., Kalinowski, H.-O. and Berger, S. 150 and More Basic NMR Experiments, Wiley, VCH, Weinheim, 384–387, 1998. 58. Hurd, R. E. and John, B. K. J. Magn. Reson. 91:648–653, 1991. 59. Willker, W., Leibfritz, D., Kerssebaum, R. and Bermel, W. Magn. Reson. Chem. 31:287–292, 1993. 60. Cavanagh, J., Fairbrother, W., Palmer, A. and Skelton, N. Heteronuclear NMR Experiments from Protein NMR Spectroscopy Principles and Practice, Academic Press, San Diego, 1996. 61. Bax, A. and Summers, M.F. J. Am. Chem. Soc. 108:2093–2094, 1986. 62. Ruiz-Cabello, J., Vuister, G. W., Moonen, C. T., Van Gelderen, P., Cohen, J. S. and Van Zijl, P. C. M. J. Mag. Reson. 100:282–303, 1992. 63. Martin, G. E. and Zektzer, A. S. Two-Dimensional NMR Methods for Establishing Molecular Connectivity. VCH, Weinheim, 267–273, 1988.

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64. Kogler, H., Sorensen, W., Bodenhausen, G. and Ernst, R. J. Magn. Reson. 55:157–163, 1983. 65. Kinns, M. and Sanders, J. K. M. J. Mag. Reson. 56:518–520, 1984. 66. Neuhaus, D. and Williamson, M. The Nuclear Overhauser Effect. VCH, Weinheim, 1989. 67. Derome, A. E. Modern NMR Techniques for Chemistry Research. Pergamon Press, Oxford, 97–127, 1987. 68. Noggle, J. H. and Schirmer, R. E. The Nuclear Overhauser Effect—Chemical Applications. Academic Press, San Diego, 1972. 69. Jeener, J., Meier, B., Bachmann, P. and Ernst, R. J. Chem. Phys. 71:4546–4563, 1979. 70. Bothner-By, A., Stephens, R., Lee, J.-M., Warren, C. and Jeanloz, R. J. Am. Chem. Soc. 106: 811–813, 1984. 71. Harris, R. K. Nuclear Magnetic Resonance Spectroscopy. Longman Scientific & Technical, NY, 119–131, 1990. 72. Warren, W. and Silver, M. Adv. Magn. Reson. 12:247–384, 1988. 73. Freedman, R. Prog. NMR Spectrosc. 32, 59–106, 1998. 74. Harris, R. K. Nuclear Magnetic Resonance Spectroscopy. Longman Scientific & Technical, NY, 26, 1990. 75. Abragam, A. Principles of Nuclear Magnetism. Clarendon Press, Oxford, UK, 480–481, 1989. 76. Williams, T., Blinov, K., Elyashberg, M. and Martisrosian, E. Recent Advances in the Use of NMR Prediction and Automated Structure Elucidation Software for Natural Product Characterization, presented at the SMASH NMR Conference. Argonne, IL, 2000. 77. Williams, A. Current Opinion in Drug Discovery & Development, 3(3):298–305, 2000. 78. Koch, W. and Holthausen, M. A Chemist’s Guide to Density Functional Theory, 2nd ed. Wiley, VCH, Weinheim, 2001.

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13 HYPHENATED CHARACTERIZATION TECHNIQUES THOMAS N. FEINBERG Cardinal Health, ResearchTriangle Park, NC 27709

I. INTRODUCTION II. EXPERIMENTAL EXAMPLES A. Absorption Spectroscopy B. Mass Spectrometry C. Nuclear Magnetic Resonance (NMR) Spectroscopy III. CONCLUSIONS REFERENCES

I. INTRODUCTION The pharmaceutical industry is required by the federal Food, Drug, and Cosmetic Act1 to establish the identity and purity of the products it wishes to market in the United States. The most common techniques used for these purposes are chromatography based, coupled with detectors whose output varies with either the total amount of an eluted species (mass) or the concentration of the eluted species in the ﬂowing stream. For detectors to provide useful identity or purity measurements, the characteristic time constant of the detector signal must be less than the peak width. These basic techniques can be considered single-dimensional, as the detector varies only in time, and the resultant data is a two-dimensional plot (i.e., a chromatogram). The two most common chromatographic techniques, high-performance liquid chromatography (HPLC) and gas chromatography (GC) can provide both identity measurements by retention time and purity measurements by area (or area under the curve, AUC) when coupled with the two most widely used detectors, ultraviolet (UV) absorbance and ﬂame ionization detection, respectively. Retention time matching can be validated to establish identity, but is insufﬁciently speciﬁc for general use. Retention times are not necessarily unique for chemically distinct species; even isomers may or may not co-elute. A change in synthetic process may introduce a different impurity proﬁle; any of these new process impurities has a non-zero chance of co-elution with

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another impurity or the desired product. A change in storage condition can also invalidate a previously used retention time identity test as different degradation pathways (heat, light, etc.) may have different intermediates and/ or products. It is possible to determine, a priori, the probability of species co-elution in a chromatogram, using some basic relationships from chromatography. It is easiest to show how this probability can be calculated using the case of isocratic or isothermal chromatography, although it can be generalized by appropriate mapping techniques.2–4 Consider the basic relationship between retention time (t), peak width (w), and chromatographic plates (N) for an isocratic (isothermal) separation:5 N ¼ 16

h t i2 w

ð1Þ

Over an observation window from tf to ti, the average peak width waverage for peaks with similar interaction with the column (constant N), is given by waverage ¼

2 ðtf þ ti Þ pﬃﬃﬃﬃﬃ N

ð2Þ

Dividing the observation time (tf ti) by the average peak width, the total number of separated peaks (peak capacity, nc) can be deduced as follows: pﬃﬃﬃﬃﬃ ðtf ti Þ ðtf ti Þ N ¼ nc ¼ waverage 2 ðtf þ ti Þ

ð3Þ

Given an arbitrarily long ﬁnal observation time (limit of nc as tf approaches inﬁnity), the total peak capacity of a separation system is approximated as pﬃﬃﬃﬃﬃ N ð4Þ nc ðtf ! 1Þ ¼ 2 Table 1 lists some common separation techniques and typical peak capacities. Cursory examination would suggest that a carefully chosen separation technique and a single dimension detector would be sufﬁcient to separate mixtures containing up to nc components. This conclusion however would be incorrect. Components in mixtures will have a distribution of retention times with no a priori way to avoid coelution. The probability of coelution is much like the old party trick of showing that in a roomful of 40 or more people, two people are very likely to share the same birthday (try it!). The statistical argument can be presented for the birthday trick (or for chromatography) as follows: In a room with only two people, the second person has 365 out of 366 (remember leap years) chance of not having the same birthday. Adding an additional person to the room, the chance that the third person does not share either of the other two birthdays is given by 364 divided by 366. But the chance that none in the room share the same birthday is joint, i.e., the

343

13 HYPHENATED CHARACTERIZATION TECHNIQUES

TABLE1 Calculated Peak Capacities for Common Chromatographic SeparationTechniques Separation technique

Theoretical plates (N)

Thin-layer chromatography

10

Flash chromatography Packed column gas chromatography High-performance liquid chromatography Capillary gas chromatography Capillary electrophoresis

Peak capacity (nc) <2

100

5

1,000

16

10,000

50

100,000

160

1,000,000

500

probabilities are multiplied! Generalizing to the situation where there are nc unique positions (birthdays) and S components (people) with randomly assigned positions, the probability, of nc components not sharing positions (purity) is given by:

Purity ¼

nc ! nSc ðnc S þ 1Þ!

ð5Þ

Using Stirling’s approximation,6 Equation (5) can be more conveniently calculated as Purity ¼

ð1 S=nc ÞðSnc 0:5Þ expðSÞ

ð6Þ

with the numerical result that there is only an 11% chance that two people out of a group of 40 will not share the same birthday.

The ﬁrst three columns of Table 2 show typical results for the case of a separation with 10,000 theoretical plates (e.g., HPLC) and S varying from 2 to 20. Even for the situation with only 2 components, there is only a 98% chance that any peak is pure. Typical drug substance acceptance criteria require at least 98% purity. Therefore, in the absence of any process or degradation information, a typical HPLC assay with a single-dimensional detector cannot ensure adequate purity or speciﬁcity without further investigation. Historically, the procedure for the establishment of a chromatographic method’s speciﬁcity, in addition to the characterization of impurities and degradants, has relied on the simple concept (yet complex laboratory procedures) of isolation and recharacterization using orthogonal techniques (commonly referred to as ‘‘ofﬂine’’ analysis). The orthogonality of these other techniques implies multidimensionality, i.e., the resultant data (e.g., retention time versus absorption maxima) will not be easily presented on a single sheet of paper, but may consist of two or more separate traces as depicted in Figure 1, or a multidimensional contour plot. Referring back

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TABLE 2 Calculated Statistical Purity Probabilities for Individual HPLC Peaks with Single Wavelength and Diode Array Detection Peak capacity (nc)

Single-dimension purity probability (%)

Distinguishable UV absorbance maxima

Two-dimension purity probability (%)

2

50

98.0%

83

100.0%

4

50

88.4%

83

99.2%

6

50

73.2%

83

95.5%

8

50

55.4%

83

86.9%

10

50

38.2%

83

73.3%

12

50

23.8%

83

56.9%

14

50

13.4%

83

40.5%

16

50

6.8%

83

26.6%

18

50

3.0%

83

16.3%

20

50

1.2%

83

9.4%

Number of components

FIGURE 1 HPLC-UV chromatogram (229 nm) of a 1-mg sample of ranitidine HCl spiked with its related substances A^C as noted (left-hand side). 1H NMR spectra acquired in stopflow mode for each peak are presented on the right-hand side.

13 HYPHENATED CHARACTERIZATION TECHNIQUES

345

to Equation (6), if there were a second independent dimension to sort through the co-eluters, the accidental coincidence probability would be greatly diminished. Take, for example, UV absorption, which is, by far, the most common hyphenated technique in any analytical laboratory7 (i.e., HPLC-DAD, diode array detection). A typical scan range might be from 200 to 450 nm. Band pass values for common laboratory equipment are usually about 3 nm to distinguish absorption maxima. The resultant ‘‘capacity’’ of the system can be calculated as 450 nm200 nm=3 nm 83: The last three columns of Table 2 show the accidental coincidence probabilities for a typical HPLC-DAD experiment. Even with such a common laboratory system, the power of hyphenated techniques becomes clear. By collecting the necessary data (impurity proﬁling,8 purposeful or forced degradation studies,9,10 and impurity/degradant characterization11), it can be shown that a particular assay has the necessary speciﬁcity to satisfy regulatory requirements coupled with hyphenated techniques. The number of types of hyphenated techniques, which have been used for problem solving is enormous. Numerically speaking, there are an inﬁnite number! It is possible to repeatedly analyze a sample ad inﬁnitem by such techniques as GCxGC or 2D-PAGE or LC-MSn. Hyphenated techniques can be categorized into two distinct groups: extrinsic (or mixture) and intrinsic characterizations. The former group includes the multiple separation techniques such as two-dimensional chromatography, where the sample is repeatedly isolated into distinct chemical species. The latter group includes those methods where spectroscopic analyses of single molecular properties are performed, such as HPLC-DAD, LC-MSn, and LC-NMR. Prior to the advent of fast microcomputers, multidimensional chromatography methods were the most common techniques for the analysis of complex mixtures. As foreseen by Bertsch (1990)12 over 10 years ago, ‘‘Since the presence or absence of drugs has far-reaching implications, most analysts feel compelled to rely usually on additional information such as provided by spectrometry. It can be expected that capillary columns with selective detectors, especially mass spectrometry will be favored for the foreseeable future over twodimensional GC.’’ Collecting an additional dimension alone does not, by itself, allow unambiguous differentiation between co-eluting species or proper characterization information for a peak. An example of an HPLC-UV absorption detection (204 nm) chromatogram of a pharmaceutical product is shown as Figure 2.13 A mass spectrum for the largest peak (13.7 min) is shown in Figure 3, as collected online. If the peak were collected and analyzed ofﬂine, the same results as Figure 3 would have been obtained, but this is not the mass spectrum of the peak at 13.7 minutes. Rather, the mass spectrum shown in Figure 3 is related to an excipient (a disaccharide). This can be conﬁrmed by plotting the base peak in the mass spectrum as a function of time, as shown in Figure 3 (m/z 145) as function of time. Here it is evident that no rise and fall at a retention time (with expected time lag between detectors) of the target can be discerned (see Figure 4). Online techniques are most powerful when the second dimensional signal is modulated by the chromatographic signal to highlight the important coincidences between

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FIGURE 2 HPLC-UV (204 nm) chromatogram of nitroglycerin tablet.

FIGURE 3 Positive-ion atmospheric pressure chemical ionization mass spectrum at retention time approximately 13.7 minutes for nitroglycerin tablet sample.

the two data sets. If the technique were changed to one more suited to the analysis (in this case, negative ion attachment atmospheric pressure chemical ionization), the mass spectrum of the retention time will appear as Figure 5. The lower spectrum shown in Figure 5 has been manipulated to subtract contributions from species eluting after the peak of interest. Compare Figure 6 to Figure 4, where a correct correlation has been noted in the extracted ion

13 HYPHENATED CHARACTERIZATION TECHNIQUES

347

FIGURE 4 Extracted ion chromatogram of base peak for 15 minutes (m/z 145, top) and HPLC-UV chromatogram (204 nm, bottom). Note no correlation between signals at 13.7 minutes.

chromatogram using negative ion attachment LC-MS. The signal is deﬁnitely correlated between the two data sets, which provides characterization data for the unknown. As previously mentioned, there are two ways in which multidimensional data can be collected: ofﬂine or online. Prior to the advent of inexpensive data-collection systems, the common techniques for multidimensional characterization involved isolation from one chromatographic or chemical (qualitative organic analysis) separation technique and subsequent analysis by a second spectroscopic or chromatographic or chemical separation technique. This method minimizes the total computational and memory requirements (whether analog circuit and strip chart recorder or microcomputer) by collecting second dimensional data for only one or two data points as was illustrated in Figure 1. Online or hyphenated analyses such as the common HPLC-DAD system require much greater computational and memory resources. Consider the HPLC-DAD scan range of 200–400 nm; if the resolution were set for 1 nm and the absorption data were represented by an 8-bit value, the collection device will be required to collect 62 kilobits per second. Over one megabyte of memory would be required for a single 30-minute chromatographic run. Compared to HPLC-DAD, hyphenated mass spectrometry (gas chromatography, GC-MS, or liquid chromatography, LC-MS) techniques have both greater memory and computational requirements because of higher scan rates and increased mass axis resolution. In today’s computation environment, personal computer’s computation speed is measured in gigahertz and memory is measured in gigabytes; personal computers having 256 kilobytes of memory were quite uncommon in 1985.

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FIGURE 5 Negative ion attachment (Cl - ) mass spectra at retention time13.7 minutes (top), and with background subtraction (bottom). Note chloride ion attachment pseudomolecular ion at m/z 262 with correct isotope ratio for a single chlorine.

Recent reviews for hyphenated techniques have also been published,14,15 and the ﬁeld continues to grow rapidly. As of writing the present chapter, Chemical Abstract Services lists over 2600 entries for LC-NMR alone! The possibility of presenting an authoritative work on the whole topic of hyphenated characterization techniques in a single chapter would be, therefore, impossible. Instead, a few techniques have been chosen, based on the author’s experience, to be common and useful. The vast majority of these examples are organic qualitative analyses at trace levels to address the commonly asked question in the pharmaceutical industry: ‘‘What is it ?’’.

13 HYPHENATED CHARACTERIZATION TECHNIQUES

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FIGURE 6 HPLC-UV (204 nm) chromatogram (top) and extracted ion chromatogram (m/z 262) using negative ion attachment (Cl- ) atmospheric pressure chemical ionization for nitroglycerin tablet.

Advances in analytical instrumentation and techniques are such that the amount of an impurity available in a single injection can be used to obtain a multitude of data, from chromatographic retention time to nuclear magnetic resonance (NMR) spectra. The introduction of hyphenated techniques to the arsenal available to pharmaceutical scientists has led to increased efﬁciency for elucidation of organic chemical structures of impurities and degradants. The International Conference on Harmonisation16 guidelines have set the acceptable limit for unknown related compounds of an active pharmaceutical ingredient (API) in both drug substance and drug product at no greater than 0.1% levels. Many ethical pharmaceutical manufacturers require identiﬁcation at levels approaching 0.05%. All of these reporting levels depend on the assay or stability-indicating methodology chosen and may be only semiquantitative, usually area percent in an HPLC-UV method. These assay methods are not developed to provide optimal sensitivity for all unknown processes or degradants; rather they are developed to be robust in the manufacturer’s release laboratory. Mobile phases generally consist of easily prepared, stable pH organic solvents; inexpensive columns are used; pumps and detectors are chosen for ruggedness and ease of repair. It also should be noted that the absolute amount analyzed per reporting value is always very small, i.e., individual injections contain only a few micrograms, which are meant to represent batches of many kilograms. Finished and marketed drug products (formulations) may contain active ingredients at vastly different levels, from almost entirely pure drug substance to parts-per-million (ppm) levels. Therefore the characterization of drug product impurities in formulations is always a challenging project. Before any characterization technique is chosen, the amount available for analysis

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T. N. FEINBERG

should be considered. If the project is to identify a 0.1% impurity in a drug product with an active level at 10 ng per mL, then in a sample of 500 mL of the drug product only 5 ng of the impurity are available. The amount of information that can be obtained on such a small sample is limited. Table 3 provides a rough guide to the needed amount (per injection or analysis) of an unknown to obtain useful information. Fortunately, these necessary target levels are decreasing as instrument manufacturers push the detection limits of their instruments lower and lower.

II. EXPERIMENTAL EXAMPLES Many problems faced during the characterization or identiﬁcation of unknown impurities or degradants do not need full proof of structure (molecular connectivity) information; in most cases, all that is needed to resolve a problem is to observe (or not observe) a speciﬁc structural feature. When faced with an identiﬁcation (or elucidation) of an unknown it is useful to decide whether consistent or conﬁrmed data is required. As shown previously, there is a ﬁnite probability that any two chemical compounds will share similar retention times. Thus, if an unknown compound is retained at a speciﬁc retention time and a sample of a probable match is obtained with a similar retention time, the data are considered consistent. If both the mass and UV spectra also match, then the identity is conﬁrmed. A conﬁrmed structure uses the minimum number of data sets to exclude all other possibilities. Currently, there is no consensus among pharmaceutical scientists as to the amount of additional data necessary to conﬁrm a structure. As will be discussed later, when an authentic standard of an impurity is available, the ‘‘three ion rule’’ can be used with mass

TABLE 3 Minimum Target Mass Levels for Usable Hyphenated Method Data Using Different Separation and Characterization Modes Separation mode

Characterization method

Destructive?

Minimum level (kg)

Capillary GC

IR

No

Capillary GC

Electron ionization MS

Yes

Capillary GC

Chemical ionization MS

Yes

0.01–1

Capillary GC

AES

Yes

0.0001–0.01

RP-LC (Analytical)

DAD

No

0.01–1

RP-LC (Analytical)

Fluorescence

No

0.001–0.1

RP-LC (Analytical)

API MS

Yes

0.01–1

RP-LC (Microscale)

API MS

Yes

0.001–0.1

RP-LC (Analytical)

IR

No

0.1–10

RP-LC (Analytical)

NMR

No

Polyacrylamide gel electrophoresis (PAGE)

MALDI-MS

Partially

0.01–1 0.001–0.100

0.1–10 0.001–0.1

13 HYPHENATED CHARACTERIZATION TECHNIQUES

351

spectrometric detection.17 This is the only recognized standard for conﬁrmation by the FDA at this time.

A. Absorption Spectroscopy Absorption spectroscopy is the simplest type of second-stage analysis for hyphenated techniques to understand. The inherent problem with absorption spectroscopy is that the desired signal is presented as a difference from a reference amount, i.e., the transmission is less than the incident. Since at low levels of impurities (the usual case) absorption is small, there is a limit to the amount of signal available to interpret. UV, IR, and NMR are all forms of absorption spectroscopy; but NMR, because of its widespread use as the ‘‘ultimate’’ structural characterization tool will be treated in a separate section. 1. Ultraviolet Absorbance Spectroscopy The ﬁrst exposure to spectroscopy for most scientists is ultraviolet/ visible absorbance. As virtually every HPLC chromatograph employed in the pharmaceutical industry uses UV absorbance as the detection method, it is no wonder that the most popular hyphenated technique is HPLC-DAD. DAD spectrographs have been coupled to all liquid-based chromatographic systems including HPLC (preparative, analytical, and microbore), capillary electrophoresis (CE) and supercritical ﬂuid chromatography (SFC). There have been several successes with TLC plates,18 but it is more common for developed plates to be scraped and the sample analyzed ofﬂine. The quantitative aspects of UV absorption are dependent on the molar absorptivity, path length, the incident light, and the stability of the detector. HPLC with UV detection can detect very small amounts (10 pg) of compounds with extremely high molar extinction coefﬁcients ("100,000 M1 cm1). The detection limits for other chromatographic modes is often hampered by the small size of the columns (capillary HPLC, and CE) and the resultant small path length. Several manufacturers have designed bubble and Z cells to increase the sensitivity, but the enhancement gained is at the expense of mixing and potential band broadening. As a characterization technique, UV absorption can be used as an important piece of conﬁrmatory evidence for a proposed structure. Simple rules for the prediction of absorption maxima for structures can be found in introductory texts.19 More advanced techniques for the uses of HPLC-DAD have been previously presented.20 Often, HPLC-DAD data are used to show that an impurity or degradant is similar (electronically) to the drug substance by virtue of the same chromophore.21 2. FourierTransform Infrared Spectroscopy (FTIR) Fourier transform infrared spectroscopy (FTIR) provides a useful ‘‘ﬁngerprint’’ of an organic molecule.19 FTIR is insufﬁciently speciﬁc for a de novo identiﬁcation but provides useful starting data. Unfortunately, as

352

T. N. FEINBERG

molecular mass increases above 100 Da, the resolution and utility of FTIR decreases dramatically. Consequently, de novo structure proofs for multifunctional pharmaceutical compounds rarely begin with infrared spectroscopy. FTIR is useful when the presence or absence of a strongly absorbing functional group (e.g., carbonyl) can be used to conﬁrm or deny a proposed structure. A recent review of the use of LC-IR in pharmaceutical analysis has been published.22,23 Fourier transform infrared spectroscopy spectrometers have been coupled to chromatographs by either gas-phase interfaces or deposition interfaces.24 These instruments used a high temperature oven with a large (approx. 20 cm) path length to achieve modest sensitivity (1–100 mg).25 There are a number of present-day couplings that rely on the deposition of the efﬂuent of chromatographs (gas phase via cryofreezing, liquid phase by direct deposition) onto plates, either transparent or reﬂecting. As the chromatogram proceeds, the deposition point is continuously moved to a new position. The spectrum is read back by stepping the plate across the focal point of a spectrometer, as depicted in Figure 7.24 As gas chromatographs typically use helium as the carrier gas, the peak of interest will always have a lower boiling point than the carrier and, at a suitable temperature, will be deposited on the plate without interferences. Liquid chromatographs have been interfaced with FTIR spectrometers using both supercritical ﬂuid, nonaqueous, and reversed-phase mobile phases. SFC can also use a restrictor prior to deposition, where the mobile phase is gaseous but the analytes are semivolatile. Reversed-phase chromatography interfaces rely on mobile phases made of volatile components, as will be discussed for LC-MS. Not all methods are compatible with the technique, and chromatographic conditions need to be redeveloped for optimal separation and volatilization. An example of the type of data capable of being acquired by hyphenated FTIR techniques is presented in Figure 8, which shows the chromatography (top) and absorption mode spectroscopy of a mixture of acetaminophen, caffeine, and dodecanolactam.

FIGURE 7 Optical design of hyphenated FTIR transmission detection instrument (reproduced with permission from Bourne, 1998).

13 HYPHENATED CHARACTERIZATION TECHNIQUES

353

FIGURE 8 Example of HPLC-FTIR chromatograms for three drugs, acetaminophen, caffeine and dodecanolactam (top). Corresponding FTIR spectra are shown at the bottom (reproduced with permission from Bourne, 1998).

B. Mass Spectrometry Because of the variety of interfaces and the low detection limits, hyphenated mass spectrometers are among of the fastest growing markets for scientiﬁc instrument manufacturers in the 1990s and beyond. Every available type of chromatographic separation has been analyzed by mass spectrometry from desorption methods for TLC plates26 to online CE-MS.27,28 Not all techniques are routinely used for the characterization of small organic molecules of pharmaceutical interest (Table 4). The two workhorse instruments for the characterization of small molecules are GC-MS and

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TABLE 4 Suggested Ionization and Detection Methods Based on Analyte Polarity Polarity

MW

pKa

pKb

Ionization source

Detection

Nonpolar

< 500 Da

Low

Low

EI FI/FD

Positive

Polar

< 500 Da

Low

Low

CI (GC) APPI (HPLC)

Positive

Nonpolar

< 500 Da

High

Low

CI (GC) Electrospray (HPLC) APCI (HPLC)

Negative

Polar

< 500 Da

High

Low

CI (GC) Electrospray (HPLC) APCI (HPLC)

Negative

Nonpolar

< 500 Da

Low

High

CI (GC) Electrospray (HPLC) APCI (HPLC)

Positive

Nonpolar

> 500 Da

Low

Low

FI (GC)

Positive

Polar

> 500 Da

Low

Low

Electrospray (HPLC) APPI (HPLC) APCI (HPLC)

Positive (usually)

Nonpolar

> 500 Da

High

Low

Electrospray (HPLC)

Negative

Polar

> 500 Da

High

Low

Electrospray (HPLC) APCI (HPLC)

Negative

Nonpolar

> 500 Da

Low

High

Electrospray (HPLC) APCI (HPLC)

Positive

Polar

> 500 Da

Low

High

Electrospray

Positive

Note: EI is electron ionization, CI is chemical ionization, FI/FD is field ionization/field desorption, APPI is atmospheric pressure photoionization, and APCI is atmospheric pressure chemical ionization.

LC-MS. In addition to the present volume, there have been a number of recent reviews of MS in the ﬁeld of pharmaceutical science.29 The starting point for mass spectrometric characterizations is often the determination of the molecular weight. If the impurity arose after exposure to oxygen and a mass shift of 16 is observed between the drug substance and the impurity, it can be concluded that the impurity is oxidized drug substance. High mass accuracy mass spectrometry may also be used for molecular formula determination. After observation of a molecular or pseudomolecular ion, the most important feature of a mass spectrum is the fragmentation behavior, i.e., the differences between the observed ion mass-to-charge ratios and their relative abundances. The rules for structural elucidations for electron ionization (EI) are well known and accessible. The reader is referred to McLafferty and Turacek (1993) either as an introduction or as a refresher.30 It is remarkable that EI mass spectra have been compiled for use to identify unknown compounds. Over 130,000 different compound spectra have been compiled and are easily accessible by computer software. Sphon (1978) published results showing that one could retrieve a correct identiﬁcation for the veterinary drug residue diethylstilbestrol (DES) out of the then 10,000 or so library of EI mass spectra by referencing only 3 ions and their relative abundances with 5% tolerances.17 These rules still hold true

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today with over 138,000 available spectra! This ‘‘three ion rule’’ is still the only FDA – recognized standard for structural conﬁrmation.31,32

C. Nuclear Magnetic Resonance (NMR) Spectroscopy The newest instrument in the arsenal of the pharmaceutical scientist is the combination of chromatography with nuclear magnetic resonance (NMR) spectroscopy.33 The ability to perform these types of experiments has only recently been made possible with the advent of advanced solvent suppression routines and high ﬁeld strength (greater than 400 MHz) spectrometers. In addition to online data acquisition for LC-NMR, there are two alternative arrangements for data acquisition: stopped-ﬂow chromatography and automated loop collection/reinjection. Stopped ﬂow LC-NMR is performed by stopping the HPLC pump, after a predetermined delay, at the apex of peak as determined by an inline UV detector. The delay accounts for the transfer from the UV detector to the NMR ﬂow cell. The entire range of multidimensional NMR techniques is subsequently possible. The former has the advantage of simplicity, but with the possibility of adversely affecting the chromatographic integrity (diffusion and band spreading) especially for longer retained compounds. Atoms that have isotopes with non-zero nuclear spin (such as 1H and 13C) split their energy levels in an applied magnetic ﬁeld. A resonant radio frequency (rf) burst of energy can drive transitions between these levels, giving rise to an NMR signal (see Chapter 12). Local magnetic ﬁelds (nearby nuclei and ring currents) in a molecule perturb the resonant frequency from that expected for a free nucleus. Sophisticated NMR experiments allow the transfer of magnetization from one nucleus to the next, allowing the spectroscopist to virtually walk along the molecule, one atom at a time, giving a complete picture of all of the molecular connectivities. The uses of NMR in pharmaceutical science have recently been reviewed in detail by Holzgrabe et al. (1998). There are compatibility issues for chromatographic methods and NMR. Protic solvents such as water or methanol are usually replaced with deuterated solvents although solvent suppression algorithms can be used with shallow gradient methods. Organic modiﬁers such as ion-pair reagents must be avoided, and mineral acid based buffer systems are preferred (the opposite of the LC-MS situation).34 An example taken from Kolbert et al. (in press) is presented below.35 A sample of the drug substance ranitidine was spiked with three commercially available impurities at three different levels relative to the drug substance, 10, 1, and 0.1%. As an example of the possibilities for LC-NMR, Figure 1 shows the resulting UV absorbance chromatogram (229 nm) of a 1-mg injection of ranitidine and the corresponding impurities. Note that the chromatography over the course of the acquisition (stopped ﬂow) shows well-separated peaks. To the right of the chromatogram, individual proton spectra are shown. The spectrum for the 0.1% level impurity was averaged using 23,880 acquisitions. An example of an on-ﬂow experiment is shown in Figure 9. A two-dimensional (total correlation spectroscopy-TOCSY)

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FIGURE 9 On-flow HPLC-UV chromatogram (229 nm) and 1HNMR detection of a 1mg sample of ranitidine HCl spiked with its related substances A^C. UV absorbance data are presented on the y-axis, and proton chemical shift data are presented on the x-axis.

spectrum, acquired in stopped-ﬂow mode, was obtained for the 1% level impurities as shown in Figure 10 on page 357.

III. CONCLUSIONS The future of the pharmaceutical industry rests on the ability to discover and develop new molecules into products. The faster that information can be delivered to product managers and regulatory scientists, the greater the potential for shortening the time to market. Consequently, there is an economic pressure to devise instruments able to deliver many data sets at once. Coupling of instruments has become easier with expanded capabilities (HPLC-DAD-MS is almost routine, HPLC-DAD-NMR-MS is now a common commercial instrument). Another pressure on pharmaceutical scientists is the promise of biopharmaceuticals and high-potency APIs. These compounds often have complicated impurity and degradation proﬁles at low absolute concentrations. Fortunately, instrument manufacturers have been quick to attempt to satisfy both of these needs. The challenge now to the pharmaceutical scientist is the organization of the many data sets into presentation quality so that scientists and managers can make correct decisions quickly. Software has been developed to speed the collation, analysis, and presentation of the many spectroscopic characterization techniques necessary. Detection limits for mass spectrometers are now approaching the zeptomole level, and NMR spectrometers have recently seen dramatic increases in the sensitivity down

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FIGURE 10 TOCSY spectrum of ranitidine related compound A at the 1% level relative to ranitidine HCl (10 mg on column).

to the nanogram level. Instrument designers seem to enjoy the challenges that the industry presents and are willing to bring to market instruments with the pharmaceutical scientist in mind.

ACKNOWLEDGMENTS The author wishes to thank Drs. Charlotte Carroll, Alan Hendricker, and Andrew Kolbert for their help in reviewing this manuscript.

REFERENCES 1. FDA, Federal Food, Drug, and Cosmetic Act, 21 USC Chapter V (2 January 2001). 2. Giddings, J. C. Maximum number of components resolvable by gel filtration and other elution chromatographic methods. Anal. Chem. 39:1027–1028, 1967. 3. Giddings, J. C. and Dahlgren, K. Resolution and peak capacity in equilibrium-gradient methods of separation. Separ. Sci. 6:345–356, 1971.

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4. Giddings, J. C. and Davis, J. M. Statistical theory of component overlap in multicomponent chromatograms. Anal. Chem. 55:418–424, 1983. 5. Snyder, L. R., Kirkland, J. J. and Glajch, J. L. Practical HPLC Method Development, 2nd ed., John Wiley and Sons, NY, Chapter 2, 1997. 6. Kreyszig, E. Advanced Engineering Mathematics, 5th ed., John Wiley and Sons, NY, p. A-56, 1983. 7. George, S. A. Historical Developments. In Diode Array Detection in HPLC (L. Huber and S.A. George, eds.), Marcel Dekker, NY, Chapter 1, 1993. 8. Go¨ro¨g, S., Babja´k, M., Balogh, G., Brlik, J., Csehi, A., Dravecz, F., Gasdag, M., Horva´th, P., Lauko´, A. and Varga, K. Drug impurity profiling strategies. Talanta. 44:1517–1526, 1997. 9. Reynolds, D. W., Facchine, K. L., Mullaney, J. F., Alsante, K. M., Hatajik, T. D. and Motto, M. G. Available guidance and best practices for conducting force degradation studies. Pharm. Tech. 26:48–56, 2002. 10. Alsante, K. M., Friedman, R. C., Hatajik, T. D., Lohr, L. L., Sharp, T. R., Snyder, K. D. and Szczesny, E. J. Degradation and impurity analysis for pharmaceutical drug candidates. Sep. Sci. and Tech. 3:85–172, 2001. 11. Lohr, L. L., Sharp, T. R., Alsante, K. M. and Hatajik, T. D. Isolation and identification of process related impurities and degradation products from pharmaceutical drug candidates: Part II—the roles of NMR and mass spectrometry. American Pharmaceutical Review 4:70–78, 2001. 12. Bertsch, W. Multidimensional gas chromatography. In Multidimensional Chromatography: Techniques and Applications (H.J. Cortes, ed.), Marcel Dekker, NY, Chapter 3, p. 139. 1990. 13. Norwood, D. L., Feinberg, T. N., Cain, T. C. and Lennon, J. D. Applications of chloride ion attachment LC/MS in pharmaceutical development. Presented at the 50th Southeastern Regional Meeting of the American Chemical Society, November 4–7, Research Triangle Park, NC, 1998. 14. Bruno, T. J. A review of hyphenated chromatographic instrumentation. Separation and Purification Methods, 29:63–69, 2000. 15. Albert, K., Krucker, M., Glaser, T., Schefer, A., Lienau, A. and Zeeb, D. Hyphenated techniques. Anal. and Bioanal. Chem. 372:25–26, 2002. 16. International Conference on Harmonisation (ICH) Guideline: Impurities in New Drug Products, Federal Register, 62, No. 96, pp. 27454ff. (May 19, 1997). 17. Sphon, J. A. Use of mass-spectrometry for confirmation of animal drug residues J. Assoc. Off. Anal. Chem. 61:1247–1252, 1978. 18. Gocan, S. and Cimpan, G. Compound identification in thin layer chromatography using spectrometric methods. Reviews in Analytical Chemistry. 16:1–24, 1997. 19. Dyer, J. R. Applications of Absorption Spectroscopy, 2nd ed. Prentice-Hall, Englewood Cliffs, NJ, Chapter 2, 1965. 20. Guiffre, R. W. The use of diode array detectors in the pharmaceutical industry. In Diode Array Detection in HPLC (L. Huber and S.A. George, eds.), Marcel Dekker, NY, Chapter 6, 1993. 21. Gorog, S. The role of the diode-array UV spectra in the identification of impurities. Prog. Pharm. Biomed. Anal. 4:252–265, 2000. 22. DiNunzio, J. E. Pharmaceutical applications of high-performance liquid chromatography interfaced with Fourier transform infrared spectroscopy. Journal of Chromatography. 626:97–107, 1992. 23. Geldart, S. LC-IR of pharmaceuticals. American Laboratory. 32:32–36, 2000. 24. Bourne, S. An online direct-deposition FTIR detector for chromatographs. American Laboratory. 30:17F-17J, 1998. 25. Smith, S. L. and Adams, G. E. Chromatographic performance and capillary gas chromatography–Fourier transform infrared spectroscopy. Journal of Chromatography. 279:623–630, 1983. 26. Chang, T. T. and Andrawes, F. Direct analysis of TLC spots by FAB mass spectrometry In Proc. Int. Symp. Instrum. High Perfom. Thin-Layer Chromatogr 3 (Kaiser, R. E, ed.), 427–433, 1985.

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27. Hamdan, M. Pharmaceutical applications of liquid chromatography, capillary electrophoresis coupled to mass spectrometry. Process Control Qual. 10:113–127, 1997. 28. Cherkaoui, S., Rudaz, S., Varesio, E. and Veuthey, J.-L. Online capillary electrophoresis electrospray mass spectrometry for the analysis of pharmaceuticals Chimia. 53:501–505, 1999. 29. Ermer, J. and Vogel, M. Applications of hyphenated LC-MS techniques in pharmaceutical analysis. Biomedical Chromatography. 14:373–383, 2000. 30. McLafferty, F. W. and Turacek F. Interpretation of Mass Spectra, 4th ed., University Science Books, Sausalito, CA, 1993. 31. Federal Register 42:10412–10437. 32. FDA (draft 2001) Guidance for industry: mass spectrometry for confirmation of the identity of animal drug residues. 33. Holzgrabe, U., Diehl, B. W. K. and Wawer, I. NMR spectroscopy in pharmacy. J. of Pharm and Biomed Anal. 17:557–616, 1998. 34. Lindon, J. C., Nicholson, J. K. and Wilson, I. D. Directly coupled HPLC-NMR and HPLCNMR-MS in pharmaceutical research and development. Journal of Chromatography B. 748: 233–258, 2000. 35. Kolbert, A. C., Carroll, C. A. and Huff, L. A. Nuclear magnetic resonance in pharmaceutical development. In Biopharma Guide to Analytical Methods (in press).

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14 SOLVING IMPURITY/DEGRADATION PROBLEMS: CASE STUDIES KAREN M. ALSANTE,TODD D. HATAJIK, LINDA L. LOHR, DINOS SANTAFIANOS, AND THOMAS R. SHARP Pfizer, Inc.,Groton,CT 06340

I. INTRODUCTION AND BACKGROUND A. The Drug Development Time Line B. The Process C. The ICH Guidelines D. Evaluation of Known Standards E. Cases with No Standard Match F. LC/MS Analysis G. Preparative IsolationVersus Small-Scale Synthesis H. NMR Characterization II. CASE STUDIES A. Degradation Case Studies B. Process-Related Impurities Case Studies III. SUMMARYAND CONCLUSIONS APPENDIXLESSONS LEARNED REFERENCES

I. INTRODUCTION AND BACKGROUND The objective of this chapter is to provide guidance for isolating and identifying process-related impurities and degradation products from pharmaceutical drug candidates using actual case studies. The identiﬁcation of process-related impurities and degradation products can provide an understanding of impurity formation and deﬁne degradation mechanisms. If the identiﬁcation process is performed at an early stage of drug development, there is adequate time to address the drug substance process and drug product formulation to prevent or control these impurities and degradants from forming long before the ﬁling stage. Impurity and degradant structure elucidation is a collaborative effort involving the analytical chemist, process chemist and/or formulator, as well as the degradation, mass spectrometry, and NMR experts. The process described in this chapter uses a designed approach for the impurity and/or degradant identiﬁcation, which focuses on

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efﬁciency so that the success of data collection is maximized and project time lines are met. There are a number of activities other than collecting experimental data, even though the experiments are central to the process. Some of these key activities include collecting project background information prior to pursuing experimental work, asking the right questions, and meeting with project analysts and structure elucidation experts. The activities associated with the overall process are captured in the process ﬂowchart provided in Figures 1–3.1

FIGURE1 Impurity/degradant isolation and Identification process flowchart (RRT ¼relative retention time; PRI ¼process-related impurity).

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FIGURE 2 Impurity/degradant isolation and identification process flowchart.

A. The Drug Development Time Line One of the most important aspects of the project that determines approach is where the pharmaceutical drug candidate is in the drug development time line. Typically, the focus will be on collecting LC/MS data only through the phase 1 clinical stage because of limited analytical resources compared to the number of compounds in early development. At the phase 2 clinical stage and beyond, more time is invested in isolation, synthesis, and structural identity using NMR characterization of forced degradation products and impurities of concern. At this stage, the pharmaceutical drug candidate has a better chance of making it to market. If an unknown degradant or impurity is at a critical threshold and drug substance and/or drug product release for clinical supplies could be impacted, the most efﬁcient technique for identiﬁcation will be used. For these cases, isolation, synthesis, LC-NMR, and/or traditional NMR are used no matter what stage of development: phase 1 all the way through registration.

B. The Process As already emphasized, the process of identiﬁcation of impurities and/or degradants begins early in drug development. Early brainstorming sessions should involve the analytical chemist, process chemist, formulator, and degradation chemist, as well as the mass spectrometry and NMR experts. It is imperative to involve all that are familiar with the project of interest. The

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FIGURE 3 NMR spectroscopy identification process flowchart.

group meets to assess the time lines for completion and to gather all pertinent information. This initial planning and discussion effort can save signiﬁcant time in the experimental stage. A few questions that need to be answered at this early stage are . . . . . .

Is this an impurity or degradant problem? Is it a drug substance, drug product, or excipient-related problem? At what level is the impurity/degradant present? Is it a process-related impurity (PRI); and if so, at what step of the process is it formed? Is it a degradant; and if so, under what degradation condition is it formed? Are enriched samples with the unknown impurity/degradant available?

By gathering all relevant information, the most efﬁcient method of isolation and identiﬁcation can be selected. Many more unexpected questions

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and insights can arise at the early meetings that can improve the efﬁciency and quality of the identiﬁcation process.

C. The ICH Guidelines The ﬁrst step of the process is to determine at what level the unknown is present. According to the ICH Guidelines on Impurities in New Drug Substances:2 The studies conducted to characterize the structure of actual impurities present in the new drug substance at a level greater than 0.1% (depending on the daily dose, calculated using the response factor of the drug substance) should be described. Note that all speciﬁed impurities at a level greater than the identiﬁcation threshold in batches manufactured by the proposed commercial process should be identiﬁed. Degradation products observed in stability studies at recommended storage conditions should be similarly identiﬁed. When the identiﬁcation of an impurity is not feasible, a summary of the laboratory studies demonstrating the unsuccessful effort should be included in the application.

According to the ICH Guidelines on Impurities in New Drug Products:3 Degradation products observed in stability studies conducted at recommended storage conditions should be identiﬁed when present at a level greater than the identiﬁcation thresholds (1% for a maximum daily dose of <1 mg to 0.1% for a maximum daily dose of > 2 g).

Identiﬁcation of impurities below the 0.1% level is generally not necessary unless the potential impurities are expected to be unusually potent or toxic.2 Therefore, it is imperative to determine the level of the unknown impurity and/or degradant early in the process. If the unknown is below the 0.1% threshold, then a discussion will need to take place among the project team members in order to determine if isolation and identiﬁcation are necessary. However, if the unknown is at or above the 0.1% limit, then effort should be put forth for identiﬁcation.

D. Evaluation of Known Standards Once a decision has been made to identify an unknown, the next logical step is to evaluate all known process-related impurities, precursors, intermediates, and degradation products. By observing the relative retention times (HPLC) of all known process-related impurities, precursors, and intermediates (if available), one can quickly determine whether or not the impurity of interest is truly unknown. If the relative retention time of the unknown impurity matches that of a standard, the unknown can be identiﬁed using HPLC with ultraviolet (UV) photodiode array detection as well as mass spectrometry (MS) detection. The identity can be considered conﬁrmed by correlating the retention time, UV spectra, and mass spectra of the unknown impurity with that of the standard. The time and energy saved by analyzing data that may be already available can be considerable.

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E. Cases with No Standard Match Identifying an unknown by using a standard, as described in the above paragraph, is a quick and easy process. However, what happens when the relative retention time of an unknown does not match that of a standard? The next step is to obtain molecular mass and fragmentation data via HPLC/ MS. It is essential to determine the molecular mass of the unknown. Not only does the molecular mass help in the identiﬁcation of the unknown, but it also enables one to track the correct peak by HPLC if isolation becomes necessary. In order to run LC/MS, a mass spectrometry compatible HPLC method must be available. The mobile phase should contain volatile buffers that are HPLC/MS-compatible. If such a method is not available, then one must be developed, which adds time to the identiﬁcation time frame.

F. LC/MS Analysis When a new unknown impurity is observed, as a ﬁrst experiment, the sample is analyzed by LC/MS. If the structure of the unknown impurity cannot be conclusively elucidated by LC/MS data, LC-NMR can be employed to analyze the sample. If the sample is not suitable for LC-NMR analysis, the impurity needs to be puriﬁed for NMR characterization. If the mass spectrometry data evaluation yields sufﬁcient structural information, this eliminates the need to isolate the impurity in question. If standards of the proposed structures are available, they can be correlated with the unknown as previously described. If standards are not available, which is usually the case, the proposed structures can be discussed with the project team. The project team can then decide if the information is suitable for their needs or if isolation is required. It is essential for a thorough analysis of the pros and cons of each ionization technique. For example, knowing that a degradant might be unstable enables the selection of a delicate ionization technique that will not destroy the degradant prior to mass spectral detection.

G. Preparative Isolation Versus Small-Scale Synthesis Preparative HPLC is typically the technique of choice for impurity puriﬁcation. It is often necessary to enrich the impurity before preparative HPLC puriﬁcation. Various techniques such as solid-phase extraction can be used to enrich the low-level impurity. To ultimately conﬁrm the structure of a new impurity, it may be necessary to synthesize the compound and compare its spectroscopic characteristics to those observed in the original sample. A very effective means of getting useful structural information is to conduct a degradation study on the puriﬁed impurity. An alternative to isolation is small-scale synthesis. If possible, structures have been proposed from the mass spectrometry data, one can study the synthetic chemistry and determine at which step of the process the impurity

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and/or degradant is most likely to be formed. By knowing the synthetic chemistry, the feasibility of the proposed structures can be evaluated. Proposed structures can then be synthesized if a reasonable synthesis is available. It is easier to synthesize and identify the unknown if the chemistry works quickly (i.e., one-step/straightforward chemistry). If small-scale synthesis is chosen, the synthesis must be the most efﬁcient route. At this stage of the process, it is frequently necessary to isolate and characterize the unknown. One of the most important factors to consider when approaching an isolation experiment is the sample origin. It is vital to determine whether the unknown is an impurity and/or degradant and to locate a sample that contains an enriched quantity of the unknown. Isolating low-level impurities can prove to be very cumbersome and timeconsuming. Therefore, the ultimate goal is to ﬁnd a sample that contains an enriched quantity of the unknown. Two great resources of enriched samples are retained mother liquor samples and purposeful degradation/ stability samples. If the unknown is a drug substance degradant, then the degradation reaction can be scaled up to generate a large quantity of the unknown. If it is a drug product degradant, then effort should be put forth to form the degradant in the drug substance so that extraction from the excipients is not required. Whenever enriched samples are not available, the unknown must be isolated from the bulk drug substance or drug product. A number of methods can be used for isolating impurities and/or degradants. Three of the most utilized techniques discussed earlier in Chapters 9 and 10 are thin-layer chromatography (TLC), ﬂash chromatography (column chromatography), and preparative high-performance liquid chromatography (HPLC). The actual technique used depends on the nature of the impurity and/or degradant, including the amount present in the original material from which it must be isolated. A good starting point is to assess the separation that is currently being used by analytical chemists. Does the current methodology provide optimum resolution of the impurity/degradant from the main band and other impurities, and if so, is that method by TLC or HPLC? This is a key factor in determining which technique to utilize.

H. NMR Characterization Nuclear magnetic resonance (NMR) spectroscopy is used as a complementary technique to LC/MS. It is a powerful analytical tool for structural elucidations, particularly for small molecules. It provides key structural information and intramolecular connectivity not readily obtainable by any other analytical methods. While mass spectrometry typically uses fragmentation patterns and total mass to suggest a general family of plausible structures, NMR spectroscopy reﬁnes the molecular connectivity to elucidate a single, speciﬁc structure and, if desired, stereochemistry. Recent technology advancements in the ﬁeld of magnetic resonance have made signiﬁcant strides in improving sensitivity limits of this technique. This becomes particularly critical in the structural elucidation of pharmaceutical drug impurities and

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degradants, which are often extremely mass- and sometimes solubilitylimited. The nondestructive nature of NMR makes it an especially valuable tool for impurity and degradant characterization, since these are typically valuable samples that may need to be revisited in the future. The NMR strategy previously outlined serves as a guideline for the approaches taken in the following case studies.

II. CASE STUDIES Actual case studies using these isolation and syntheses as well as mass spectral and NMR approaches are outlined in the remainder of this chapter. This collaborative multidisciplinary strategy is applied to the structure elucidation of all the impurity and degradant case studies presented. The successful elucidation of these structures was essential to predict potential toxicity, set threshold limits, and identify ways to prevent or greatly reduce their formation. The case studies have been organized into degradation and process-related impurities examples.

A. Degradation Case Studies 1. Case Study A.1: N-OxideVersus Sulfoxide Differentiation Using Preparative HPLC, LC/MS, and NMR Characterization

In this ﬁrst case study, HPLC analysis of multiple drug substance samples (bulk release, stability, and forced degradation) indicated the presence of a very polar impurity/degradant, eluting just after the solvent front. A twomonth time frame was allotted for identiﬁcation. In collecting background information on the project, it was noted that the degradant steadily increased in stability samples as well as in oxidative forced degradation experiments.1 LC/MS data indicated a molecular weight of Mþ16. LC/MS could not conﬁrm the site of oxidation (N-oxide versus sulfoxide); therefore, NMR work was required. Since forced degradation generated the degradant, a scaled-up oxidative challenge was performed on the drug substance to prepare an enriched quantity of [Mþ16] degradant for isolation and identiﬁcation. Reaction conditions for the scaleup were selected based on previous forced degradation oxidative data. Oxidative degradation for 10 days, with an anhydrous methanol solvent system, yielded the most

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abundant quantity of the degradant (20%). Reversed-phase preparative HPLC isolation was problematic since the Mþ16 degradant was extremely polar and eluted in the void volume. The LC/MS data conﬁrmed that the isolated degradant had a molecular mass of Mþ16. Removal of the solvents by evaporation unfortunately resulted in a high level of residual ammonium acetate in the NMR sample that greatly compromised the detection of the target degradant signals. Even more important, the 87:1 salt:degradant content dramatically changed the magnetic susceptibility of the sample, making the NMR probe untuneable. Running the NMR experiments using an untuned probe would greatly compromise the achievable sensitivity. Therefore, the sample was further puriﬁed. The sample was placed in a lyopholizer to try to remove the ammonium acetate, but the attempt was not successful. An alternate method of isolation was developed to obtain a cleaner sample. Normal-phase chromatography was attempted because of problems experienced with reversed-phase chromatography. A drug substance oxidation solution was analyzed by TLC using an ethyl acetate/methanol/triethylamine solvent system. This mobile phase was found to be suitable for a large-scale puriﬁcation by column chromatography on silica gel. The solution was puriﬁed by medium pressure (40 psi) column chromatography on silica gel using ethyl acetate/methanol/triethylamine as the eluent. Fractions containing the desired material were concentrated by evaporation and dried under high vacuum, to give a clear colorless oil. The oil was dissolved in a small amount of methanol/ ethyl acetate and concentrated by evaporation. This step was repeated twice. The oil was dissolved in methanol/ethyl acetate cooled to 0 C (affording traces of a white solid) and treated with sufﬁcient hexanes (added slowly) to effect slow precipitation of a white solid. The solid was collected by ﬁltration and dried under high vacuum overnight to give a white solid (142 mg). A second crop was obtained and dried overnight under high vacuum to give (52 mg). Samples were both demonstrated to have the same Rf by TLC analysis. The LC/MS data conﬁrmed that the isolated degradant had a molecular mass of Mþ16, a critical step to conﬁrm no further degradation on isolation/ concentration prior to detailed NMR analysis and interpretation. The integrity of the sample was conﬁrmed by correlating the retention time and mass spectra with that of the unknown acid Mþ16 degradation sample. A complete NMR characterization1 was performed on this puriﬁed sample, as well as on the parent free base and related sulfone degradant for comparison. Methanol was chosen as the solvent, since solubility in methanol was demonstrated in the LC method development. The parent was also soluble in methanol, which allowed for a straightforward comparison of chemical shifts. Enough sample was isolated (30 mg) to acquire a carbon spectrum, which was particularly useful to detect the quaternary carbon resonances in the proposed oxidation region. Compared to the parent carbon spectrum, signiﬁcant downﬁeld chemical shifts were observed for both quaternary carbons neighboring the sulfur entity. In addition, upﬁeld chemical shifts were observed for the aromatic methines immediately next to these quaternaries. The NMR data therefore conﬁrmed oxidation had occurred at the sulfur site, and hence the sample was identiﬁed as the sulfoxide of the parent structure.

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2. Case Study A.2: N-OxideVersus Sulfoxide Differentiation Using Synthesis/Preparative HPLC

In case study A.2, a degradant was observed at a greater than 0.1% level in drug product stability studies using ICH storage conditions. A oneweek time frame was allotted for the identiﬁcation process to meet the project time line. Oxidation of the compound was suspected by LC/MS by the presence of a Mþ16 degradant, suggesting the addition of oxygen; however, as in case study A.1, LC/MS analysis could not differentiate between an N-oxide or sulfoxide. Further NMR analysis was required to characterize the degradant. Since time was an important factor, the most efﬁcient route to obtaining a pure sample for NMR was determined to be a synthetic reaction. When considering synthesis as an option in the identiﬁcation of impurities, having a high degree of certainty in the structure of the impurity is necessary. A one- or two-step reaction can afford grams of material sufﬁcient for mass spectrometry and NMR analyses, correlation by HPLC, and for use as a standard for HPLC method development. Most important, the synthesis has to be short and efﬁcient. Multistep syntheses can become extensive research projects when complicated reactions do not work as expected. With this example, a single-step mild oxidation was planned in order to selectively synthesize the sulfoxide or the N-oxide. HPLC analysis of the crude reaction mixture would conﬁrm if the correct degradant was being formed. An aggressive oxidant would probably afford both degradants nonselectively; hence, a mixture of the drug substance was treated with a less aggressive excess of hydrogen peroxide–urea complex. Analysis of the reaction mixture indicated that the desired degradant was forming. The reaction afforded 94 mg of crude degradant. The purity was lower than desirable for NMR analysis, so further puriﬁcation was undertaken to facilitate structure elucidation by NMR. The existing analytical methodology contained perchloric acid that was not suitable for preparative HPLC isolation and LC/MS analysis because of safety concerns with concentration of perchloric acid and high probability of damage to the mass spectrometer over time. A mass spectrometry compatible method using a 0.1% acetic acid buffer was selected as a starting point. Minor method development produced a suitable method to separate the reaction components: the drug substance, the sulfoxide, and the N-oxide.

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By determining the maximum analytical load (load that maintains adequate resolution of the unknown component), the chromatographic conditions were scaled up and a semipreparative chromatographic method was developed for the isolation of the degradant. The degradant of interest was then isolated by preparative HPLC. Concentration of the isolate by evaporation and drying under high vacuum afforded 153 mg of degradant. The theoretical yield was 42 mg; therefore, the remaining 111 mg was attributed to ammonium acetate and solvent impurities from the mobile phase, a complication to be aware of in preparative HPLC. Analytical HPLC analysis of the concentrated sample showed a single peak with the retention time of the proposed N-oxide. It is essential to conﬁrm the retention time with the actual analytical method for purity and potency used by the project team before further NMR and mass spectrometry structure elucidation begins. Despite the presence of extremely large amounts of ammonium acetate, structure elucidation of the N-oxide by NMR spectroscopy was successful with no further cleanup. This is the exception rather than the norm in the isolation process. Of the 150 mg of sample yielded from the isolation process, less than 11 mg of isolate was attributed to the degradant. The remaining mass was due to residual ammonium acetate. As with Case A.1, this extremely high salt content signiﬁcantly shifted the magnetic susceptibility of the NMR probe, thus making probe tuning quite difﬁcult. In addition, the presence of a sizeable background component put high demands on the dynamic range of the NMR spectrometer’s receiver, making detection of the relatively small amount of degradant challenging. Methanol was chosen as the preferred solvent because it is commonly used for chromatography and has the ability to remove the solvent easily if needed. Unfortunately, the sample demonstrated poor solubility in methanol, thus reducing the sample concentration and hence the detected signal size. Methanol also prohibits the detection of labile protons, although this was not forecasted as an issue for this project. The proposed N-oxide structure is a charged species, so spectral simulations would not be typically reliable, since usually only neutral species are included in simulation databases.4 Despite these challenges, a full set of NMR experiments was performed. The data were interpreted and the N-oxide structure was successfully elucidated within 24 hours. Most noteworthy NMR observations included major chemical shift changes near the nitrogen site plus a broadening of the N-methyl resonance. This NMR evidence facilitated a subsequent structural elucidation of another degradant of the same parent species, which was determined to be a chloromethyl adduct binding at the same nitrogen site. This clearly demonstrates the potential reactivity of the N-methyl site. In terms of impact on the project, the N-oxide structural elucidation allowed for an appropriate speciﬁcation of the degradant, and clinical time lines were not impacted. It is advised in all preparative HPLC isolations that an analytical or preparative scale reinjection is performed to clean up the analyte of interest from the salt. This can include washing the analyte by reversed-phase HPLC (preparative or analytical scale depending on isolate amount) with aqueous phase and ramping up the organic phase to elute the desalted analyte of interest.

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3. Case Study A.3: Degradant Isolation Using Preparative HPLC, LC/MS, and NMR Characterization

Case study A.3 involves the same drug substance detailed in case study A.2. The drug substance was stored at ICH storage conditions of 40 C/75% relative humidity for six days. Samples analyzed by HPLC indicated the presence of an unknown degradant at the 0.6% level. The aim of this project was to isolate and identify this degradation product. This required a team effort from the following groups: Degradation, NMR, Mass Spectrometry, and Discovery. The molecular weight of the degradant was conﬁrmed by LC/ MS (M-262); however, very little structural information was obtained from LC/MS/MS fragmentation. As a result, isolation and further NMR analysis were required to complete the identiﬁcation project. In order to obtain a pure sample for traditional NMR, the degradant was isolated by preparative HPLC. The existing analytical method had the advantage of being mass spectrometry–compatible from the start with a mobile phase of 0.1% acetic acid pH adjusted with ammonium hydroxide solution and acetonitrile as the organic modiﬁer. Additional method development was not required, which can be time-consuming and adds to the isolation and characterization time-frame. On the preparative HPLC scale, degraded material at a concentration of 50 mg/ml gave a split peak. This was determined to be the result of the high concentration of the material loaded on the preparative HPLC column, another occurrence to keep in mind in the scale-up process. Sufﬁcient preparative runs were conducted to afford 6 mg of the degradant. LC/MS analysis on the pure isolated sample afforded greater sensitivity and conﬁrmed the molecular weight of the degradant (M-262). NMR structure elucidation indicated that a cyclized compound had formed. In particular, COSY-type proton–proton correlations gave the general ring structure shown. Key observations included a change from the parent proton spectrum of a methylene multiplet to a broad singlet, in addition to the maintenance of an electron-rich methine singlet. Parallel to this characterization effort, the Discovery project team had resource capacity and synthesized the proposed molecule. The NMR data for the isolated degradant and synthesized compound were in excellent agreement and hence supported the structure for the isolated degradant.

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Proposed Mechanism of Formation

(a) Formation of a radical cation at the sulfur atom, (b) allylic radical can exist in two canonical forms, (c) the second form can undergo a [1,5] hydrogen abstraction to transfer the radical to the side chain, (d) at some point loss of a proton can regenerate the vinylic sulﬁde, (e) the radical is well positioned to cyclize onto the vinylic double bond and the resultant radical can be quenched by radical abstraction from solvent, (f) the sulfur atom can oxidize to the sulfoxide, (g) the sulfoxide can form a double bond by synelimination resulting in loss of the sulfur containing side chain. 4. Case Study A.4: Scaled-up Oxidative Degradation and Isolation Using Solid-Phase Extraction and Preparative HPLC

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A degradant at a critical level ranging from 0.6% to 2.8% was observed in drug product stability samples stored at 30 C/60%RH for six weeks. The drug product vehicle was an acidiﬁed polymeric matrix making this case study A.4 particularly challenging. Identiﬁcation was required for the following reasons: (1) this degradant increased at 5 C, the storage condition for the drug product, (2) after six weeks at 5 C, the degradant was 0.1%, and (3) after 12 weeks at 5 C, the degradant was 0.18%. In order to set a higher speciﬁcation for this degradant so that a longer expiration date may be obtained for the drug product, isolation and identiﬁcation were needed. LC/MS indicated that the molecular weight of the unknown degradant was Mþ14, a possible ketone functionality. Since the oxidation could reasonably occur at different benzylic positions that are present in the parent molecule, further isolation and NMR analysis were required to identify the exact site of oxidation. This degradant was observed only during stability and forced degradation studies of the drug substance in the presence of the polymeric matrix. All stress testing studies on the drug substance ‘‘as is’’ without the polymer showed that the drug substance was stable. Therefore, isolation work could not be conducted on the drug substance alone, without the complex polymeric matrix. It is always advantageous when a drug product degradant can be replicated in drug-substance-only stress samples, eliminating the need to further isolate the degradant from the excipients. In this case, it required cleaning up the degradant from the complex polymeric matrix. To generate adequate amounts of the degradant of interest and decrease isolation time, a pressurized forced degradation oxidation reaction was performed on the polymer formulation in the presence of a free radical initiator. Approximately 14% of the degradant was generated by intentionally degrading the formulation. To isolate the degradant from the polymer formulation, Waters Oasis MCX ion-exchange solid-phase extraction (SPE) cartridges were utilized to separate the degradant from the complex polymers. Oasis MCX cartridges contain a mixed-mode polymeric sorbent with reversed-phase and cation exchange functionalities that are highly selective for basic compounds. The oxidation solution was placed on 100 Waters 6cc Oasis MCX cartridges prewashed with 0.1% hydrochloric acid. The loaded cartridges were washed sequentially with 0.1% hydrochloric acid, water, and ﬁnally methanol to remove the polymer. The remaining organic material was then eluted from the cartridges by washing with a solution of 5% ammonium hydroxide in methanol. Combining the ammonium hydroxide in methanol fractions afforded a clear colorless solution, which was concentrated by evaporation to give a white semisolid (1.64 g). This SPE cleanup successfully extracted the drug substance–related compounds from the polymer. The desired degradant was then isolated by preparative chromatography. The isolated degradant was analyzed by NMR and mass spectrometry for structural identity. Based on LC/MS data collected, the isolated degradant was conﬁrmed to have an addition of 14 Da to the molecular weight of the drug substance, indicative of a ketone moiety. NMR analysis conﬁrmed that the Mþ14 degradant contained a carbonyl group at the benzylic site shown.

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Comparing the proton and carbon spectra of the parent and degradant, it was apparent that the proton resonances at the benzylic site had disappeared, and the carbon resonance was no longer observed in the aliphatic region. Additionally, the proton and carbon resonances of the adjacent carbon atom were deshielded, with respect to the parent, in the degradant. The ketone oxidation product was conﬁrmed by both NMR and mass spectrometry, and the degradation mechanism outlined in Scheme 1 was proposed. Since the degradant was formed at higher levels with pressured oxygen/free-radical forced degradation conditions, a free-radical autoxidation mechanism involving Russell termination was proposed.5 Proposed Mechanism of Formation

5. Case Study A.5: Isolation of a Degradant with Same Molecular Weight as the Drug Substance Requiring NMR Characterization for Structure Elucidation

The current manufacturing process for the synthesis of the drug substance in case study A.5 generated an impurity at the 1–2% level. Identiﬁcation was required for a drug safety assessment on this impurity within a 1-month time line. LC/MS analysis suggested that the impurity had

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a different HPLC retention time but had an identical molecular weight. It was decided to isolate and thoroughly identify this impurity, using NMR in order to elucidate the structure. Isolation and identiﬁcation of the degradant was complicated by the structural similarity of the impurity with the parent compound, having the same molecular weight. This resulted in very similar HPLC retention times for the impurity and the main band. An enriched process-retained mother liquor sample containing approximately 4–5% impurity was obtained, and a new preparative-scale HPLC method was developed. The impurity was isolated by preparative-scale HPLC. According to LC/MS analysis, an early step in the bulk manufacturing process for the synthesis of the drug substance generated a ‘‘precursor impurity’’ with an identical molecular weight to the desired product. This precursor impurity was tracked through the synthesis starting at a step in which a cyclobutyl precursor was coupled to a dichloro substituted intermediate. It was assumed that the impurity was the result of the cyclobutyl precursor displacing the undesired chloro group. The completion of the synthesis led to the presence of impurity in the ﬁnal drug substance. This assumption suggested that the impurity was a structural isomer of the desired product. The 1-month time line allowed method development for isolation. Normal-phase silica gel TLC was investigated brieﬂy. However, a suitable separation was not achieved. The analytical HPLC method was not suitable for preparative-scale HPLC chromatography. The method utilized a Symmetry Shield RP-8 packing material that was not readily available as a preparative-scale column. A sample containing 5% of the desired impurity was obtained and used for HPLC method development and impurity isolation. Due to the similarity of the retention times, this problem was not trivial. The ﬁrst preparative HPLC method using 0.05% triﬂuoroacetic acid/ acetonitrile/methanol appeared to work well on an analytical scale; however, when the method was scaled up on a Symmetry C-8 column, adequate resolution was not achieved. This method was attempted using a Waters radial compression technology as well as a stainless steel preparative column containing Symmetry C-8. The method was also adjusted to 0.1% triﬂuoroacetic acid; however, no separation was achieved. A new method was developed on an analytical scale using 0.1% formic acid in water/ tetrahydrofuran/methanol. This method worked well on a preparative scale using a radial compression Symmetry C-8 column. A solution containing the impurity dissolved in 50% tetrahydrofuran/50% water was puriﬁed by preparative HPLC. The combined fractions were concentrated by evaporation to give a white solid (47.5 mg). This sample was analyzed by HPLC and found to match the retention time of the impurity. The sample was dissolved in tetrahydrofuran and puriﬁed by preparative-scale HPLC using a longer elution time in order to effect a ﬁnal analytical cleanup. This sample was analyzed by HPLC and found to match the retention time of the impurity with a purity of 97.4%. A sample of the impurity was analyzed by LC/MS and NMR. Based on 1 H and 13C NMR spectroscopy, the unexpected ring enlargement structure was assigned. To facilitate spectral comparisons to the parent, DMSO was

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used as the NMR solvent for both the parent and the impurity. This has the additional advantage over protic solvents of permitting detection of labile protons. Unfortunately, DMSO is difﬁcult to remove from a sample if an alternative solvent has to be used. The NMR experiments were acquired on only 1.7 mg of sample. Since this predated our purchase of microprobes optimized for such smaller sample sizes, a standard 5-mm probe was used, thus reducing potential sensitivity gains from concentrating the sample. The presence of several consecutive quaternary carbons and heteroatoms made long-range correlations to protons difﬁcult to observe. Therefore, a carbon spectrum was desired, but was challenging to acquire because of sample mass limitations. Both hetero- and homonuclear correlation experiments were used to elucidate the ﬁnal structure. After isolation and identiﬁcation of the impurity, the structure was found to be different from the originally proposed structure. This example demonstrates the need to obtain the scientiﬁc evidence rather than rely on suggestions and proposals. Fortunately, in this case study the time line allowed ample time for method development, and this is not always the case. It is impossible to predict the complications that may arise in the isolation or characterization. Proactive involvement prior to crisis mode is desired. 6. Case Study A.6: Scaled-Up Oxidative Degradation, Preparative HPLC, and Characterization by LC/MS and NMR

Oxidative forced degradation of the drug substance generated an unknown degradant that was observed in ICH stability samples of both the drug substance and drug product (tablets). This pharmaceutical drug candidate was at a later stage of development; therefore, it was decided to proceed through the identiﬁcation process in a proactive manner under no strict deadlines. From forced degradation studies, it was determined that the unknown degradant was present at approximately 29% in the oxidative challenge degradation sample. Without having a proposed structure, preparing an enriched sample and isolation by preparative HPLC was concluded to be the most efﬁcient route. A large-scale oxidation degradation using a free-radical initiator and molecular oxygen was performed on the drug substance in order to produce a large amount of the unknown oxidative degradant for preparative HPLC isolation. By determining the maximum analytical load of the unknown oxidative degradant, the chromatographic conditions were scaled up and a semipreparative chromatographic method was developed. Thirty-two injections of the scaled-up oxidative challenge degradation sample were performed.

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The collected fractions were combined and evaporated to dryness yielding a yellow solid. The isolated material was submitted for LC/MS and NMR analyses. LC/MS of the primary degradant indicated a molecular mass increase of 16 Da. Initial thoughts suggested an N-oxide, but the fragmentation induced by collisional activation in the tandem mass spectrometer was inconsistent with this proposal, and two other structures were proposed. The initial NMR results indicated that the sample was not pure and contained a large amount of residual ammonium triﬂuoroacetate from the mobile phase, which greatly effected the electronic environment and dominated the experimental spectra. This made both probe tuning and impurity detection extremely challenging. Therefore, the sample was further puriﬁed. HPLC reinjection was performed to remove any residual TFA (triﬂuoroacetic acid) salt that might have been present. The sample was dissolved in a minimum amount of methanol and reinjected onto the semipreparative column. In order to avoid forming a TFA salt, TFA was not used in the mobile phase during the cleanup procedure. The cleanup procedure yielded 170 mg of the unknown oxidative degradant, which was analyzed by NMR. Of the three proposed structures, the NMR data supported only the N-oxide. The standard set of NMR experiments was acquired. Based on comparison of corresponding proton and carbon chemical shifts, the local environment of the degradant was more electron-rich than the parent at the proposed oxidation site, as is expected for an N-oxide. This observation is consistent with oxygen addition. The remaining proton and carbon resonances were essentially the same for the parent compound and the degradant, thus demonstrating no other structural differences. The N-oxide structure is also consistent with the parent MS fragmentation, but inconsistent with the daughter fragmentation. This is a case where interpretation of the fragmentation led to false conclusions about the position of the oxygen atom. 7. Case Study A.7: Scaled-Up Acid Degradation and Isolation Using Preparative HPLC: Characterization by LC/MS, NMR, and IR Spectroscopy

Acid-forced degradation of the drug substance generated an unknown degradant that was not observed using ICH storage conditions (40 C/75% RH), so identiﬁcation was not time-critical. This pharmaceutical drug candidate was at a later stage of development; therefore, it was decided to proceed through the identiﬁcation process in a proactive manner under no

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strict deadlines. Based on LC/MS data, the molecular mass of the unknown acid degradant was determined to be Mþ30. Degradant structures matching the molecular weight were proposed, but further structural information based on NMR analysis was required. From forced degradation studies, it was determined that the unknown degradant was present at approximately 6% in the acid challenge degradation sample. Without having a proposed structure, preparing an enriched sample and isolation by preparative HPLC was concluded to be the most efﬁcient route. A large-scale acid degradation using 1.0 N HCl at 70 C was conducted using the drug substance in order to produce a large amount of the drug substance unknown acid degradant for preparative HPLC isolation. By determining the maximum analytical load of the unknown acid degradant, the chromatographic conditions were scaled up and a semipreparative chromatographic method was developed for the isolation of the unknown acid degradant. Ninety injections of the scaled-up acid-challenged degradation sample were performed. The collected fractions were combined and evaporated to dryness, yielding a yellow solid. HPLC reinjection was performed to remove any residual TFA (triﬂuoroacetic acid) salt that might have been present. It was dissolved in a minimum amount of methanol and reinjected onto the semipreparative column. In order to avoid forming a TFA salt, TFA was not used in the mobile phase during the cleanup procedure. The cleanup procedure yielded 50 mg of the unknown acid degradant. The isolated sample was analyzed by LC/MS, and the LC/MS data conﬁrmed a Mþ30 degradant after isolation and concentration conﬁrming the pure isolated degradant sample was the correct species. The identity was conﬁrmed by correlating the retention time and mass spectra with that of the unknown acid degradant peak in the ten-day acid degradation sample. Based on NMR analysis, the structure shown was proposed for the drug substance–related unknown acid degradant. In order to provide further evidence for the presence of the carbonyl in the proposed structure, infrared spectroscopy was performed. The IR spectrum showed a C ¼ O stretch at 1670 cm1 (consistent with an aryl ketone as in the proposed structure of the acid degradant). The IR and NMR spectral data indicated no signiﬁcant TFA in the acid degradant isolate. The MS, NMR, and IR data were all consistent with the acid degradant structure proposed. 8. Case Study A.8: Scaled-Up Oxidative Degradation and Preparative Isolation: NMR Characterization Critical to Differentiate Between Two Possible Structures

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An unknown degradant formed in the drug product during stability studies using ICH conditions (40 C/75% RH) and was projected to be present at approximately 0.1% at the end of shelf-life date. Hence, the identiﬁcation team proactively tackled the identiﬁcation effort. LC/MS of the unknown degradant indicated a molecular mass decrease of 44 Da. Several M-44 structures were proposed. In evaluating the degradation data, the degradant was also observed in forced degradation oxidative challenge. An oxidative degradation study was performed on the drug substance in order to generate the unknown drug product degradant. The unknown degradant was formed by free-radical oxidation of the drug substance only, a signiﬁcant advantage. This eliminated the need to isolate the low-level degradant from the drug substance as well as the excipients. HPLC analysis of the pressurized oxygen/ radical initiator–forced degradation study indicated that 5% of this degradant was produced after 20 days. The degradant was identiﬁed by analytical HPLC as being the drug product degradant. A large-scale oxidative degradation was performed on drug substance in order to produce a large amount of unknown degradant for isolation. A suitable semipreparative HPLC method was developed for isolation of the unknown degradant from the drug substance. Fractions containing the desired degradant were combined and concentrated by evaporation. The isolated material was puriﬁed in a ﬁnal analytical cleanup to yield the pure desired degradant for NMR analysis. A complete NMR characterization was performed to elucidate the structure. For comparison, the same set of experiments was collected for the parent drug substance. Initial NMR characterization data were consistent with a proposed structure, but reevaluation was performed when a new reaction pathway was proposed. A second structure was proposed that was also consistent with the NMR data. Additional NMR experiments were performed to differentiate between the two structures. These included nOe difference experiments to probe spatial relationships within the molecule.

9. Case Study A.9: Scaled-Up Light Degradation and LC/MS and NMR Characterization

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In a related case study (A.9), an unknown degradant formed in the drug product during stability studies using ICH conditions of 40 C/75% RH and was projected to be present at approximately 0.1% at the end of shelf-life date using ICH conditions. LC/MS of the unknown degradant indicated a molecular mass increase of 14 Da, indicative of oxidation to a ketone moiety. Since several ketone products were possible, isolation of the major degradant was required to conﬁrm the exact structure. A light degradation study was performed on a mixture of drug substance N-oxide and polymer excipient (1:9) in an attempt to generate an enriched quantity of the unknown degradant that had been observed in drug substance and drug product stability samples. HPLC analysis of the UV-challenged forced degradation sample indicated that 4% of the unknown degradant was produced. Therefore, a large-scale UV degradation was performed so that the degradant could be isolated using semipreparative chromatography. A suitable semipreparative HPLC method was developed for isolation of the unknown degradant from the drug substance N-oxide/polymer excipient mixture. Fractions containing the desired degradant were combined and concentrated by evaporation. The isolated material was puriﬁed in a ﬁnal analytical cleanup to yield the desired impurity. The structure of the degradant was determined by LC-MS and NMR spectroscopy. Two possible structures were proposed based on the molecular weight. NMR characterization was performed to elucidate the structure. For comparison, the same set of experiments was collected for the parent drug substance. Unfortunately, because of solubility differences, the parent was dissolved in dimethyl sulfoxide, while the degradant was dissolved in methanol. This somewhat complicated comparison of the corresponding proton resonances. Sensitivity was an issue with this sample because of mass limitations; only 0.6 mg were available for the NMR experiments. Preliminary interpretations of the NMR data revealed an inconsistency between the NMR and MS results. As always, a proton spectrum was acquired at the start and end of the NMR experiments to ensure sample integrity during the full acquisition period. A synthetically plausible structure was proposed based on the NMR observations. This structure differed from that originally expected by the project team. The mass spectral data were then revisited. The recorded total molecular mass of the impurity was exactly 28 Da higher than that of the proposed structure. This suggested that a sodium adduct had formed during the LC/MS experiment, a relatively common anomaly. Repeating the LC/MS work with this in mind demonstrated consistency between the NMR and MS data, thereby conﬁrming the proposed structure. 10. Case Study A.10: Adduct Degradant Synthesized and Characterized by LC/MS and NMR

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In case study A.10, the drug substance generated a degradant at greater than 0.6% under ICH conditions of 40 C/75% RH for 10 days. Mass spectral analysis indicated the degradant had a molecular weight of Mþ100, leading to the suggestion that the succinic acid had condensed with the drug substance to form an amide (succinic acid adduct). In deciding the most efﬁcient identiﬁcation route, only one adduct degradant structure could be proposed with a high degree of conﬁdence. In evaluation of isolation versus synthesis, a synthetic one-step route with well-precedented chemistry was available; hence, the synthetic route was selected. A mixture of the drug substance free base from the succinate salt in chloroform was treated with succinic anhydride and heated at reﬂux. The solution was cooled to room temperature, concentrated by evaporation, and puriﬁed by column chromatography on silica gel to give the product (0.95 g, 69% yield) as an off-white foam. The synthesized material was shown by HPLC to elute at the same relative retention time as the impurity and co-eluted when spiked with a sample of degraded drug substance containing the degradant. When synthesizing, the impurity/degradant spiking experiments are a key piece of conﬁrmation data. The molecular weight and LC/MS fragmentation data were consistent with a succinamide derivative. A complete NMR characterization was performed to elucidate the structure. Chloroform was chosen as the preferred solvent since it allows for detection of labile protons, and it is easily removed if necessary. However, NMR experiments on the parent species were run, using dimethyl sulfoxide as the solvent. This therefore made comparison of proton resonances between parent and adduct degradant less straightforward. The presence of two new carbonyl entities strongly supported the proposed succinamide structure. Long-range proton–carbon correlations observed between the methylenes and the carbonyl neighboring the nitrogen of interest conﬁrmed the covalent bonding of the succinate moiety. The NMR data obtained have been informative for comparison to subsequent adduct degradants and impurities of this drug substance requiring identiﬁcation. One such example is the analogous tartrate adduct. Archiving of data and capturing lessons learned have saved valuable time in minimizing duplication of effort. 11. Case Study A.11: Adduct Degradant Synthesized and Characterized by LC/MS and NMR

In case study A.11, a degradant was observed in drug product–forced degradation (thermal/humidity) studies of the drug substance salt. This compound was at a later stage of development and proactive efforts were

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devoted to identiﬁcation. LC/MS evidence indicated that the degradant had a molecular weight of Mþ132 Da, which corresponded to the condensation of the tartaric acid with the drug substance to form a tartrate adduct. Since there was a reasonably high degree of conﬁdence in the proposed structure, synthesis was determined to be the most efﬁcient route. Ideally, a one-step condensation of tartaric acid and amine would be the shortest route. However, since tartaric acid contains two carboxylic acid groups, it would be difﬁcult to stop the amine from condensing with both groups to form an unwanted bis adduct in which one tartaric acid condenses with two drug molecules. It was decided to investigate the reaction of tartaric acid anhydride. An acid anhydride can react with an amine to give the desired adduct. The second acid group is hidden in the anhydride as a leaving group; therefore, it did not interfere with the reaction.

However, it is possible for a hydroxyl group to undergo the same reaction to form an ester group instead of an amide. For this reason it is necessary to remove or protect any hydroxyl group in the reaction mixture.

The hydroxyl groups of tartaric acid anhydride as shown above had to be protected. Diacetyl tartaric anhydride was available from Aldrich. It was determined that this anydride would react to form the desired diacetyl adduct, and the removal of an acetyl group tends to be a relatively trivial task in organic chemistry. The degradant was prepared in two steps by the condensation of the drug substance and (þ)-diacetyl-L-tartaric anhydride followed by hydrolytic removal of the acetate groups. The ﬁrst step was conducted in chloroform at 50 C and was catalyzed by the addition of dimethylaminopyridine. The reaction was complete in approximately 2 days. The solvent was then removed by evaporation, and the crude organic material was puriﬁed by column chromatography on silica gel using 1:1, methanol:ethyl acetate as the mobile phase to afford 634 mg of the diacetate. A solution of the diacetate adduct in methanol was treated with potassium carbonate. The resultant hazy solution was stirred at room temperature for 4 hours. HPLC analysis of the reaction mixture indicated that the reaction was complete. The solution was concentrated by evaporation to remove methanol, treated with water, and neutralized to approximately pH 7 by the addition of formic acid. The resultant solution was concentrated by

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evaporation, and the condensate puriﬁed by preparative scale HPLC. The main peak was collected and the solvent was removed from the combined fractions by evaporation to give a glassy solid. The solid was dissolved in methanol, ﬁltered through a short plug of cotton wool, and concentrated by evaporation to give an oil. The oil was treated with hexane and manipulated to give a white solid that was dried under high vacuum overnight. Once a proposed degradant/impurity has been synthesized, it is important to take extra care to ensure that the correct compound has been made. It is vitally important that the sample is analyzed by HPLC and UV and shown to co-elute with the degradant to ensure that the compound is identical to the degradant. The sample was analyzed by LC/MS and found to have a molecular weight of Mþ132, corresponding to the molecular weight of the proposed tartaric acid adduct. HPLC and 1H and 13C NMR spectroscopy were also used to fully characterize the compound. The standard set of NMR experiments were collected using deuterated methanol as the chosen solvent. This provided consistency with the corresponding parent spectra, yet prohibited the detection of exchangeable protons such as N–H and O–H groups. The spectra were complicated by the presence of a sample impurity. In addition, rotamers were observed, as evidenced by pairs of peaks apparent for several resonances. This work supported the hypothesis that the tartrate salt generated an adduct in stability studies. Unexpectedly, based on interpretation of the NMR results, the complexation of tartrate disrupted the molecular symmetry observed for the parent. Assignment of 1H and 13C NMR resonances demonstrated a clear match with the corresponding impurity data, thus conﬁrming the proposed tartrate adduct formation. 12. Case Study A.12: Adduct Degradant Synthesized and Characterized by LC/MS: Description of NMR Complications

This case study (A.12) describes the synthesis and identiﬁcation of an adduct degradant that had been observed in degradation (thermal/humidity) studies of the amorphous drug. For this project, there was plenty of notice provided and resource allocated since the compound was at a later stage in development. LC/MS data showed the adduct degradant had a molecular weight of Mþ204, suggesting addition of succinic acid (the drug substance salt) to the parent. However, LC/MS data was unable to assign which of the three hydroxyl groups had reacted with succinic acid groups. Since this was not time-critical, synthesis was evaluated as the optimum route rather than isolation at a low level, which would have been extremely difﬁcult and time-consuming.

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It was decided that the most efﬁcient method of identiﬁcation was to couple succinic anhydride with the drug substance and isolate the peak of interest by preparative-scale HPLC. A solution of the drug substance was treated with dimethylaminopyridine and succinic anhydride. The resultant solution was stirred at room temperature for 48 hours. The reaction mixture was partitioned between ethyl acetate and water, and the aqueous layer was then treated with 1N HCl. The two layers were shaken well, and the aqueous layer was removed. The organic layer was then washed with water, saturated sodium chloride solution, dried with magnesium sulfate, and concentrated by evaporation to afford a clear colorless oil (1.81 g). A suitable preparative HPLC method using a volatile mobile phase of 0.1% formic acid in water/ methanol was developed, and the crude reaction mixture was puriﬁed by preparative-scale HPLC. The solution was concentrated by evaporation and the water was removed from this solution by freeze-drying to afford a white lyophilate (40 mg). Analysis of the isolated compound by LC/MS determined that the succinic acid had added to a hydroxyl group on the pyrollidine ring. The two hydroxyl groups on the 5-membered pyrollidine ring are so similar that they were considered the same reactivity. The isolate was analyzed by mass spectrometry and found to have a molecular weight corresponding to the succinic acid adduct. MS/MS analysis of this sample indicated that a fragment with a molecular weight of 204 Da was being formed. This LC/MS evidence suggested that the succinic acid had added to a hydroxyl group on the pyrollidine ring; however, it was not possible to determine which of the two hydroxyl groups reacted with succinic acid. NMR spectral analysis did not yield a deﬁnitive answer because of complications caused by limited sample mass, contaminants present in the sample, and most signiﬁcantly, a rotamer effect arising from two separate amides. This gave rise to very complex NMR spectra that were not easily interpreted. In this study, the structure of the impurity was based on the LC/MS/MS data only.

B. Process-Related Impurities Case Studies 1. Case Study B.1: Identification Using a Combination of Enriched Forced Degradation Samples and Normal-Phase HPLC

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A drug substance sample contained three unknown impurities in case study B.1. Impurity A was present at 0.3% and Impurities B and C were both present at 0.1%. The time line for completion of the identiﬁcation process was 1 month. At the initial stage of the identiﬁcation project, it is critical to look at the degradation data and process-related impurities to determine if unknowns are actually known standards. From forced degradation studies, Impurities A and B were identiﬁed in the acid degradation experiment (3 hours in 1 N HCl) at the 13% and 5% level, respectively, based on HPLC relative retention time comparison. Acid degradation provided a key enriched sample. At the 5% and 13% level, this made the isolation task an order of magnitude less complex. The identities of Impurities A and B were conﬁrmed by correlating retention times and mass spectra of the unknown impurities in the acid degradation sample with the retention times and mass spectra of known standards. LC/MS of the acid degradant gave molecular weights that were found to be identical to the standards. The standards were run on LC/MS and spiked into the sample. Impurity A was conﬁrmed as the carboxylic acid analog of the amide drug substance, and Impurity B was conﬁrmed as the aryl portion of the drug substance (as shown above). This background work saved valuable intensive isolation efforts. Impurity C did not have a standard match. Additionally, it could not be enriched by degradation or found in retained synthesis mother liquor samples, so isolation at the 0.1% level was required. In order to identify Impurity C, a normal-phase TLC method was modiﬁed for isolation since it gave adequate separation of all three impurities. The analytical TLC method utilized a number of solvents. One of the major components of the mobile phase was acetic acid. Since acetic acid is an aggressive acidic solvent that can degrade compounds, it was deemed necessary to develop a method in the absence of acetic acid. Acetic acid is also difﬁcult to remove in the isolation process. If evaporation is used to remove solvents from the isolate, less polar solvents can evaporate preferentially, leaving the isolated compound in concentrated acetic acid solution. Bearing this in mind, it was decided to develop a milder mobile phase. The stability of the impurity should always be a concern in developing an isolation/identiﬁcation strategy. The isolate should be checked by HPLC and LC/MS at many different stages of the process. Previous TLC analysis of the drug substance afforded two method solvent systems that were able to separate the desired impurities. The old conditions involved a methyl ethyl ketone (or methyl butyl ketone)/acetic acid/water solvent system. Hence, after experimentation, it was found that the solvent system of methyl ethyl ketone/water/acetic acid (80/19/1 by volume) afforded separation of the main band from the desired impurities and reduced the amount of acetic acid present. Using this new solvent system, the impurity band of interest was separated from the main band and the silica gel containing the impurity was removed from the plate by scraping with a spatula. Extraction of the compound from the silica gel was accomplished using acetonitrile. The isolated material was analyzed by HPLC, and the Impurity C isolate was identiﬁed.

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A major concern was the presence of silica gel in the sample. It was found that the presence of silica contaminants in crude unﬁltered solutions was complicating the LC/MS identiﬁcation/conﬁrmation of the presence of Impurity C. It was decided to attempt to remove the silica gel by ﬁltering the extraction solution through a pad of celite. In a subsequent experiment, it was found that extraction of the impurities from the silica gel using methanol/dichloromethane, followed by removal of the silica gel by ﬁltration through a short pad of celite afforded samples with very little organic material present. It was presumed that the organic impurity adhered to the celite and did not elute from the solid phase. Normally an organic chemist can remove solid contaminants such as silica gel by ﬁltration through celite without any problems; however, the extreme difference in the scale involved in this case study complicated the procedure. Synthetic organic chemists normally deal with gram-to-milligram quantities of material, and sometimes techniques do not transfer from the milligram scale to the submilligram scale. Hence, the experiment was repeated and celite was eliminated in the removal of the silica gel from the sample. The extraction solution was ﬁltered through a pin/head-sized plug of prewashed cotton wool wedged into a pipette. This example demonstrates the need to perform isolation and extraction experiments at a small scale before committing precious isolate samples and the need to check for the presence of the desired impurity/degradant at multiple stages along the isolation process. After completing the TLC method development, a concentrated solution of the drug substance was applied to 10 TLC plates (20 20 mm). The plates were eluted using 80% methyl ethyl ketone/19% water/1% acetic acid (using top phase only). The band of silica gel containing the impurity of interest was removed from the TLC plates by identifying the band under UV light and carefully scraping the band. The silica gel was extracted by stirring with methanol/dichloromethane followed by crude ﬁltration through glass ﬁlter, then ﬁltration through a short prewashed plug of cotton wool. The resultant clear, colorless solution was concentrated by evaporation to give a sample of the impurity for mass spectral analysis. Because of the weak intensity of the bands (more polar than the main band by TLC) according to UV light, a highly concentrated solution was applied in order to observe the impurity bands. LC/MS analysis of the sample was aided by the greater abundance and purity of the sample, and more sophisticated techniques were utilized to elucidate the structure. Impurity C showed an abundant doubly charged ion with a molecular weight consistent with addition of an extra nucleus. Based on this LC/MS evidence, Impurity C was proposed to originate by incomplete protection of a synthetic precursor. Further conﬁrmation of structure by traditional NMR was not possible because of limited amount of sample. This project demonstrated the need to bring in new microsample NMR technologies. These technologies have been purchased and utilized successfully to solve subsequent degradation issues as demonstrated in some of the following case studies.

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2. Case Study B.2: Isolation Using Normal-Phase Preparative HPLC and Characterization by LC/MS and NMR Characterization Within 1 Week

For case study B.2, an impure starting material gave rise to an impurity at the end of the drug substance synthesis. An unknown impurity at the 0.3% level was discovered after process colleagues prepared the freebase form of the drug from the salt form. The time line for completion of the identiﬁcation was 1 week and posed quite a challenge. There was no time available for lengthy HPLC method development and isolation, as well as challenging spectral structure elucidation experiments and data interpretation. As illustrated in this series of case studies, these factors are very difﬁcult to predict at the beginning of the project. Because of the difference in polarity, normal-phase chromatography was identiﬁed as the technique of choice under the strict time line. A sample of the drug substance was analyzed by a normal-phase TLC technique already developed, which detected the impurity. A suitable solvent system for column chromatographic separation was developed based on the analytical TLC method (10% ethyl acetate/90% hexane). The drug substance sample was eluted on two large (20 20 cm) TLC plates using 10% ethyl acetate/90% hexanes as the mobile phase. The silica gel containing the impurity was removed from the TLC plates, and the compound of interest was extracted by washing with 1% triethylamine/99% acetonitrile. The material was analyzed by HPLC and found to be identical to the impurity of interest. The stability of the impurity on silica gel was investigated, and no problems were identiﬁed — another critical step after a lengthy isolation process. It is essential that the degradant does not further degrade in the concentration step. Up-front stability investigation is a worthwhile investment and can save valuable time reisolating an impurity or degradant, especially in cases in which the project is sample-limited. The impurity was then isolated at a larger scale by column (170 45 mm) chromatography on silica gel using 10% ethyl acetate/90% hexane as the mobile phase. Fractions containing mostly the required impurity were combined and concentrated by evaporation to give a clear, colorless oil (0.9 mg). By LC/MS, the isolated impurity showed an abundant doubly charged ion. The molecular weight suggested that one of the starting materials was contaminated by a dimer, which propagated through the synthesis. Less than 1 mg of degradant was isolated for NMR characterization. Hexanes present in the sample further complicated the analysis. Therefore, traditional 5-mm tube NMR would have been excessively time-consuming.

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A 3-mm NMR ‘‘microprobe’’ was used to obtain high-quality data in a fraction of the time. In addition, the enhanced sensitivity for mass-limited samples afforded by microprobe NMR technology enabled the acquisition of a carbon spectrum, which would not have been readily achievable using a traditional 5-mm probe. This was advantageous for this project because of the large number of nonprotonated carbons present. The symmetry of the molecular structure led to pairs of magnetically equivalent carbon sites giving rise to single peaks in the carbon spectrum. Proton integration showed that the unusual ratio of a bridging methylene to the remaining groups was due to a single methylene group that joins the two identical ends of the dimer. An alternative proposed structure included an additional oxygen neighboring the bridging methylene, which was disproved by the observed proton and carbon chemical shifts in this molecular region. Simulations of the proton and carbon spectra (using ACDLabsTM software) further supported the reported dimer structure. Based on NMR and MS analyses on the isolated impurity, an unexpected dimer structure was conﬁrmed within the one-week time frame required. Ideally, one should allocate a larger amount of time to characterize such mass-limited samples.

3. Case Study B.3: Isolation Using Preparative HPLC: LC/MS and NMR Characterization Required to Differentiate Between Four Proposed Structures

A bulk lot sample of drug substance contained an unknown impurity present at 0.96% in case study B.3. At the request of the project team, this impurity was isolated for structural identiﬁcation within a 1-month time frame. Previous mass spectral data indicated that the impurity had a molecular weight of M-18. Two possible structures were proposed based on the mass spectral data: a dehydration product and a process-related –CF2H product. NMR data were required to differentiate between these proposed structures. Given the deadline, the most efﬁcient technique for sample isolation was reversed-phase preparative HPLC isolation, scaling up the existing analytical reversed-phase method. With two or more possible structures, synthesis is typically too time- and resource-intensive. This would be twice the effort in that the CF2H and dehydration products would both need to be synthesized.

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Since the existing analytical method contained phosphate buffer, method conditions needed to be modiﬁed for preparative isolation requiring a volatile mobile phase (0.1% formic acid in methanol). The bulk drug substance lot was puriﬁed by preparative HPLC using the modiﬁed preparative chromatographic conditions. The component of interest eluted from 5–6 minutes and was collected over multiple runs. The fractions containing the impurity were combined and concentrated by evaporation to give a crude oil. The oil was further puriﬁed in a ﬁnal analytical cleanup to afford 1 mg of sample for NMR analysis. Despite the mass limitations and other challenges faced, a full set of NMR experiments were acquired on the sample, using a 3-mm microprobe. Methanol was chosen as the NMR solvent-based on solubility determinations from the chromatography analysis. Unfortunately, this prohibited the detection of exchangeable protons, including N–H and O–H groups. It also yielded relatively poor proton spectral resolution. In addition, this differed from acetone used for the parent, hence complicating chemical shift comparisons of the proton spectra. Racemization at the chiral center for both the parent and the impurity led to pairs of resonances in the carbon spectra. Peaks arising from a sample contaminant were also apparent in both the proton and carbon spectra of the impurity. Signal-to-noise ratios for carbon resonances near the ﬂuorinated group were low because of 13C–19F splittings. Carbon quartets observed in the parent spectrum for the ﬂuorinated quaternary and two nearby methylenes appeared as triplets in the impurity carbon spectrum. This clearly demonstrated that the triﬂuoro group had become a diﬂuoro group in the impurity. 1H and 13C NMR and mass spectral data were consistent with the –CF2H product, and so the dehydration product was ruled out. This approach yielded the identiﬁcation required within the time frame allotted. 4. Case Study B.4: Identification Using a Combination of Preparative HPLC and Synthesis

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For case study B.4, a bulk lot sample of drug substance contained an unknown Impurity A present at 0.82% and an unknown Impurity B present at 1.12%. Identiﬁcation of both impurities was requested within a 1-month time frame. It was conﬁrmed that the impurities did not match any known standards. LC/MS data indicated that Impurity A had a molecular weight of Mþ60, suggesting propanol addition, and Impurity B had a molecular weight of Mþ32, suggesting methanol addition. The next step was to look into the synthesis to ﬁnd possible propanol and methanol addition reactions. In this investigative step, it was found that the reagent 2-propanol was utilized in step 11 of the process synthetic route. For Impurity A, a process-enriched sample was not available; therefore, reversed-phase preparative HPLC was used to clean up the analyte. The drug substance bulk lot was puriﬁed by preparative HPLC, using a chromatographic method modiﬁed for isolation with a volatile mobile phase containing a formic acid buffer and methanol organic modiﬁer. Impurity A was collected over multiple runs. The fractions were combined and concentrated by evaporation to give a crude oil that was further puriﬁed in a ﬁnal analytical cleanup to provide a clean sample for NMR. HPLC reinjection was performed to remove residual peaks that were present and could complicate the NMR analysis. The isolate was dissolved in a minimum amount of solvent, and reinjected onto the semipreparative column. The resultant formic acid salt sample was converted to the freebase by an analytical reversed-phase HPLC cleanup using water followed by a water/methanol gradient as the mobile phase. This process afforded 1.9 mg of Impurity A. The molecular mass of the isolated Impurity A was conﬁrmed to be 60 Da higher than the drug substance. This mass spectral conﬁrmation on the isolate is critical prior to NMR analysis to assure that the correct peak has been isolated and that there are no changes that are due to contamination or degradant of the sample. Typically, this should be performed on the fraction collected from ﬁrst preparative injections to conﬁrm peak identity. NMR characterization was performed on approximately 1 mg of sample, which was particularly challenging because of contaminants present in the sample. Methanol was used as the solvent based on chromatographic solubility studies. Exchangeable protons were thus not observable, although this was not deemed to be of consequence in this project. Comparison to the corresponding parent proton spectrum was somewhat difﬁcult because of the solvent change from dimethyl sulfoxide. It was observed that most of the structure of the impurity was identical to the parent and that the acetylene of the parent had changed. The observation of a dimethyl methine resonance exhibiting a long-range proton–carbon correlation to a methine was key to the structural elucidation. The observed proton–proton coupling constant of 7 Hz for the protons on either side of a double bond pointed to a cis conﬁguration, consistent with vinylic coupling constant values reported in the literature.6 For Impurity B, process colleagues synthesized methanol adducts of the drug substance, using a literature cesium hydroxide–catalyzed method.7 NMR analysis of the synthesized Mþ32 indicated that the impurity was the methyl (versus isopropyl) vinyl ether analog of the drug substance. The

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synthesis yielded a cis–trans mixture of enol ether isomers according to NMR and LC/MS. One of these isomers corresponded to the Mþ32 impurity. Based on NMR and mass spectrometry, Impurity B was identiﬁed as the methyl vinyl ether analog of the drug substance. 5. Case Study B.5: Preparative HPLC Isolation from a Retained Process Sample and Characterization Using LC/MS and NMR Characterization

A bulk lot sample of the drug substance contained an unidentiﬁed process-related impurity present at 0.2% in case study B.5. Based on mass spectral data, the impurity had a molecular weight of Mþ92, suggesting addition of phenol. Mass spectral fragmentation data suggested that phenol added in the left-hand portion of the molecule and the right-hand portion of the molecule was intact. At this low level, it was critical to look for an enriched sample. Review of forced degradation data had not produced this impurity. Fortunately, a retained process sample containing the desired impurity at the 2.6% level was available. This sample, enriched in the desired impurity, was used for isolation by preparative-scale HPLC. A suitable preparative HPLC method using 0.1% formic acid in water and methanol afforded 10 mg of the desired impurity. The NMR analysis conﬁrmed the phenol addition proposed from mass spectral evidence. NMR spectral interpretation was complicated because of the absence of an expected long-range proton–carbon correlation peak pertaining to the complicated right-hand portion of the molecule. This suggested a potential inconsistency between the NMR and MS observations. Since a lack of an observation is not the preferred approach for a structural proof, the NMR experiments were repeated in an alternative solvent. The sample previously run in deuterated acetonitrile was dried and then redissolved in deuterated methanol. The new long-range heteronuclear correlation spectrum showed the anticipated proton–carbon coupling, hence supporting the proposed impurity structure. The original version of the NMR experiment in question was optimized for a standard long-range coupling constant of 6 Hz, as observed in the parent drug substance. However, the long-range coupling for the impurity structure was observed to be weaker than this, perhaps because of changes in molecular geometry. As noted, mass spectral analysis of the impurity suggested that the righthand portion was intact, and more in-depth NMR experiments proved consistent with this conclusion as well. The source of this impurity was subsequently investigated. Mass spectral analysis of the process intermediates indicated that the Mþ92 impurity was present at the start of the synthesis,

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before the right-hand portion of the molecule is added to the molecule. This case study is a prime example that no technique can stand alone. NMR and mass spectrometry are critical techniques in structure elucidation, and any apparent discrepancies must be further probed. 6. Case Study B.6: MultidisciplinaryTeam Approach Using LC/MS, Process Enrichment, Scaled-Up Forced Degradation, Preparative HPLC, Synthesis, and NMR

A multidisciplinary team approach was used to conclusively elucidate the unknown impurity in case study B.6. A bulk sample of drug substance contained an unknown impurity at the 0.15% level. The identiﬁcation required a number of techniques already discussed in this chapter (all incorporated into this case study): LC/MS, enrichment of a process sample, scale-up of forced degradation, preparative isolation, NMR characterization, and small-scale synthesis. The instability of the impurity under acidic chromatographic conditions presented a tremendous challenge in purifying and identifying the impurity. However, this acidic instability problem turned into an advantage, conducting a degradation study of the impurity that provided very rich information about its structure. Extensive LC/MS analysis suggested that the molecular weight was Mþ371. Several structures were proposed with a low degree of conﬁdence; therefore, isolation and spectral characterization were required to conclude the exact structure. Isolation and identiﬁcation of the impurity was complicated by the acid instability of the drug substance and impurity. Standard workup of the preparative HPLC fractions decomposed the impurity before spectral data could be recorded. A new high pH isolation strategy was developed. The drug substance main band was removed from the bulk drug substance containing the impurity by solid-phase extraction technology, and the impurity was isolated by preparative-scale HPLC. Attempts to directly isolate this impurity using preparative HPLC proved to be unsuccessful. Solid-phase extraction technology was employed to remove the main band and more polar impurities resulting in a concentrated solution of the desired impurity. The NMR analysis of this compound was extremely challenging both experimentally and in data interpretation. Only 3 mg of sample were available, which was low given the relatively high molecular weight. Sample concentration was further limited by very poor solubility. The ﬁnal solvent chosen was deuterium oxide, doped with a small amount of sodium hydroxide to adjust the pH to a more basic environment. The use of a protic solvent eliminated the ability to detect labile protons, including what proved to be a critical N–H group involved in a newly formed molecular bridge. The parent

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species was run in dimethyl sulfoxide, so comparison of proton spectra between parent and impurity was not straightforward. Spectral resolution was not ideal because it was later shown to be a pseudo-dimer structure yielding very similar proton and carbon chemical shifts for the two halves of the molecule. Additionally, two-dimensional heteronuclear correlation experiments were not as useful as they may otherwise have been because of several consecutive quaternary carbons and heteroatoms. Sample integrity during NMR acquisitions was veriﬁed by acquiring a proton spectrum both initially and at the conclusion of the full set of experiments. No change in the spectrum conﬁrmed that the sample remained intact. However, preliminary analysis of the NMR data supported a structure vastly different from the mass spectral results, conﬂicting signiﬁcantly with the determined molecular mass. Since sample integrity during NMR acquisition was veriﬁed, the mass spectral experiments were rerun to evaluate data reproducibility. It was subsequently shown that the sample integrity was compromised by acidic chromatographic conditions used for the LC/MS experiments. This explained the apparent inconsistency in results and provided a clue of the molecular structure. In addition, running LC/MS on the NMR sample veriﬁed the presence of an exchangeable proton in the middle of the molecular structure, since the molecular weight of deuterium increased the fragment size in this region by one mass unit. Combined with observed NMR heteronuclear correlations and the MS ‘‘nitrogen count rule,’’ it was determined that this was an N–H group. Combined interpretation of the NMR and mass spectral observations yielded the bulk of the molecular structure as a pseudo-dimer. However, the carbon NMR spectrum appeared to be ‘‘missing’’ an anticipated quaternary carbon resonance, based on the measured total mass. Removing the small degree of line broadening commonly used for processing carbon spectra revealed a distinct shoulder on one of the observed carbon resonances. This was the ‘‘missing’’ carbon and demonstrated the necessity of fully appreciating one’s detection and resolution limitations. Analysis of the process synthetic pathway used to prepare this molecule afforded insight into the potential mechanism of formation of this processrelated impurity. It was observed that an early step in the synthesis of the molecule was forming a small amount of side product. It was subsequently observed that the side product was still present at later stages in the manufacture of the drug. This side product in turn reacted with one of the starting materials. The impurity that was formed at this stage was not the isolated impurity. This emerging impurity was modiﬁed by the subsequent steps of the synthesis to form the impurity that was ﬁnally isolated and identiﬁed.

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This multidisciplinary team approach toward impurity identiﬁcation was successfully applied to the identiﬁcation of this impurity. Each and every discipline played a very important role. Without SPE enrichment to remove the drug substance from the impurities, the impurity could not be puriﬁed by preparative HPLC. The novel acidic degradation study of the impurity provided very valuable information of the structure of the impurity. Mass spectrometry and nuclear magnetic resonance spectroscopy were the ultimate tools in this structure elucidation. Furthermore, the formation mechanisms were concluded by a careful examination of the process. 7. Case Study B.7: Preparative HPLC on an Enriched Drug Substance Lot and Characterization by LC/MS and NMR

A bulk lot sample of drug substance contained an unknown processrelated impurity at 0.4% in case study B.7. To meet project time lines, it was necessary to identify the impurity. Mass spectral data indicated that the impurity had a molecular weight of Mþ12 (compared to the parent). LC/MS/ MS data indicated that the change was located in the sugar portion of the molecule. Isolation and NMR analysis was required to provide more structural information on the impurity. This again was a process-related impurity not observed in forced degradation studies; therefore, effort was placed on ﬁnding an enriched mother liquor sample. An enriched bulk lot containing 16% of the impurity of interest was identiﬁed and used to reduce the time required for isolation by a factor of 40. Isolation was required for further NMR analysis. The enriched bulk lot containing 16% impurity was used for isolation by preparative HPLC using 45 500-mL injections. A suitable reversed-phase preparative HPLC method using a volatile mobile phase (0.1% formic acid in water and acetonitrile) was developed based on the analytical purity and potency assay. The fractions containing the impurity were combined and concentrated by evaporation. A ﬁnal analytical cleanup was performed to remove salts and

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solvent impurities for NMR analysis. The resultant formic acid salt sample was converted to the freebase by the approach used in case study B.4. Preparative scale reversed-phase HPLC using water followed by a water/ acetonitrile gradient as the mobile phase afforded 12 mg of the impurity. It was not possible to determine the nature of the counterion; therefore, X has been used to denote a counterion of unknown composition. The process-related impurity structure was conﬁrmed by LC/MS and NMR spectroscopy. The mass spectral data pointed to an unusual Mþ12 total mass, implying an implausible ‘‘raw’’ carbon addition. Fragmentation patterns and comparison with the parent data showed the sugar ring to be the altered region of the molecular structure. NMR experiments were run on a mass limited sample, which was challenging because of the relatively high molecular weight. Deuterated methanol was chosen for high solubility and ease of removal. This also facilitated comparison of proton spectra between impurity and parent, since the parent was also run in methanol. Unfortunately, the proton and carbon spectra were quite complex, since virtually all of the resonances were clustered in the aliphatic spectral region. The two methyl groups were observed to be magnetically equivalent in the parent structure, yet were unequivalent in the impurity, demonstrating a change in the electronic environment at the attached nitrogen site. A new methylene resonance was observed, which accounted for the expected carbon addition. However, the total mass was not readily explained. Long-range heteronuclear correlations provided the necessary insight. Correlations were observed between the two methyl groups and the methine neighboring the nitrogen. The dimethyl entity also showed a correlation to the new methylene, establishing proximity between the two species. Finally, the new methylene showed a long-range correlation to the oxygenated methine, thereby conﬁrming the unexpected closed-ring structure. Chemical shift changes compared to the parent spectra provided further support for this novel structure. 8. Case Study B.8: Preparative HPLC Isolation from Retained Process Samples and Characterization by LC/MS and NMR

A bulk lot sample of drug substance in case study B.8 contained an unknown impurity at the 0.3% level. This unknown process-related impurity was less polar than the parent in the chromatographic assay. Based on mass spectral data, the impurity had a molecular weight of Mþ4, suggesting over-reduction of the benzene ring. Attempts to obtain mass spectral

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fragmentation were unsuccessful; therefore, isolation was required for structural identiﬁcation. Considering the low level of impurity, it was critical to look for an enriched sample. A retained process mother liquor containing approximately 13% of the unknown impurity was prepared, and a suitable semipreparative HPLC method was developed for isolation of the unknown impurity from the mother liquor sample. Fractions containing the desired impurity were combined and concentrated by evaporation. The isolated material was puriﬁed in a ﬁnal analytical cleanup to yield a 162 mg of the desired impurity for NMR analysis. A complete NMR characterization was performed to elucidate the structure. Assignment of the 1H and 13C spectra was able to determine the placement of the double bond in the partially saturated ring. 9. Case Study B.9: Conversion to Freebase and Preparative HPLC Isolation, Followed by LC/MS and NMR Characterization

A bulk lot sample in case study B.9 contained an unknown impurity present at 0.2%, and identiﬁcation was required. An enriched sample was not available. In addition, the drug substance was a succinate salt. In deciding the isolation approach and in order to avoid contamination of the isolated impurity with succinic acid, the impurity was isolated from the freebase. To prepare the freebase, the succinic acid salt was dissolved in dichloromethane and treated with a 1 N solution of sodium hydroxide in water. The aqueous layer was removed, the organic layer was then washed with brine and dried with sodium sulfate; and the solvent was removed by evaporation to give a pale orange solid. The solid was dried in vacuo at room temperature overnight to give the freebase as a pale orange solid (6.15 g, 96%). HPLC analysis against a standard indicated that the sample was 99.4% freebase. A semipreparative HPLC method was developed for isolation of the unknown impurity from the freebase. Fractions containing the desired impurity were combined and concentrated by evaporation. The isolated material was puriﬁed in a ﬁnal analytical cleanup to yield 25 mg of the desired impurity. LC/MS of the isolated impurity indicated the presence of two peaks, corresponding to a dimer-like degradant and the parent drug. A full set of NMR experiments was performed to elucidate the structure. Despite having an adequate amount of sample, sensitivity was limited by low solubility. Deuterium oxide as the solvent eliminated the ability to detect exchangeable protons, although this was not deemed to be a signiﬁcant limitation for this structural elucidation. The NMR analysis conﬁrmed that there were two primary components present in a ratio of approximately 2.5 to 1,

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respectively. The spectra were consistent with the parent and the structures given above.

III. SUMMARY AND CONCLUSIONS The process of characterization of impurities described in this chapter uses a designed approach for the isolation of unknown impurities and degradants in pharmaceutical drug substances. This approach focuses on efﬁciency, so that the success of data collection is maximized. The isolation of pure material is crucial when trying to identify the structure of an unknown impurity/degradant. Once the unknown has been isolated, it can be submitted for structure elucidation using mass spectrometry and NMR spectroscopy. Identiﬁcation of degradants and impurities aids in the understanding of degradation mechanisms and impurity formation. Identiﬁcation conducted at an early stage allows for improvement in the drug substance/drug product development process to prevent these degradants/impurities long before the ﬁling stage. The multidisciplinary team-based approach outlined in this chapter is most efﬁcient for solving impurity/degradant problems. One can often be misled by a single technique, and multiple characterization techniques are essential. The following appendix summarizes key lessons learned in the process of isolating and characterizing degradant and impurities.

APPENDIXLESSONS LEARNED Isolation/Synthesis .

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Utilize all available sources of information such as TLC, LC/MS, NMR archive data, project lab background, as well as process, formulation, and discovery chemistry. Ask questions and conduct meetings when needed; even the projects that look easy can be hard. To collect all background data can be of significant value with tight time lines. Find out what methodologies are in place. If a method without a volatile mobile phase is not available, development time is required which adds time to the identification. The mobile phase used for isolation should be selected carefully to avoid salt complexation. It is advised to perform isolation and extraction experiments at a small scale before committing precious isolate samples. Shortcuts should not be taken in isolation efforts for NMR analysis. An analytical HPLC cleanup/desalting is required for NMR analysis. This improves the sample purity and quality of NMR data. It is especially difficult to characterize dimers and oxidations of heteroatoms if a sample is not of high purity. Clean glassware is essential in the isolation process when isolating mg/mg-sample amounts. Glassware impurities can contaminate precious isolation samples and complicate NMR analysis.

14 SOLVING IMPURITY/DEGRADATION PROBLEMS: CASE STUDIES .

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Up-front stability investigation is a worthwhile investment and can save valuable time reisolating an impurity/degradant, especially in cases in which the project is sample-limited. Attention should be given to HPLC and collection tubing selection as well: Tygon tubing that can leach phthalates should not be used in HPLC and fraction-collection equipment. In a last step prior to NMR analysis, the isolated/synthesized sample should be washed with deuterated solvents several times to purify the sample for NMR. Recovery is never what you expect it to be . . . assume 50%. Also, the project completion time line is never what you expect it to be. Project teams need to be aware of this and notify the isolation/characterization experts as soon as possible to ensure there is adequate time to complete the project. Use chemistry knowledge to assess the stability of a compound prior to isolation, and determine if special collection conditions may be necessary. For preparative TLC and column chromatography, it can be critical to remove silica gel and other interferences in a filtration step.

Mass Spectrometry .

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For mass spectrometry–compatible mobile phases, volatile acid salts are acceptable (i.e., TFA, formic acid, acetic acid). The ammonium counterion works best. TEA and other organic amines as mobile-phase additives tend not to work well and suppress ionization. Less than 1 mg of sample can be analyzed, and with LC/MS, timeintensive isolation is generally not required.

NMR Spectroscopy . . .

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Deuterated solvents are required for NMR analysis. For drug substance analysis, the freebase is preferable to avoid excessive salt levels, which complicate data interpretation. Greater than 5-mg sample is preferred, but less than 1 mg is possible in some cases. A microprobe can be used for the less than 1-mg scale sample cases. Nonprotic organic solvents minimize undesirable proton exchange (i.e., CDCl3, d6-DMSO). Molecular weight and proposed structures based on LC/MS analysis expedite the NMR structure elucidation.

REFERENCES 1. Alsante, K. M., Friedmann, R. C., Hatajik, T. D., Lohr, L. L., Sharp, T. S., Snyder, K. D., Szczesny, E. J. Degradation and Impurity Analysis for Pharmaceutical Drug Candidates. In Handbook of Modern Pharmaceutical Analysis (Ahuja, S. and Scypinski, S., eds.), Academic Press, 2001.

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2. International Conference on Harmonisation, ‘‘Draft Revised Guidance on Impurities in New Drug Substances,’’ Federal Register 65(140):45085–45090, 2000. 3. International Conference on Harmonisation, ‘‘Draft Revised Guidance on Impurities in New Drug Products,’’ Federal Register 65(139):44791–44797, 2000. 4. ACDLabsTM Software. 5. Russell, G. A. Deuterium-isotope Effects in the Autoxidation of Aralkyl Hydrocarbons. Mechanisms of the Interaction of Peroxy Radicals. J. Am. Chem. Soc. 79:3871, 1957. 6. Bellucci, G. and Chiappe, C. Crown Ether Catalyzed Stereoselective Synthesis of Vinyl Ethers in a Solid Liquid Two-Phase System. Synlett. 880, 1996. 7. Knochel, P. Cesium Hydroxide Catalyzed Addition of Alcohols and Amine Derivatives to Alkynes and Styrene. Tetrahedron Lett. 40:6139, 1999.

INDEX

Abbreviated new drug applications (ANDA), 7, 28, 30, 31 Absorption spectroscopy in hyphenated techniques, 351–356 see also speciﬁc methods Accelerated solvent extraction (ASE), 189–193 Accelerated testing, 102–103, 250, 253 Acceptance limits, 91 ACCORD-HMBC, 322, 324 Acetaldehyde, 79 Acetaminophen, 44, 352, 353 Acetic acid, 82 Acetonitrile, 30 deuterated, 309, 311 Achiral stationary phase functional groups, 233–236 Acid degradation, case study, 378–379 Active moiety, deﬁnition, 120 Active pharmaceutical ingredient (API), 2 characterization and structure elucidation, 287 concomitant components, 5–6 deﬁnition, 120, 162

degradation products, 4, 150, 250, 251 high potency, 356 interaction with excipients, 253 isomeric purity proﬁle, 150 peak purity assessments, 153 related compounds, 349 terminology, 5 see also drug substance Adamantane, 59 Additives, 84–85 Adduct degradants, case studies, 381–385 Adducts, 283, 381 Afﬁnity solid phase extraction, 169 (L)-Alanine, solid-state proton spectra, 58 Alcohols, as impurities, 83 Aldehydes, 79–80 Aldonic acid, 82 Aluminum oxide (alumina), 168, 233, 234 Amino acid residues, basic functional groups, 266 Ammonium, as counter-ion, 399 Amorphous phase, 44, 68 Amoxicillin, 40 Ampicillin, 40, 49, 56

Amylodextrin tablets, 77 Amylose derivatives, in column chromatography, 236–237 Analytical methods development, 14–16, 91, 251 protocols, 232 for study of impurities, 33–36 Analytical reference standard (ARS), 120–121, 135–138 Antibody methods, 169 Antimicrobials, 85 Antioxidants, 79, 80, 85 Appearance (physical description), 128–129 Aqueous infusions, extraction of polar substances, 189 Area under the curve (AUC), 341 Arsenic, 81 Artifacts, 160, 215, 216, 222–223 Artiﬁcial neural network models, 52 Ash chromatography see column chromatography

401

402

INDEX

Assay methods reference standards, 134–135 robustness, 349 Association complexes, 271–272 Atmospheric pressure chemical ionization (APCI), 252, 255, 264–266, 268, 354 Atmospheric pressure photoionization (APPI), 266, 267, 354 Atorvastatin ARS, 124 Attached proton test experiment, 61, 317, 324 Attenuated total reﬂectance (ATR), 50, 52 Authentic materials (AM), deﬁnition, 121 Autoxidation, 78, 79, 82, 108, 109 Azobisisobutyronitrile (AIBN), 109

Bacteria, as impurities, 83 Base, free, 397, 399 Basicity of biomolecules, 264 gas phase, 261 Batches, scaled-up and laboratory scale, 35 Beam-type mass spectrometric analyzers, 273–274 Benzaldehyde, 79 Benzene, 30 Benzoate, as counter-ion, 147 Benzyl alcohol, oxidation, 79 Best practice documents, 120 Besylate, as counter-ion, 147 Bioburden, 84 Biological impurities, 83–84 Biological samples extraction by ASE, 189 SPE, 172 Biomolecules basicity, 264 ESI analysis technique, 264 Birthday problem, 342–343 Bismuth, 81 Body ﬂuids and tissues see biological samples

Boltzmann factor, 53 Bovine serum albumin (BSA), as chiral stationary phase, 237 Bragg’s law, 41, 42 Bridging standards, 139 British Pharmacopeia, 31 Broadband probes, 312, 313 ‘Brush-like’ chiral stationary phases, 237 Bulk pharmaceutical chemicals, 5 Bulk solid, impurities, 39–73 n-Butyl chloride reactant, 100–101 Butylated hydroxytoluene (BHT), 79, 80, 85 By-products deﬁnition, 4 discovery and identiﬁcation, 95 as source of impurities, 11–12, 91 unanticipated, 253

Cadmium, 81 Caffeine, 44, 49, 352, 353 Calcium chloride, 181 Calcium phosphate, 77 Capillary electrophoresis (CE), 17, 20, 133 coupled with MS, 262–263 with DAD, 351 peak capacity, 343 Carbamazepine, 42, 43, 49 Carbon graphitized, 167–168 isotropic shifts, 62 spectrum acquisition, 315 Carbon dioxide, in SFE, 182, 186 Carbon tetrachloride, 30 Carbons, quaternary/methine/ methylene/methyl, 317, 319, 325, 327, 390 Carbonyl groups, 336, 352 Carboxyl groups, 379 Carboxylic acids, 82, 264 Carryover, in ASE, 192 Cartridges liquid–liquid extraction, 179–180 SPE, 169–170, 172 Case studies approach used, 22–24

degradation, 368–385 LC-MS, 372–397 NMR, 338–385, 389, 393–397 oxidative degradation, 377–380 preparative HPLC, 368–375, 385, 388–397 PRIs, 385–387 process enrichment, 392–395 Catalysts, as source of impurities, 91 Catecholamine, 260 Cefepine dihydrochloride, 43, 51 Celiac disease, 84 Cellulose derivatives, 77, 79, 84, 236–237 ﬁlter membranes, 196, 197 Centrifugation, 194–195 Cephalexin, 44 Characterization methods, 20–22 reference standards, 127–135 for related substances, 254 Chemical ionization (CI), 255, 268, 354 Chemical shielding effect, 58 Chemical shifts database, 332 differences, 62 sertraline, 333, 335 trovaﬂoxacin, 333–334, 335 ziprasidone, 333–334, 335 Chemical stability, effect of water, 11, 76 Chemiluminescent nitrogen (CLN) detectors, 113 Chemistry-guided approach, 104–105, 112, 399 Chiral compounds active moiety, 120 NMR investigation, 322 screening, 154 stereoisomers in KPSS, 150 Chiral methods, 15, 93, 134 Chiral stationary phases, 236–237 Chirality, 10 see also stereoisomers Chloramphenicol palmitate, 44

403

INDEX

Chloroform, 30 Chlorpropamide, 44, 53 Chromatographic methods co-elution, 341–342, 345 ‘heart-cut’ portion, 245 multidimensional, 345 with multiple detection schemes, 92 online detection, 239–240 peak capacity, 342 preparative, 244–247 reference standards, 131–134 speciﬁcity, 343 see also speciﬁc methods Chromium, 81 Chromophores, 153, 260, 310 Cilastatin sodium, 44 Clearance rate, 288 Clinical trials, 122, 123, 363 Clustering reaction, 264 Coaxial liquid-sheath CE interface, 263 Cocktails, 161–162 Co-elution, 341–342, 345 Collection devices, SFE, 188 Collision gas, 277, 278, 279 Collision-induced dissociation (CID), 269, 277, 278, 280–282 Collision-induced excitation energy, 279, 280 Collisionally activated decomposition (CAD) see collision-induced dissociation (CID) Colony forming units (CFU), 84 Color, 305 see also chromophores Column chromatography, 20, 231–248 combination, 236 equipment, 237–240 for isolating impurities, 367 normal phase, 234–235 preparative method, 244–246 reversed-phase, 235–236, 242–244 screening methods, 241–244 Column trapping techniques, 308, 311

Combined rotation and multiple pulse spectroscopy (CRAMPS), 58–59 Compendia, role of, 30–31 Compounds identiﬁcation, 154, 252 isolation by MS, 258 Concomitant components, USP terminology, 5 Conditioning, in SPE, 170, 172–173 Continuous-ﬂow fast atom bombardment (CFFAB), 251, 263 Copper, 81, 82, 109 Corn starch, 79 Correlation spectroscopy (COSY), 318, 324, 325, 327, 328 Co-solvent system, ASE, 191 Coulometric KF-titration, 131 Counter-ions, 147, 399 Countercurrent LLE, 177–179 Coupling constants, 327, 332 Coupling of instrumentation methods, 22, 259–264, 356 Coupling parameters, 320–321 Coupling-based editing techniques, 61 Creams and ointments, extraction of polar substances, 189 Creutzfeldt-Jacob disease, 84 Critical point (Cp), 182 Crixivan, 135 Crospovidone, 77, 79 Cross-linking agents, 79 Cross-polarization build-up curve, 68 Cross-polarization magic angle spinning (CPMAS), 57, 67 Crystalline sample, amorphous state, 44 Crystallization-related impurities, 9–10 Cycle time, in ASE, 192 Cyclodextrins, 237, 264

Data acquisition, 275, 282–286, 356, 398

multidimensional, 282–283, 343, 347 set, 287 Decision trees, 32, 33, 34 Deconvolution methods, 55 Degradants see degradation products; Impurities Degradation case studies, 368–385 data interpretation, 154 increased resistance, 253 kinetics, 13–14 pathways, 107 source of impurities, 8, 102–115 see also purposeful degradation Degradation products API, 150–151, 152 co-elution, 152 deﬁnition, 4 elimination from KPSS, 154–155 identiﬁcation, 255–256 potential and signiﬁcant, 105–106 source, 91 thresholds, 35 by TLC methods, 211 toxicological evaluation, 253 UV detection, 152, 153 Delavirdine mesylate, 51 Deprotonation, 264 Derivatizing agents, 204, 211, 215 Detection methods/detectors differential response, 113 inverse or indirect, 318 for molecular formula determination, 354 simultaneously employed, 282 for TLC/HPLC, 204, 205, 208, 242, 260 universal, 20 Deuterated solvents, 305–306, 309, 311, 356, 399 Deuterium gradient shimming, 310, 314 Diastereomers, 150 Diatomaceous earth, 179, 190 1,1-Dichloroethane, 30 1,2-Dichloromethane, 30

404

INDEX

Dielectric constant, supercritical ﬂuids, 184 Diethylhexylphthalate (DEHP), 82 Diethylstilbestrol (DES), structural identiﬁcation, 354 Differential scanning calorimetry (DSC), 47–49 reference standards, 131 Differential thermal analysis (DTA), 47, 48 Diffuse reﬂectance (DRIFT), 50–51 Dimer species, 283, 331, 336, 389 case study, 389 Dimethyl sulfoxide (DMSO), 306, 323, 366–377, 381, 382, 391, 394 Dioctyltin isooctylmercaptoacetate, 82 Diode array detection (DAD), 16, 132, 283, 310, 311, 345, 351 1,4-Dioxane, 30 Disks, SPE, 171, 172 Distortionless enhanced polarization transfer (DEPT), 316–317, 325 Distortionless enhanced polarization transfer (DEPT)-HSQC, 319, 320, 324, 325 Distribution ratio, LLE, 176, 177, 179 DNA, absence in ﬁnished products, 3 Dodecanolactam, 352, 353 Dosage forms effect of water, 11, 76 inactive ingredients, 35 lyophilized, 76–77, 78 properties, 77 see also drug product Double focusing sector mass spectrometers, 270, 274, 275 Double quantum ﬁltered COSY (DQFCOSY), 318, 324, 327, 328 Drug candidates, stress testing, 253 Drug development life cycle, 121–125, 250–251 preclinical stages, 252, 262

time line, 363 TLC applications, 206–207 Drug Master File (DMF), Type II, 30, 31–32 Drug product deﬁnition, 120 impurities, 33, 75–88, 349 launch, 123–124 LOD/LOQ/low level linearity, 218–219 maturation, 124–125 peak set, 146 stress testing, 113–115 TLC method development and validation, 216–220 Drug substance deﬁnition, 2, 120 impurities, 28–30 intrinsic stability, 113 LOD/LOQ/low level linearity, 209–210 peak set, 146 peak tracking example, 152 phase pure, 40 separation from excipients, 217–218 thresholds, 35 TLC method development and validation, 208–216 Drug synthesis, generalized scheme, 95 Drug-excipient compatibility studies, 106, 114 Drying agents for ASE, 191 for LLE, 180–181, 182 Duloxetine hydrochloride, 114–115 Dyes, 79

EFGR tyrosine kinase inhibitor, 56 Elastomeric stoppers, source of aldehydes, 79 Electrochemical detection (ECD), 240, 260 Electron ionization (EI), structural elucidations, 354 Electron-induced excitation in organics (EIEIO), 277

Electrophoretic separations, 133 Electrospray ionization (ESI), 252, 255–256, 261, 264–268, 272 Electrostatic mass spectrometric analyzers, 273 Elemental analysis, 129 Elution, in SPE, 170, 171, 174 Emulsions, 180 see also chiral compounds Enantiomers, 44, 150, 243 Endotoxins, 83, 84 Environmental conditions, 76, 91, 253 Essential Component Set, 146 Ethers, polymeric, 78 Ethylene oxide–based materials, 78 European Pharmacopeia, 31, 95 Evaporative light scattering (ELS) detectors, 113 Excipients additives, 84–85 animal derived, 84 interaction with API, 33, 91, 253 in KPSS, 150 microbial limits, 84 moisture content, 77, 78 peroxide impurities, 78–79 polymeric, 85 polyvinyl pyrrolidone (PVP)–based, 79 as source of impurities, 11, 12 SS-NMR, 63–64 storage, 84 water content, 76 Extractables from ﬁlter membranes, 198–199 from packaging, 82–83 Extraction exhaustive or selective, 176 in LLE, 174 Extraction cells, SFE, 185–186

Factor analysis, 55 Fast-atom bombardment (FAB), 255

405

INDEX

Fats and oils hydrogenated, 81 unsaturated, 79, 82 FDA see United States Food and Drug Administration Feed material, preparative chromatography, 244–245, 246 Field ionization/ﬁeld desorption (FI/FD), 354 Film coatings, 77 Filter membranes afﬁnity for analyte, 198 extractables, 198–199 materials, 196, 197 pore size, 196, 198 Filters choice of, 196–199 housing material, 196 size (diameter), 196, 198 Filtration, 195–199 Finished product see drug product Flame ionization detection (FID), 341 Flash chromatography, peak capacity, 343 Flash silica, 234–235 Flavinoids, 266 Flavoring agents, 79 Flow rate, preparative chromatography, 244, 247 Flowcharts impurity/degradant isolation and identiﬁcation process, 362, 363 NMR spectroscopy identiﬁcation process, 364 Fluconazole, extraction by SFE, 189 Fluorine, NMR studies, 66 Fluoxetine hydrochloride, synthesis, 94 Focused methods, 105 Food, Drug, and Cosmetics Act (USA), 7–8, 10, 341 Forced degradation see purposeful degradation Foreign substances, USP criteria, 5, 6, 29 Formaldehyde, 79 Formic acid, 82 Formulations, SS-NMR analysis, 63–64

Fourier transform infrared spectroscopy (FTIR), 50, 351–353 Fourier transform infrared spectroscopy (FTIR)-ATR spectroscopy, 52 Fourier transform mass spectrometer, 275 Fourier transform methodology, 49 Fraction collection, 160 Fragmentation spectrum, 279, 354 Freebase, 399 case study, 397–398 Free radicals, 374, 375 Functional groups basic, 266 mass spectral data, 285 Fungi, as impurities, 83, 84 Furfural, 79, 82

Ganciclovir polymorphs, FTIR-ATR spectroscopy, 52 Gas chromatography (GC) common technique, 341 minimum target mass levels, 350 peak capacity, 343 quantiﬁable, 17 reference standards, 134 tandem methods, 345 Gas chromatography-mass spectrometry (GCMS), 251, 347, 353 Gas-phase basicities, solvents, 261 Gelatin, 80, 84 Glass borosilicate, 196, 197 extractables, 82 for packaging, 78 transition temperature (Tg), 68, 78 Glassware, clean, 306–307, 398 Glucose, 80 Gluten, 84 Glyoxal, 79 Glyoxalic acid, 82 Goals, for the investigation of impurities, 91–92 Gold standard see analytical reference standard

Good Manufacturing Practices (GMP), 33, 207–208 Gradient coherence selection, 318 single or triple, 311, 312 Guanidine hydrochloride, quantitation by TLC, 221

Handbook of Sorbent Extraction Technology, 172 Hartman-Hahn matching condition, 59 ‘Heart-cut’ portion of chromatography, 245 Helium, as collision gas, 278 Hemicellulose, 79 Heteroatoms, 327, 330 Heteronuclear correlation (HETCOR), 319, 392 Heteronuclear multiple bond correlation (HMBC), 320–321, 327, 329 Heteronuclear multiple quantum coherence (HMQC), 317, 319, 325, 328 Heteronuclear single quantum coherence (HSQC), 317, 319, 324, 325, 327, 328 Hexamethylenetetramine, 79 High-performance liquid chromatography (HPLC) common technique, 254, 341 coupled with MS see liquid chromatography-mass spectrometry (LC-MS) coupled with tandem mass spectrometry (LC-MS-MS), 289 with DAD, 344, 345, 347, 351, 356 detectors, 260 instrumentation, 311 methods, 33, 35 mobile phase, 260, 261 with MS detection, 113 normal phase, case study, 385–387, 388–389 peak capacity, 343

406

INDEX

preparative, 258–259, 366, 367 case studies, 368–375, 385, 388–397 reference standards, 131–132 relative retention times, 365 reversed-phase see reversed-phase HPLC semipreparative, 258–259 case study, 380–381 solvent effects, 260 solvents, 260, 261, 265 superior technology, 31 thermally labile compounds, 251 with UV detection, 122, 344, 345, 346, 365 vs TLC, 205–206 High-performance thin layer chromatography (HPTLC), 204 High-pressure liquid chromatography, 17 High-pressure liquid chromatography see mainly highperformance liquid chromatography High-resolution mass spectrometry (HRMS), 130 Homonuclear decoupling experiment, 325 Hydrates, water content, 48–49 Hydrocarbons, halogenated, ionization, 256 Hydrogen peroxide, 78, 85 Hydrolysis, 76, 79, 107–108 Hydroxyethylcellulose, 79 Hydroxypropylmethylcellulose, 77, 79 Hyphenated techniques, 254, 289, 304, 309, 341–359

see also LC NMR Ibuprofen, enantiomeric composition, 44 Identiﬁcation (proof of structure), 129 Identity measurements, chromatographic techniques, 341

Imipenem, quantitative XRPD, 44 Impurities achiral, 240–244 analytical methodology, 33–36 artifacts, 222–223 bacteria, 83 characterization, 3–4, 220–228, 251, 252–258, 301–339, 345, 349, 367–368 chemical classiﬁcation, 22–23 ‘chemistry-guided’ approach, 92–93 chiral, 243–244 classiﬁcation, 90 common, 253 common names, 4–5 control, 94, 95–96 crystallization-related, 9–10 deﬁnitions, 2–3 degradation-related, 91, 92, 102–115 designations, 4–6 in drug products, 33–36, 75–88 enhancement, 157 evaluation, 31–32 formation mechanism, 373, 375 formulation-related, 12, 114, 349 fungi, 83, 84 ICH terminology, 6 identiﬁcation, 350, 361–365 identiﬁcation threshold, 20–30, 250 intermediates, 5, 11–12, 91 inorganic, 6, 29, 91 investigation strategies, 89–117 isolation, 3–4, 220–228, 232, 241–244, 251, 362, 363, 367 case study, 372, 373–375 metals, 81, 172 molecular mass, 366 new, 32–33 NMR characterization, 301–339, 367–368 organic, 6, 29, 90, 91 organic volatile (OVI), 2, 5, 6, 11, 30, 134 polar and nonpolar, 93

process-related, 91, 92 puriﬁcation, 366 qualiﬁcation, 32–33, 250 quantitation, 36, 209, 218–219 polymorphs, 39–40 prions, 83, 84 regulatory guidance, 27–37 salt-related, 223, 224 similarity to parent, 325, 326, 331 sources, 8–14, 75, 91, 96, 303, 304 stereochemistry-related, 10–11 storage-related, 13–14 structure, 231 technique-oriented approach, 92 threshold values, 20–30, 32, 90 TLC analysis, 221–227 toxic, 93, 101, 365 USP criteria, 5, 6, 30 types, 28–30 unknown, 223–227, 350, 363, 366 case study, 390–392 USP terminology, 5–6 Viruses, 83 Impurity grid, 151, 162 Impurity proﬁle, 341–342 In-house standard see working standard In-source skimmer/cone, CID, 280 Inclusion complex stationary phases, 237 Indinavir sulfate, 135 Indomethacin, 44, 55 Inductively coupled plasma (ICP), 131 Information sources, 304–305, 398 Infrared (IR) spectroscopy, 16, 50–52, 129, 305 Ingredients, inactive, 35 Instrument Qualiﬁcation (IQ), 207 Instrumentation advances, 349 coupling, 22 interfacing, 260, 263 Interaction product, 4 Intermediates, 5, 91 as source of impurities, 11–12

407

INDEX

International Conference on Harmonisation (ICH) deﬁnition of stress testing, 106 guidance on New Molecular Entities (NMEs), 33 guidelines, 208, 250, 349 guidelines on impurities, 8, 90, 209, 222, 365 guidelines on stability, 102 regulations, 146 Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), 2, 36 guidance, 27, 28–29 terminology, 6 Investigational new drug (IND), 36, 122 Iodine detection, 242 Ion activation methods, 277–279 Ion chromatography (IC), 132–133 Ion cyclotron resonance (ICR) spectrometer, 270, 275, 279, 283 Ion-exchange materials, 167, 172 Ionization techniques, 255, 260, 261, 264–272, 354, 366 Ions characterization, 255–256, 276 doubly charged, 268, 269, 388 formula determination, 255–256 fragmentation and spectral interpretation, 256, 257 inorganic, eliminated from ﬁlter, 199 isomerization (rearrangements), 278 multiply charged, 266–272 structural interrogation, 277–282 Iron, 81, 82, 109 Isobaric species, resolving, 275–276 Isocratic methods, 93, 94, 342 Isolation choice of methods, 18–20

key lessons, 398–399 preparative, 366–367 case study, 379–380 process, 397–398 Isomers cis–trans mixture, 392 optically active and nonoptically active, 150 purity proﬁle of API, 150 Isotopes, naturally occurring, 284, 286 J-coupling, 61, 62 Japanese Pharmacopeia, 31 Kaolin, 81 Karl Fischer (KF) titration, 131 Key predictive sample set (KPSS), 146–149, 153 Kubelka-Munk units, 51 Laboratory standard see working standard Lactose anhydrous, 77 in excipient, 80 monohydrate, 77, 84 phase composition, 56 spray-dried, 79 TG analysis, 46, 47, 48 Lanolin and derivatives, 84, 85 Latex dispersions, 85 Lead, 81, 122 Lead declaration, 122 Lecithin, 85 Levothyroxine, 135–136 Light degradation, case study, 380–381 Limit of detection (LOD), in TLC, 208–210, 218–219 Limit of quantitation (LOQ), in TLC, 208–210, 218–219 Lipitor, 124 Liquid automated handling, 176 direct introduction, 251 Liquid chromatography (LC) low-pressure, 238–239 reversed-phase, 350 see also high-performance liquid chromatography (HPLC)

Liquid chromatography-mass spectrometry (LC-MS), 289, 304, 366 advantages, 253–254 case studies, 372–397 choice of methods, 259–262 common technique, 353, 356 with DAD, 356 interface devices, 251–252 ionization modes, 267 memory and computational requirements, 347 molecular mass and fragmentation data, 366 tandem methods, 289, 345 trace components, 253 Liquid chromatographynuclear magnetic resonance (LC-NMR), 289, 302–303, 305, 307, 325, 345, 366 equipment, 310–312 on-ﬂow, 311 online data acquisition, 356 peak collection, 311, 312 probe, 313 samples, 307–309, 310–311 solvents and buffers or modiﬁers, 309 stopped-ﬂow, 311–312 see also high-performance liquid chromatography (HPLC-NMR) Liquid chromatographynuclear magnetic resonance-mass spectrometry (LC-NMR-MS), 254, 309 Liquid-junction CE interface, 263 Liquid–liquid extraction (LLE), 174–181 Loop collection/reinjection, 310, 356 Loss on drying, 130–131 Lumaxis analog, 44 LY297802, 97–102, 110–111 Lyophilization, 76–77

Magic angle spinning (MAS), 56, 57, 66 Magnesium salts, as drying agents, 181

408

INDEX

Magnesium silicate (Florisil), 168 Magnesium stearate, 77, 84 Magnetic sectors mass spectrometric analyzers, 273, 274 Maltose, 80 Mannitol, 77 Manufacturing process control, 91 Mass balance, 109–112, 113, 152–154 Mass spectrometry (MS) analyzers, 256, 273–277 artifacts, 265 characterization/structural information, 16, 21–22, 249–299 complex spectra, 264 coupled with capillary electrophoresis (CE), 262–263 detection/detectors, 259, 356 differential sensitivity, 260 fragmentation behavior, 354 in hyphenated techniques, 255, 311, 353–355 see also gas chromatography-mass spectrometry (GC-MS) liquid chromatography-mass spectrometry (LC-MS) internal environment, 261 interpretation, 284–285 isolation technique, 258 isotopic patterns, 271, 284, 286 key lessons, 399 molecular weight determination, 16, 277, 354 online detection, 240, 254 peptides/proteins, 266–269 pharmaceutical applications, 286–288 quadrupole instruments, 274–275 qualitative, 252 in reference standard characterization, 130 reverse geometry instruments, 278 sample size, 255, 256 sector instruments, 270, 273, 274–275

software, 285 structure elucidation, 254, 255 tandem methods, 274, 277, 283, 284, 288 trapping instruments, 274, 279, 282 triple quadrupole methods, 274, 279, 280, 282, 283 ‘universal’ detector, 113, 259, 260 Mass spectrometry-highperformance liquid chromatography (MS-HPLC), 255 Mass transfer properties, supercritical ﬂuids, 184 Mass/charge (m/z) accurate determination, 275 and molecular mass, 283 Material balance see mass balance Matrix, polymeric, 374 Matrix components deﬁnition, 162 removal, 171 see also excipients Matrix-assisted laser desorption ionization (MALDI), 268, 350 Membrane ﬁlter materials, 196, 197 Membrane ﬁltration see ﬁltration 2-Mercaptobenzothiazole, 82 Mercury, 81 Mesylate esters, as impurities, 102, 103 Metabolites, 286–288 Metal catalysts, 81 Metal chelates, SPE, 172 Metal impurities, 80–82 Metal oxides, 82 Metals heavy, 81 leached, 81 trace, SPE, 172 Methanol, deuterated, 309 Methine/methylene/methyl groups, 317, 319, 325, 327, 390 Method development 14–16, 91, 251 peak set, 162 process, 146

sample solutions, 159 Methylcellulose, 77 Methylene chloride, 30 Michelson interferometer, 50 Microcrystalline cellulose (MCC), 77, 84 Microprobes, 313, 389, 399 Miller index, 41 Misoprosotol, 188 Mobile phase effects, 260, 261 mass spectrometry compatible, 399 selection, 398 Modifying solvent, for SFE, 186–187 Moisture content, 76, 77, 78 see also water Molecular formula, determination, 255–256, 354 Molecular mass analysis, 283 determination by MS, 16, 277, 354, 366 and m/z value, 283 Monoisotopic precursor ion selection, 284 Mother liquor deﬁnition, 163 samples, 151–152, 258, 304, 308, 367, 395, 397 Moving belt, 251 Mull techniques, 50–51 Multidisciplinary approach, 363–365, 398 case study, 393–395 Multiple quantum magic angle spinning (MQMAS), 66

Near-infrared (NIR) spectroscopy, 55–57 Nedocromil metal salts, hydration, 49 Negative ion attachment mass spectra, 347, 348, 349 Neurotransmitters, electrochemical detection, 260 New chemical entity (NCE), 11, 120 see also drug substance New drug application (NDA), 7, 28, 30, 31, 123

409

INDEX

New molecular entities (NMEs), ICH guidance, 33 Nickel, 81 Nitrogens count rule, 271, 273, 394 tertiary, 109 Nitroglycerin, 345, 346, 349 NOESY experiments, 323 Normalizing factors (response factors), 259 Notice of Claimed Investigational Exemption for a New Drug, 7 Nuclear magnetic resonance (NMR) methods case studies, 338–385, 389, 393–397 chirality investigation, 322 choice of experiment set, 324–325 consistency with MS or IR data, 336 data interpretation, 325–334 detection limit, 325 deuterated solvents, 305–306 experiments, 314–324 forced degradation products, 363 heteronuclear two-dimensional experiments, 319–321, 324–325 homonuclear two-dimensional experiments, 318–319 in hyphenated methods, 355–356 identiﬁcation process ﬂowchart, 364 impurities, 301–339, 367–368 case study, 391–392 information gathering, 304–305 instrumentation, 309–314 key lessons, 399 magnetic ﬁeld strength, 310 microprobes, 313, 389, 399 molecular mass, 304 nondestructive nature, 21, 302, 368 on-line capability, 254

one-dimensional experiments, 315–317, 325–330 peak assignments, 59–62, 325–330 probe speciﬁcations, 312–314 quantitation, 302 redundancy, 327 in reference standard characterization, 130 safety precautions, 307 samples, 301, 305, 309–310 secondary interpretation, 331 sensitivity, 65, 301–302, 367–368 solid state (SS-NMR), 57–69 solvents, 305–306, 309–310 spectral simulations, 331–334 stop ﬂow mode, 344 structural elucidation, 16, 325–330, 367 case study, 375–377 tube-based, 303, 307, 311 two-dimensional experiments, 317–324, 325–330 ‘universal detector’ for hydrogen and carbon, 302 Nuclear Overhauser effect (nOe), 322–323, 325, 331, 336 Nylon ﬁlter membrane, 196, 197

Ofﬂine analysis, 343, 347 see also orthogonal techniques Online analysis, 239–240, 251, 252, 254, 347, 356 Operational Qualiﬁcation (OQ), 207 Optical activity, 150 Optical microscopy, 129 Optical rotatory dispersion (ORD), in reference standard characterization, 130

Ordinary impurities, USP criteria, 5, 6, 30 Organic compounds as impurities, 6, 29, 90, 91 ionization, 261 trace level identiﬁcation, 256 Organic peroxides, 78 Organic volatile impurities (OVI), 2, 5, 6, 11, 30, 134 Orthogonal techniques, 20, 92, 154, 163, 206, 207, 208, 231, 343 Ottawa sand, 190 Oxidation peroxide-mediated, 108, 109 photochemically induced, 108, 109 radical-mediated, 108 Oxidative catalysis, 82 Oxidative degradation, 108–109, 368, 373–375 case studies, 377–380 peroxide-mediated, 108, 109 N-Oxides differentiation from sulfoxide, 368–371 oxidation site, 326, 331

Packaging appropriate, 91 composition information, 82 extractable chemicals, 82–83 glass, 78 interaction with, 249 metal impurities, 81 protective effect, 76, 78 stress testing, 114, 250 Packing material for column chromatography, 233–237 polymeric, 167, 168, 233 Palladium, 81 Parent species impurities similar to, 325, 326, 330 spectra, 285, 305 Paroxetine HCl, impurity determination, 101–102

410

INDEX

Partial least squares regression, 51, 55 Particle size, 247 Patient safety, 122 Peak capacity, Chromatographic separation, 343 Peaks assignment (NMR), 325–330 characterization, 345 identiﬁcation, 154 tracking, 157, 160, 161, 162, 366 unresolved, 271 width, optimizing, 308–309 Peanut allergens, 84 PENDANT, 317 Penicillins, 3 Peptides/proteins as chiral stationary phases, 237 molecular mass determination, 268, 269 MS, 266–269 sequencing, 269 Performance Qualiﬁcation (PQ), 207–208 Permitted daily exposure (PDE), 30 Peroxides impurity in excipients, 78–79 organic, 78 in oxidative degradation, 108, 109 pH adjustment, in LLE, 174–175 Pharmaceutical industry applications of MS, 286–288 applications of SFE, 188–189 applications of TLC, 206–207 regulatory requirements, 341 role of manufacturers, 36 Pharmaceuticals degradation pathways, 107 development see drug development post-approval changes, 36 shelf life or expiry date, 249–250 see also active pharmaceutical ingredient drug

product; drug substance; speciﬁc techniques Pharmacopeia harmonization, 31 primary guidance on impurities, 7 see also United States Pharmacopeia (USP) Pharmacopeial Convention, 124 Pharmacopeial Forum (PF), 31, 124 Pharmacopeial methods, purity monitoring, 94–95 Pharmacopeial speciﬁcations, heavy metal toxicity, 81 Phase diagram, 182, 183 Phase equilibrium puriﬁcation see phase solubility analysis Phase separation, in LLE, 174 Phase solubility analysis, 134–135, 156–157, 160 Phenanthrene, phase transformations, 44 Photodiode array (PDA) detection, 152, 154, 157 Photodissociation (PD), 277 Photolytic degradation, 108 Plastic packaging materials, extractables, 82 Plasticizers, 85 Platinum, 81 Polar substances, extraction by SFE, 189 Polarity, supercritical ﬂuids, 184 Polyacrylamide gel electrophoresis (PAGE), 345, 350 Polyaromatic hydrocarbons (PAH), 266 Polyethylene glycols (PEGs), 78, 79 Polyethylene oxides (PEOs), 78 Polyethyltetraﬂuoroethylene (PTFE) ﬁlter membrane, 196, 197 Poly(lactide), 82 Polymers, helical, 236–237

Polymorphism/polymorphs deﬁnition, 9, 39–40 detection by SS-NMR, 63–64 determination by XRPD, 62–63 as impurities, 39–73 proof of existence, 67 quantitative analysis, 67–69 single crystal X-ray diffraction studies, 62 SS-NMR studies, 62–69 Polyoxyethylene alkyl ethers, 78 Polyoxyethylene stearates, 78 Polypropylene ﬁlter membranes, 196, 197 Polysorbates, 78, 79 Polysulfone ﬁlter membranes, 196, 197 Polyvinyl pyrrolidone (PVP), in excipients, 79 Pore size, ﬁlter membrane, 196 Pot reactions, 12 Povidone, 79 Prazosin hydrochloride, 42–43 Presaturation, 309 Preservatives, 79 Pressure effects, in ASE, 192 Pressurized liquid extraction see accelerated solvent extraction (ASE) Primary standard see analytical reference standard Principal component analysis, 55 Prions, as impurities, 83, 84 Probes cryogenic, 313–314 dual probe, 313 types and sizes, 312–313 Process enrichment, case studies, 392–395 Process-related impurities (PRIs) case studies, 385–387 deﬁnition, 163 investigation and control, 95–102, 364 in KPSS, 150–152 separation priority, 148 Pro-drugs, active moiety, 120 Product-ion MS-MS spectra, 280–282, 284

411

INDEX

Progesterone, Raman spectroscopy, 54 Proteins see peptides/proteins Proton-bound dimers, 264 Proton gradient shimming, 314 Proton solid-state nuclear magnetic resonance (SS-NMR), 57–59 Proton spectrum acquisition, 323, 324, 381, 394 integration, 315 Protons aromatic ring, 328 chemical shifts, 305, 315, 331 donation or abstraction, 265 labile, 323 transfer (ionization), 265 Pseudoephedrine hydrochloride, extraction by SFE, 189 Pumps, 185–186, 311 Puriﬁcation procedures, effectiveness, 96 Purity assignment, 136–138 reference standards, 130–134 requirements, 232 Purposeful degradation deﬁnition, 163 products, 250, 251 samples, 152–155, 385–387 scaled-up, case study, 393–395 stability samples, 367 studies, 345 Pyridine, 30

Quadrupole methods, 271, 274, 279, 282–283 Quadrupole time-of-ﬂight (QTOF) instrument, 279, 280, 282, 283 Qualiﬁcation process, reference standards, 126–127 Quantitative structure activity relationship (QSAR) models, 33

Quasi-equilibrium or RRKM theory, 284 Quinazolinium derivative, 56 5-(4-oxo-phenoxy-4HQuinolizine-3-carboxamide)-tetrazolate, 44

Racemates, active moiety, 120 Racemization, 390 Radial compression technology, 376 Raman effect, 49, 53 Raman spectroscopy, 16, 53–55, 129 Ranitidine, 44, 51–52, 344, 355–356 Reagents, as source of impurities, 91 Recovery, less than expected, 399 Reference standards appearance (physical description), 128–129 assay methods, 134–135 characterization techniques, 127–135 chromatographic methods, 131–134 control and use procedures, 125–126 deﬁnitions, 120–121 DSC, 131 GC, 134 governance, 125 HPLC, 131–132 of impurities, 32 purity, 130–134 qualiﬁcation process, 126–139 in quantitative XRPD, 43 quantity required, 232 role, 15–16 supply, 123 TGA, 131 TLC, 133–134 USFDA, 125 Reﬂectron ion mirror, 276 Refractive index (RI) detection, 240, 282 Registration of Pharmaceuticals for Human Use (ICH), 36 Regulatory requirements, 7–8, 341, 345

Related product, 5, 349 Relative centrifugal force (RCF), 194 Residue on ignition (ROI), 131 Resuspension, in LLE, 174 Retention time matching, 341, 342 relative (RRT), 154, 262, 305, 365 13-cis-Retinoic acid, extraction by SFE, 189 Reversed-phase column chromatography, 235–236, 242–244 Reversed-phase HPLC (RP-HPLC) compatible with CZE, 133 complementary to TLC, 204 reference standards, 132 with UV detection, 92, 93, 105, 109 Reversed-phase liquid chromatography (LC), minimum target mass levels, 350 Rf value, calculation, 204 ROESY experiments, 323 Rotamers, 323, 331 Rotational echo double resonance (REDOR), 66 RRKM theory, 284 Rubber stoppers, extractables, 82

Salt content, 305 Sample automated handling and preparation, 253 cleanup, 171–172 detection, in TLC, 204 enrichment, 308, 309, 366–367, 385–387, 395–396 introduction, in SPE, 170, 171, 173 isolated, 161 load, 244 matrix, SPE, 172 origin, 367 preparation, 165–201, 253, 305, 307–309 pretreatment, ASE, 190–191

412

INDEX

purity, 304, 398 quality, 258 quantity, 232, 304–305, 306, 308, 309–310, 324, 325, 350 retained, 392, 397 selection, 159–162 set, 146 spinning, 314 stability, 309, 323 Scale-up, 286, 367, 373–375, 376, 377–378, 380–381 preparative chromatography, 246–247 Scintillation detector, 42 Screening methods, 93, 96, 97, 152–154, 241–244 Secondary standard see working standard Sector instruments, MS, 270, 273, 274–275, 276 Sedimentation velocity, 194 Semisynthetic methods, 251 Separation methods, 17–18 generic, 262 registered with regulatory authorities, 253 Sertraline, 333, 335 Sheathless CE interface, 263 Shelf life, 35, 123, 249–250, 253 see also storage Signal impurities, USP terminology, 5, 6 Silica as contaminant, 387, 399 ﬁber, coating and derivitization, 174 gel, 167–168 as stationary phase, 233, 234 Silicon dioxide, colloidal, 77 Single crystal X-ray diffraction, 41–42, 62, 130 Size exclusion, 172 Skimmer/cone CID, 282 Sodium, in excipients, 81 Sodium lauryl sulfate (SLS), 77 Sodium starch glycolate (Explotab), 77 Sodium sulfate, as drying agent, 181 Software mass spectra interpretation, 285

simulated spectra, 332, 333 Solid dosage forms, extraction by ASE, 189 Solid–liquid slurries, 57 Solid-phase extraction (SPE), 166–174, 308, 309, 311, 366, 374, 395 case study, 373–375 Solid-phase micro extraction (SPME), 174 Solid-state NMR (SS-NMR), 57–69 Solutions, stability, 159 Solvation or association complexes, 265–266 Solvatomorphic impurities, 9, 39–73 Solvatomorphism, 40 Solvents ASE, 189, 191 chromotography, 160 class I-IV, 30 critical pressures, temperatures, and densities, 183 deuterated, 305–306, 309, 311, 356, 399 effects, 260 evaporation, 174 gas-phase basicities, 261 HPLC, 260, 261, 265 LC-NMR, 309 LLE, 180, 181 NMR, 305–306, 309–310 residual, 6, 11, 29 SFE, 183, 186–187 strength, 160, 243 suppression, 309, 312 TLC, 205 Solvolysis, 35 Sorbents, 167–169, 172 Soup, deﬁnition, 163 Soxhlet extraction, 193 Speciﬁcation limits, routine monitoring, 93–94 Speciﬁcation tests, 91 Spectra, database, 354 Spectral features, assignment, 59–62 Spectral simulations empirical, 332 NMR, 331–334 software, 389 theoretical, 332–334 Spectroscopy multiple wavelength, 55 useful methods, 16

see also speciﬁc methods Spiking experiments, 218–219, 382 Spin-1/2 nuclei, 315 Sponsors, role, 36 Stability characteristics, 91 intrinsic, 103–104, 112, 113 of sample, 159, 305 samples in KPSS, 155–156 studies, 13, 33, 103, 250, 388–390, 399 Stability-speciﬁc methodology, deﬁnition, 163 Stabilizers, 84–85 Standard Operating Procedure (SOP), 125–127, 208 Standards, matching, 365–366 Starting material, as source of impurities, 91 Stationary-phase column chromatography, 233–237 separation capability, 242 TLC, 204 Stearic acid, 77 Stereoisomers, 10, 147–150, 331 see also chirality Sterility standards, 83 Stokes and anti-Stokes lines, 53 Stoke’s Law, 194 Stopped-ﬂow chromatography, 311–312, 356 Storage bulk material, 40 conditions, 96, 250, 342 excipients, 84 samples, 155–156 as source of impurities, 13–14 Stress testing common problems, 109–112 deﬁnition, 102–103, 106 drug product, 113–115 studies, 104, 374 Structural analogs, 286 Structural elucidation COSY, 325 EI, 354 MS, 252, 254, 255 NMR, 318, 325–330, 367, 375–377

413

INDEX

Styrene, 82 Styrene-divinyl benzene based sorbents, 168 Submicroprobe (‘SMIDGE’ probe), 313 Succinate salt, 397 Succinic acid adduct, 381–382, 384–385 Sulfamethoxazole, 52, 56 Sulfathiazole, 56 Sulfonic acid, 262 Sulfoxide, differentiation from N-oxide, 368–371 Supercritical ﬂuid chromatography (SFC), 17–18, 20, 133 with DAD, 351 Supercritical ﬂuid extraction (SFE), 181–189 Supercritical ﬂuids, properties, 182–184 Suppliers public standards, 94 of solid-phase extraction equipment and supplies, 167 Surface-phase extractor techniques, 174 Surface-induced dissociation (SID), 277 Synthesis case study, 390–392, 393–395 cost-effective, 253 key lessons, 398–399 small-scale, 366–367

Tablets, extraction of polar substances, 189 Talc, 81 Tandem methods gas chromatography (GC), 345 mass spectrometry (MS), 274, 277, 283, 284, 288 Target analyte minimum mass levels, 350 in SPE, 172 Targeted methods, 93–94 Tartrate adduct, 382–384 Technique-oriented approach, 104–105 Temperature effects, in ASE, 191

Test methods, consolidating, 33 Tetramethylsilane (TMS), internal reference standard, 306 Theophylline, 49, 57, 77 Thermal methods of analysis, 44–49 Thermogravimetric analysis (TGA), reference standards, 131 Thermogravimetry (TG), 45–47 Thermolytic degradation, 107 Thermospray ionization (TSP), 251, 255 Thin-layer chromatography (TLC) artifacts, 215, 216, 222–223 blotter paper, 214–215 correlation with HPLC, 225–227 detection methods, 204, 205, 208, 242 history and development, 204 vs HPLC, 205–206 in isolation and characterization of impurities, 19–20, 220–228, 367 LOD/LOQ/low level linearity, 209–210, 218–219 method development and validation, 207–220 mobile-phase, 204 normal-phase, 204, 388–389 one- and two-dimensional, 206 open system, 205 orthogonal methods, 206, 207, 208 peak capacity, 343 pharmaceutical applications, 206–207 principles, 204 purity test, 31 recovery plate, 219–220 reference standards, 133–134 ruggedness/robustness, 216 sample preparation, 205 saturated/unsaturated chamber, 213–214

as separation method, 18 solvent choice, 205 speciﬁcity, 208–209 speciﬁed impurities, 221 spiking experiment, 218–219 stability considerations, 211, 212, 219 stationary-phase, 204 throughput, 205 validation summary, 216, 217, 221 Thin-layer chromatography (TLC) plates post development drying time, 211–212 pre-washing, 212–213 spraying/dipping, 215 ‘Three ion rule’, 350–351, 355 Threshold values degradation products, 35 for identiﬁcation, 20–30, 90 organic impurities, 90 for reporting, 90 for safety qualiﬁcation, 90 Time-of-ﬂight mass spectrometry (TOFMS), 130 see also mass spectrometry Time-of-ﬂight (TOF) analyzers, 270, 273–274, 276–277, 283 Time frame, short, 388–392, 399 Time to market, shortening, 356 Tissues and organs see biological samples Titration, 134 -Tocopherol, 85 Tosylate, as counter-ion, 147 Total correlation spectroscopy (TOCSY), 318–319, 324, 325, 327, 356 Total ion chromatogram (TIC), 283 Transdermal patches, 189 Transformation product, 5 Transmissible spongiform encephalopathy (TSE), 84 Trapping instruments, 274, 279, 282 Trehalose dihydrate, molecular rearrangements, 54 1,1,1-Trichloroethane, 30

414

INDEX

1,1,2-Trichloroethene, 30 Triﬂuoroacetic acid (TFA), 261, 378, 379 Trimers, distinguishing, 330–331, 336 Triple point (Tp), 182 Triple quadrupole methods, 274, 279, 280, 282, 283 Trovaﬂoxacin, chemical shift prediction, 333–334, 335 Trypsin, 269 Tubing, selection, 399

Ultracentrifugation, 194, 195 Ultraviolet (UV) spectroscopy absorbance detectors, 341 absorption spectrum, 283, 345 chromatographic detection, 205 degradation samples, 152, 153 diode array detectors (DAD), 16–17 in hyphenated techniques, 351 single wavelength detectors, 16–17, 282–283 in TLC/HPLC detection, 205 variable wavelength detector, 310 Ultraviolet-visible (UV-Vis) spectroscopy absorption detectors, 260 with diode array detection (DAD), 132 online detection, 240

in reference standard characterization, 129 United States Food and Drug Administration (FDA) guidance, 7–8, 27–28, 36 guidlelines on stereochemistry, 10 recognized standard, ‘three ion rule’, 351, 355 reference standards, 125 regulatory requirements, 7, 125, 146 United States Pharmacopeia (USP) Committee of Revision, 29 ‘General Notices’ section, 30, 31 ofﬁcial compendium, 30 regulatory requirements, 7 Standards, 124–125 terminology, 5–6, 29 United States PharmacopeiaNational Formulary (USP-NF) monographs, 3, 81, 124 revision process, 124

Validation, 35 Vanillin, 79 Variable temperature studies, 331 Variable wavelength (VW) detectors, 311 Vibrational spectroscopy, 49–57 Viruses, as impurities in excipients, 83 Visual inspection, 128–129 Vitamin B12, 13C assignments, 69 Vitamin D2 and D3, extraction by SFE, 189

Vitamins, large consumption, 3 Volatile acid salts, 399 Volatility, of mobile-phase components, 261–262

Washing, in SPE, 170, 171, 173–174 Water chemical stability issues, 11, 76 determination by NIR spectroscopy, 55 dielectric constant, 184 as impurity, 11, 76–78, 180–181, 191 pH leveling effect, 261 in preparation of dosage forms, 76 quality control, 84 WET, 309 Wet granulations, 57, 77 Wheat starch, 84 Working standard (WS), deﬁnition, 121

X-ray diffraction, 40–44, 130 X-ray powder diffraction (XRPD), 41–44, 62–63, 130

Zero pathlength difference (ZPD), 50 Zinc stearate, 82 Ziprasidone, 333–334, 335 Zirconia, 233, 234 Zone velocity centrifugation, 195