CHROMATOGRAPHY OF STEROIDS, Volume 8 (Journal of Chromatography Library)

m

Journal of Chromatography Lbrary

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Volume 8

CHROMATOGRAPHY OF STEROIDS

JOURNAL OF CHROMATOGRAPHY LIBRARY Volume 1 Chromatography of Antibiotics by G. H. Wagman and M. J. Weinstein Volume 2 Extraction Chromatography edited by T. Braun and G. Ghersini Volume 3 Liquid Column Chromatography. A Survey of Modern Techniques and Applications edited by Z . Deyl, K. Macek and J. Janak Volume 4 Detectors in Gas Chromatography by J. SevEik Volume 5 Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods by N. A. Parris Volume 6 Isotachophoresis. Theory, Instrumentation and Applications by F. M. Everaerts, J. L. Beckers and Th. P. E. M. Verheggen Volume 7 Chemical Derivatization in Liquid Chromatography by J. F. Lawrence and R. W. Frei Volume 8 Chromatography of Steroids by E. Heftmann

Journal of Chromatography Library - Volume 8

CHROMATOGRAPHY OF STEROIDS Erich Heftmann Western Regional Research Center, United States Department of Agriculture, Berkeley, Calif.

ELSEVIER SCIENTIFIC PUBLISHING COMPANY AMSTERDAM

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OXFORD - NEW YORK

1976

ELSEVIER SCIENTIFIC PUBLISHING COMPANY 335 Jan van Galenstraat P.O. Box 211, Amsterdam, The Netherlands

Distributors for the United States and Canada: ELSEVIER /NORTH-HOLLAND INC. 52, Vanderbilt Avenue New York, N.Y. 10017

L i b r a r y of Congress Cataloging in Publicalion D a t a

Heftrnann, Erich. Chromatography of s t e r o i d s .

(Journal of chromatography l i b r a r y ; V. 8) Bibliography: p. I n c l u d e s index. 1. Steroids--Analysis. 2. Chromatographic a n a l y s i s . I. T i t l e . 11. S e r i e s . ~ ~ 4 2 6 . ~ 4 547’ -731 76-24897

ISBN: 0-444-41441-x Copyright 0 1976 by Elsevier Scientific Publishing Company, Amsterdam All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the publisher, Elsevier Scientific Publishing Company, Jan van Galenstraat 335, Amsterdam

Printed in The Netherlands

TO BRIGITTE

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Contents Preface

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IX

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X

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1

Commercial Products 1. Introduction

2. Liquid column chromatography . . . . . . . . . . . . . . . . . . 2.1. Sorbents 2.2. Instrumentation . . . . . . . . . . 2.3. High-pressure liquid column chromatography .

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3 3 8 10

3 . Paper and thin-layer chromatography . . 3.1. Paper chromatography . . . . . 3.2. Thin-layer chromatography . . . . 3.3. Sorbents . . . . . . . . . 3.4. Layers . . . . . . . . . . 3.5. Development . . . . . . . . 3.6. Detection . . . . . . . . . 3.7. Quantitative and radiochemical methods 3.8. Steroid derivatives . . . . . . .

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13 13 14 14 16 17 18 24 25

4 . Gas chromatography . . . . . . . . . . . 4.1. Introduction . . . . . . . . . . . . 4.2. Steroid derivatives . . . . . . . . . . . 4.3. Packed columns . . . . . . . . . . . 4.4. Coated capillaries . . . . . . . . . . . 4.5. Instrumentation . . . . . . . . . . . 4.6. Gas chromatography-mass spectrometry combination 4.7. Quantitative and radiochemical methods . . . .

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29 29 29 33 37 41 41 43

5 . Relationsbetweenstructureandchromatographicmobility . . . . . 5.1. RM values in liquid chromatography 5.2. RM values in gas chromatography . . . . . . 5.3. Group retention factors . . . . . . . . . 5.4. Steroid numbers . . . . . . . . . . . . 5.5. Other indices . . . . . . . . . . . . .

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45 45 46 47 48 53

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55 55 57 61

6 . Sterols . . . . . . . . . 6.1. Liquid column chromatography . 6.2. Thin-layer chromatography . . 6.3. Gas chromatography . . . .

7. Bile acids and alcohols . . . . 7.1. Liquid column chromatography 7.2. Thin-layer chromatography . 7.3. Gas chromatography . . .

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VII

71 71 71 74

VIII

CONTENTS

8. Estrogens . . . . . . . . 8.1. Liquid column chromatograpRy 8.2. Thin-layer chromatography . . 8.3. Gas chromatography . . . .

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9 . Androstane derivatives . . . . 9.1. Liquid column chromatography 9.2. Thin-layer chromatography . . 9.3. Gas chromatography . . . .

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10. Pregnane derivatives . . . . 10.1. Thin-layer chromatography 10.2. Gas chromatography . .

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79 79 81 83

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87 87 87 91

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11. Corticosteroids . . . . . . . 11.1. Liquid column chromatography 11.2. Thin-layer chromatography . 11.3. Gas chromatography . . .

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12, Miscellaneous steroid hormones . . . . . . . . 12.1. Introduction . . . . . . . . . . . . . 12.2. Hormones in urine . . . . . . . . . . . 12.3. Hormones in other biological specimens . . . . 12.4. Hormones in pharmaceuticals . . . . . . . 13. VitaminsD

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93 93 96 99 99 101 103 107 107 107 109 109

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113

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115

14. Molting hormones

15. Steroid sapogenins and alkaloids

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117

16. Cardenolides and bufadienolides

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121

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125

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127

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193

List of Abbreviations References Subject index

Chromatography has become one of the most important methods in steroid research. Yet, since the appearance of Neher's booksteroid Chromatography in 1964 [769], no comprehensive review of this subject has been published. Having accumulated over 2000 reprints on the chromatography of steroids since 1964, I can well understand the reluctance of my colleagues to write such a review, but I do feel a need to organize this information for my own benefit as well as theirs. In citing the literature, I obviously had to be selective in order to keep this monograph within a reasonable size. Even after eliminating all the material presented by Neher, I found myself with more information than I could possibly use. I could have solved my problem by referring the reader to the numerous review articles, chapters, and books on individual aspects of steroid chromatography. However, I feel that the reader is entitled to enough detail so that he can at least decide which articles to look up and, preferably, so that he can repeat experiments without referring to the original papers. 1 therefore decided to omit that part of the literature which I found less original and more difficult to obtain. I am fully aware of the risk of incurring the wrath of some colleagues who may feel slighted by such omissions, but I hope that more unbiased readers will appreciate my effort at simplifying their literature search. The literature citations have been handled by the methods currently used by Chemical Abstracts and other publications of the American Chemical Society. For the more common steroids, I have used the trivial names. The systematic names (IUPAC-IUB 1967 revised tentative rules) [SO71 are shown in the subject index. It is assumed that the readers are familiar with the general terminology, theory, and techniques of chromatography. These aspects are only covered as they relate to steroids. Readers requiring further information on chromatography are referred to my book Chromatography [422]. Some background material on steroids and a relatively recent guide to the steroid literature will be found in my book Steroid Biochemistry [420]. Extensive bibliographies on chromatography are being published regularly in the Journal of Chromatography and other analytical journals, biennially in Analytical Chemistry, in several Elsevier books [233,234,681,682], as well as by various manufacturers of chromatographic equipment and supplies. Many other aspects of steroid analysis are covered in my recent bookModern Methods of Steroid Analysis [421]. Most of this book was written while I was at the Federal Institute for Lipid Research of the German Federal Republic in Munster under the terms of a U S . Senior Scientist Award by the Humboldt Foundation. I am deeply grateful to the Director of the H.P. Kaufmann Institute, Professor H.K. Mangold, and its staff as well as to the staff of the Humboldt Foundation for their most generous support and cordial welcome. ERICH HEFTMANN

Minster, October 1975

IX

Commercial Products

x

Trade designation

Chemical nature

Source

Adsorbosil-CABN Alpha-8-Metricel Amberlite IRC-50 Amberlite XAD-2 Amberlyst XN-1006 Amberlyst A-6 AN-600 Anakrom ABS ANH BDSA BSA BSTFA Celite Cellex E Centri-Chrom Chromosorb CMDMCS CMDMTMDS

AgN03 on SiOz membrane filter polyacrylic acid macroporous styrene-divinylbenzene resin anion-exchange resin anion-exchange resin 50% cyanoethyl methyl silicone silanized diatomaceous earth cyanoethyl silicone bis (dime thylsilyl) acetamide N, 0-bis (trimethylsilyl) ace tamide N, 0-bis (t rimethylsilyl) trifluoroacetamide diatomaceous earth ECTEOLA-cellulose centrifugally accelerated LC system styrene-divinylbenzene copolymer chloromethyldimethylchlorosilane 1,3-bis(chloromethyldimethyl)-1,1,3,3-tetramethyldisilazane cyanoethyl methyl silicone porous silica permanently bonded octadecylsilane Carbowax 2OM-terephthalic acid silicone sym-dichlorotetrafluoroacetone diethylene glycol succinate

Applied Science Laboratories, Stage College, Pa., U.S.A. Gelman Instruments, Ann Arbor, Mich., U.S.A. Rohm & Haas, Philadelphia, Pa., U.S.A. Rohm & Haas, Philadelphia, Pa., U.S.A. Rohm & Haas, Philadelphia, Pa., U.S.A. Rohm & Haas, Philadelphia, Pa., U.S.A. Analabs, North Haven, Conn., U.S.A. Analabs, North Haven, Conn., U.S.A. DuPont de Nemours, Wilmington, Del., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Alltech Associates, Arlington Heights, Ill., U.S.A. Bio-Rad, Richmond, Calif., U.S.A. Ivan Sorvall, Norwalk, Conn., U.S.A. Analabs, North Haven, Conn., U.S.A. Pierce Chemical Co., Rockford, Ill., U.S.A.

CNSi Corasil Corasil CIS CTpA DC-200 DCTFA DEGS

Pierce Chemical Co., Rockford, Ill., U.S.A. General Electric, Schenectady, N.Y., U.S.A. Waters Associates, Milford, Mass., U.S.A. Waters Associates, Milford, Mass., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Regis Chemical Co., Morton Grove, Ill., U.S.A. Applied Chemical Corp., Morristown, N.J., U.S.A. Regis Chemical Co., Morton Grove, Ill., U.S.A.

Dexsil-300 Diatoport S DMMCS Epon Resin 1001 F-50, etc. Factice 31-B Florisil Gas-Chrom Gas Quat L GE-F-50 HCP HFBA HI-EFF-8BP Hyamine hydroxide ITLC JXR KGn, Kalignost L45 LA-1 Lipidex Michrome No. 64 Micropak Si 60 MSTFA NGS NPGA o v - I , ov-101 OV-17,0V-25 OV-225

polycarboranesiloxane Analabs, North Haven, Conn., U.S.A. diatomaceous earth F & M Scientific Corp., Avondale, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. dime thylmonochlorosilane Alltech Associates, Arlington Heights, Ill., U.S.A. bisphenol-epichlorohy drin polymer see Versilube polymer from soybean oil Carter-Bell Manuf. Co., Springfield, N.J., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. magnesium silicate diatomaceous earth Applied Science Laboratories, State College, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. trioctadecylmethylammonium bromide Applied Science Laboratories, State College, Pa., U.S.A. see Versilube DuPont de Nemours, Wilmington, Del., U.S.A. ethylene-propylene copolymer PCR Inc., Gainesville, Fla., U.S.A. heptafluorobutyric anhydride Applied Science Laboratories, State College, Pa., U.S.A. cyclohexanedimethanol succinate diisobutylcresoxyethoxyethyl dimethyl J.T. Baker,Phillipsburg,N.J.,U.S.A. benzylammonium hydroxide Gelman Instruments, Ann Arbor, Mich., U.S.A. (Instant Thin-Laye r Chromatography) adsorbent-impregnated glass-fiber paper Supelco, Bellefonte, Pa., U.S.A. dime thylpolysiloxane sodium tetraphenylborate Eastman, Rochester, N.Y., U.S.A. General Electric, Schenectady, N.Y. methyl silicone Rohm & Haas, Philadelphia, Pa., U.S.A. n-dodecenal(trialkylmethy1)amine Pharmacia, Uppsala, Sweden hydroxyalkoxypropyl Sephadex E. Gurr Ltd., London SW 14, Great Britain primuline Varian Associates, Walnut Creek, Calif., U.S.A. silica gel N-methyl-N-trimethylsilyltrifluoroacetamide Supelco, Bellefonte, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. neopentyl glycol succinate Applied Science Laboratories, State College, Pa., U.S.A. neopentyl glycol adipate Applied Science Laboratories, State College, Pa., U.S.A. methyl silicones Applied Science Laboratories, State College, Pa., U.S.A. phenyl methyl silicones Applied Science Laboratories, State College, Pa., U.S.A. cyanopropyl phenyl methyl silicone Continued on p . XII

h icr

e

F

V

icr

0

E

Commercial products (continued) Trade designation

Chemical nature

Source

Permaphase ETH Permaphase ODS PhSi Plaskon CTFE-2300 PMPE Polygram Sil G Polyimide Poragel PN Porasil A PZ- 176 QF-1 Regisil SCX SE-30 SE-30 “ultraphase” SE-52 Sephadex Sephadex LH-20 SI-100 silanox 101 SILAR-5CP Silica Gel 1B-F Silica Gel F Silica Gel G Silica Gel HS SP-400 SP-525 SP-1000

ether-bonded controlled-porous surface beads octadecylsilane bonded to Zipax see XE-61 trifluoroethylene polymer polymetaphenoxylene silica gel on polyester sheets

DuPont de Nemours, Wilmington, Del., U.S.A. DuPont de Nemours, Wilmington, Del., U.S.A.

polystyrene gel porous silica polyphenyl ether sulfone fluoroalkyl polysiloxane BSTFA+TMCS (99: 1) strong cation exchanger methylpoly siloxane “improved” methyl silicone methyl phenyl silicone cross-linked dextran hydroxypropyl ether of dextran silica silica cyanoalkyl phenyl silicone flexible precoated TLC sheet silica plus fluorophor silica plus gypsum silanized silica chlorophenyl silicone aromatic hydrocarbon modified Carbowax 20M

Allied Chemical Corp., Morristown, N.J., U.S.A. Varian Associates, Wainut Creek, Calif., U.S.A. Macherey, Nagel & Co., Duren, G.F.R. Pennzoil United, Shreveport, La., U.S.A. Waters Associates, Milford, Mass., U.S.A. Waters Associates, Milford, Mass., U.S.A. Pennzoil United, Shreveport, La., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Regis Chemical Co., Morton Grove, Ill., U.S.A. DuPont de Nemours, Wilmington, Del., U.S.A. Analabs, North Haven, Conn., U.S.A. Phase Separation, Queensferry, Great Britain General Electric, Schenectady, N.Y., U.S.A. Pharmacia, Uppsala, Sweden Pharmacia, Uppsala, Sweden Merck, Darmstadt, G.F.R. Cabot, Boston, Mass., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Baker, Phillipsburg, N.J., U.S.A. Merck, Darmstadt, G.F.R. Merck, Darmstadt, G.F.R. Merck, Darmstadt, G.F.R. Supelco, Bellefonte, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A.

SP-240 1 Spherosil XOA-400 Supelcoport Sylon-CT TCTFA TMCBA TMDS TMSDEA Versilube Vydac XE-60 XE-6 1

Z Zipax Zorbax SIL

trifluoropropyl methyl silicone silica GC support silanizing solution 1,1,3-trichlorotrifluoroacetone tetramethylcyclobutanedioladipate tetramethyldisilazane trimethylsilyldiethylamine methyl chlorophenyl silicone porous silica layer on solid core cyanoethyl silicone phenyl methyl silicone ethylene glycol, succinic acid, and methyl siloxane copolymer porous-layer support porous silica microspheres

Supelco, Bellefonte, Pa., U.S.A. Pechiney, Saint Gobain, France Supelco, Bellefonte, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Allied Chemical Corp., Morristown, N.J., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. General Electric, Schenectady, N.Y., U.S.A. General Electric, Schenectady, N.Y., U.S.A. DuPont de Nemours, Wilmington, Del., U.S.A. DuPont de Nemours, Wilmington, Del., U.S.A.

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Chapter 1

Introduction The steroids constitute a very large group of natural and synthetic compounds with a broad range of biological activities. Because they are of considerable importance in medicine, there has been a great deal of interest in various aspects of steroid chemistry, including their analysis. In biological extracts, steroids usually occur in low concentration and invariably in association with numerous structurally related compounds. The latter may be present in considerably higher concentrations, but may not have comparable biological activities. Thus, it is desirable that analytical methods for steroids be both specific and sensitive [ 1 121. As in the analysis of other groups of products, there is a strong trend toward instrumentation and automation in steroid analysis. Because most physical methods of analysis are not sufficiently selective, the analytical samples must be purified and, preferably, fractionated. The most efficient fractionation method available to the steroid chemist is chromatography. This is one reason for the strong association between steroid chemistry and chromatography. The other reason is that scientists who develop instrumental methods of analysis, including chromatographic methods, look toward the steroid analyst as a relatively well-endowed user with many challenging problems. One of these problems is that the associated steroids in an analytical sample are often analogous or isomeric compounds with very similar physical and chemical properties. Another problem is that steroids may belong to several solubility classes, ranging from rather hydrophilic to very lipophilic compounds. Some of these compounds are highly reactive and even unstable, whereas others are extremely sluggish and may even be devoid of analytically useful functional groups. So far, no single chromatographic method has been able to overcome all of these problems, and the analyst must have the ability to select the technique appropriate to the goal [770]. Thus, in addition to the obvious constraints of his knowledge and skill, available time and facilities, the analyst is limited by the chemical nature and physical condition of the steroids in samples of biological or synthetic origin. A few generalizations about the selection of techniques are in order, although many exceptions will be found in the examples of chromatographic analyses presented in the following pages. As a rule, such methods as ion exchange and electrophoresis are only suitable for ionic or ionogenic substances and are therefore largely inapplicable to neutral steroids. Generally speaking, partition chromatography will be more successful in separating the homologs of the more hydrophilic steroids, whereas adsorption chromatography is more apt t o resolve mixtures of analogous or isomeric steroids having a more lipophilic character. For crude or bulky sam.ples, old-fashioned column chromatography is still the method of choice, although it is not as efficient with respect to resolution, labor, and time as other chromatographic techniques. Qualitative analysis is most efficiently performed by thin-layer chromatography (TLC), because a number of samples and reference compounds can be tested simultaneously. For quantitative analysis and for the best resolution, gas chromatography (GC) is preferred, but high-pressure liquid chromatography (HPLC) has several potential

1

2

INTRODUCTION

advantages over GC. It provides larger capacity and greater choice of parameters and it usually requires no derivatization, while offering advantages of speed, convenience, and sensitivity that rival GC.

Chapter 2

Liquid column chromatography 2.1. SORBENTS

As the specific examples of liquid column chromatography (LC) in this volume show, silica is by far the most useful sorbent for steroids. Some of the important work on the relation between the structure of silica gel and the chromatographic behavior of steroids goes back to 1961, but it must be mentioned here because it was not covered in Neher’s book. Klein [566] has systematically studied the effect of surface area, pore volume, and average pore diameter of different types of silica on the resolution of sterol acetates by LC. Only when the pore diameter is large relative to the size of the sterol molecule can the molecule be attracted by a flat surface. Thus, 24-dehydrosterols, which have a smaller cross-section than sterols with a nuclear double bond, are more strongly adsorbed than the latter on silica gels with small pore diameters. Activation of silica gel by heat increases the range of surface energies and results in broadening or trailing of chromatographic zones [ 5671. Deactivation, which is best accomplished by exposure to an atmosphere of controlled humidity, will improve the performance of a silica gel only if it is adjusted to the surface strilcture of that particular silica. The water content of silica depends on the water content of the solvent [69]. By gradually increasing it, one can change the chromatographic system continuously from an adsorption to a partition system. It is even possible to achieve some sort of gradient elution effect by using a wet nonpolar solvent as eluent for a dry silica column. An example of the use of silica columns is the preliminary isolation of steroids from a crude lipid extract [429]. Up to 1 g of mixture, dissolved in pentane-diethyl ether (4: 1) can be fractionated on a column, 1 cm in internal diameter (I.D.), which has been packed with 7 g silicic acid (activity grade IIB), slurried in pentane-diethyl ether. Neutral lipids, sterols, and steryl esters are eluted by 150 ml pentane-diethyl ether (4: l), other steroids by 150 ml acetone-chloroform (2: l), and more polar lipids by 150 ml methanol. Another example is the use of Silica Gel G with a water content of 10%in a 5-cm X 1.8-cm column [210]. About 10-1 5 mg of lipid mixture can be fractionated on such a column by elution with a series of solvents. Thus, 60 ml petroleum ether (b.r. 30-754 elutes the hydrocarbons, 50 m l 6 % diethyl ether in petroleum ether the cholesteryl esters, and 160 ml of 10%ethyl acetate in petroleum ether the triglycerides, followed, in the last 60 ml, by cholesterol. The activity and thus the chromatographic properties of alumina can be reversibly modified in situ by passing organic solvents with different water content through a column of alumina [283]. Useful separations can be achieved on alumina thus deactivated. Alumina, although quite selective, is now discredited by the various alterations the more active grades have been reported to produce, and it is rarely used nowadays. However, an interesting method of labeling steroids by alumina chromatography should be mentioned [569]. By taking advantage of the enolization of ketosteroids on basic

3

TABLE 2.1

P

ELUTION VOLUMES OF STEROIDS ON DIFFERENT GELS IN DIFFERENT SOLVENTS EXPRESSED AS PER CENT OF TOTAL BED VOLUME 12781 Compound

LH-20

G-25-36

Dichloromethane

Isopropanol

Propanol

Methanol

45.5 45.0 61.5 59.0 73.5 44.0 151.5 42.0 43.5 48.5 48.0 51.0 50.0 71.0 239.5 -

69.0 72.5 69.0 74.5 70.5 77.5 72.5 90.0 77.5 68.0 70.5 74.5 88.0 78.0 84.5 81.5 115.5 113.5

70.0 73.5 70.0 75.0 72.0 72.0 85.0 75.0 69.0 71.5 71.0 86.5 78.5 83.0 81.5 114.0 109.5

76.0 76.0 -

Dichloromethane

Propanol

G-15-38

G-2540

Methanol

Dichloromethane

Dichloromethane

98.0 91.0 93.0 84.5 94.0 82.5 100.5 102.5

47.0 45.0 54.0 59.0 44.0 74.5 45 .O 43.0 5 2.0 54.5 52.5 63.5 138.0 -

57.0 55 .O 64.0

~

5aCholestane 5aCholestan-3-0ne 5a€holestan-3p-o1 5pCholestan-3-one 5p€holestan-3p-ol 5 @€holestane-3,12-dione 5pCholestane-3a, 12adiol 5p€holestane-3,7,12-trione Sp€holestane-3a,7a, 12a-trio1 30-Methoxyd-cholestene 3p-AcetoxyJ-cholestene 5a-Pregnane Progesterone 5a-Androstane 5a-Androstan-17-0ne 5a-Androstan-l7p-01 Estrone Estradiol

-

73.5 73.5 76.0 76.0 77.5 83.5 83.5 99.0 98.0

54.5 53.0

67.0

-

64.5

-

80.0 52.0 132.5 53.0

-

59.5 56.5 62.5 59.5 77.0 202.5

-

75.5 79.0 71.0 -

74.0 82.0 72.5 100.5 74.0 75.5 82.5 92.5 83.5 90.5 80.5 133.0 113.0

-

93.0 -

100.5 95.0 92.0 91.0

55.0 -

68.5 53.0 87.0 55 .O 54.0 62.5 57.0 -

-

-

SORBENTS

5

alumina, HTO on the column can be made to exchange with H in such ketosteroids as 5a-cholest-7-en-3-one,and sizeable quantities of tritiated ketosteroids of high purity (5 -10 mCi/mmole) can be prepared by sirnple chromatographic development. This procedure can also be utilized for analytical purposes [568]. A mixture of ketosteroids passing through a column of tritiated alumina becomes self-labeled, and the individual compounds are readily detected and measured in submicrogram quantities by monitoring the column effluent. Ion-exchange resins are useful sorbents for partition chromatography of nonionic compounds. Seki and Matsumoto [954] have pioneered in the application of partially esterified cation exchangers to steroids (cf. p.99). Columns of neutral resins, such as polystyrene with 2% divinylbenzene cross-linkages, can be developed with benzene to fractionate mixtures of lipids, including sterols and steryl esters [ 10561. Nystrom and Sjijvall [800] began to use lipophilic dextran preparations as supports for the stationary phase in reversed-phase partition chromatography of lipids in 1964. Methylated Sephadex G-25 was mixed with lipid solvents in which the sorbent does not float, e.g., chloroform-methanol (1 : l ) , and the slurry was poured into a chromatographic tube. The same solvent mixture eluted cholesterol, some of its esters, and various bile acids from this column, in generally decreasing order of polarity, but other solvent mixtures and other lipids gave other elution orders [801,802]. The effects noted were clearly not due to reversed-phase partition alone, but gel permeation, ordinary partition, and perhaps also adsorption may have contributed to them, depending on the degree of cross-linking and methylation of the dextran preparation and the chloroform/methanol ratio of the eluent. The theoretical basis of steroid chromatography on lipophilic Sephadex gels was studied in more detail by Eneroth and Nystrom [278]. Table 2.1 shows the per cent total bed volume (PTV), i.e. the milliliters of each solvent which would have been required to elute the compounds shown from a Sephadex column, if its total volume had been 100 ml. Sephadex LH-20 is a cross-linked dextran with hydroxypropyl ether groups, and Sephadex G-I5 and G-25 are dextran gels which exclude polysaccharides above a molecular weight of 1500 and 5000, respectively. The second number in the Sephadex G series denotes the per cent methoxyl content of the dextran. Generally, the PTV value of steroids without hydroxyl groups increases with the polarity of the solvent and with the methoxyl content of the sorbent, whereas the reverse is true of steroids carrying hydroxyl groups. The elution order with nonpolar solvents, such as dichloromethane, follows the general order of increasing polarity of the samples, whereas polar solvents usually elute steroids in decreasing order of polarity. For any given sample and solvent combination, the more porous gel gives the higher PTV value. Thus, combinations of a relatively nonpolar gel with a relatively polar solvent exhibit reversed-phase partition behavior, whereas ordinary partition chromatography may be at work in combinations of relatively polar gels with relatively nonpolar solvents. Ellingboe et al. [268] have prepared a number of long-chain alkyl ethers of Sephadex and tested them as column packing materials for partition and reversed-phase partition chromatography of sterols, bile acids, and steroid hormones. The reversed-phase systems, such as Sephadex G-25 with a hydroxyalkyl (CIs-Cls) group content of 71% by weight, eluted with methanol-heptane (19: l), separated C2,, CZs, and C29 sterols; Sephadex with

6

LIQUID COLUMN CHROMATOGRAPHY

TABLE 2.2 PER CENT TOTAL BED VOLUME OF STEROIDS ON A HYDROXYCYCLOHEXYL SEPHADEX COLUMN ELUTED WITH BENZENE m i Steroid

PTV**

OH* ~

5aCholestan-3-one 4Cholesten-3-one SCholesten-3-one 5aCholestan-3p-01 (cholestanol) SaCholestan-3a-01 (epicholestanol) 5pCholestan-30-01 (epicoprostanol) 5pCholestan-3p-01 (coprostanol) 5Cholesten-3p-ol (cholesterol) SCholesten-3a-01 (epicholesterol) 3p-Hydroxydaandrostan-17-0ne (epiandrosterone) 3a-Hydroxy-5a-androstan-1"-one (androsterone) 3a-HydroxyJp-androstan-17-one (etiocholanolone) 3p-Hydroxy-5p-androstan-1 "-one 3a-Hydroxy-5p-pregnan-20-one 3p-Hydroxy-5p-pregnan-2O-one 5Cholestene-3p,7pdiol S€holestene-3p, 7adiol 5pCholan-7p-01 Sp€holan-7c~-ol 11a-Hydroxypregn-4ene-3,2Odione 1lp-Hydroxypregn-4ene-3,20-dione 5a-Androstan-l7p-01 Sa-Androstan-l7a-01 5-Pregnene-3p,2Opdiol 5-Pregnene-3@,20a-diol 4-Pregnene-3,20-dione (progesterone) 3p-Hydroxy-5-androsten-17-0ne(dehydroepiandrosterone) 3p-Hydroxy-5p-pregnan-20-one 3p-Hydroxy-5-pregnen-20-one(pregnenolone) 5a-Pregnane-3p, 20pdiol 5-Pregnene-3p,2Opdiol -

e a

e a

e a

e a

e a

e a

e a

e a

e a rL-e

ILa more hindered less hindered

55.8 56.6 55.3 88.4 82.9 87.0 87.0 94.4 70.1 114 104 107 104 99.5 99.5 170 162 67.0 67.0 115 108 103 95.2 170 183 62.9 . 108 97.2 105 193 21 1

*Conformation: a = axial; e = equatorial. **Per ceilt total bed volume,

55% hydroxyalkyl (C1l-Cl4) group content, eluted with methanol-water-l,2-dichloroethane (7:3: l), separated bile acids; and a partition column of Sephadex with 58% hydroxyalkyl (CI5-Cl8) group content, eluted with heptane-chloroform (4:l), separated various pregnane derivatives. Using the lipophilic dextran with 50% hydroxyalkyl groups in columns eluted with benzene or benzene-isopropanol(3: l), Brooks and Keates [ 1181 further investigated the chromatographic behavior of many steroids. After experimenting with various other lipophilic dextran gels [20], Anderson et al. [23] observed a considerable enhancement in selectivity when dextran gels were substituted with hydroxycyclohexyl residues. Elution with benzene gave the PTV values shown in Table 2.2. Pairs of steroids with epimeric hydroxyl groups were generally

I

SORBENTS

resolved, unless they had Rings A and B cis-fused (50-steroids). Moreover, this chromatographic system separated 5-unsaturated steroids from their saturated Sar-analogs. In both 5a- and As-steroids, the equatorial alcohols were more retarded than their axial epimers. This is illustrated in Fig. 2.1. The mechanism underlying this separation appears to be adsorption of the steroids t o the gel, more specifically, hydrogen bonding to its ether linkages.

I

A

hours

8 0

60

80

I

lw

1

SEV

Fig. 2.1. Separation of epimeric 3hydroxysteroids on a hydroxycyclohexyl Sephadex column (1 m X 3 mm I.D.), eluted with benzene. A = epicholesterol; B = epicholestanol; C = cholestanol; D = cholesterol; E = androsterone; F = epiandrosterone. SEV = standard elution volume. (Reproduced from J. Chrornarogr., 82 (1973) 340, with permission; [23].)

The use of LH-20 columns is recommended for clinical analyses, where groups of steroids must be isolated from blood or urine samples (cf. Chapter 12, Sections 2 and 3), particularly for purposes of radioimmunoassay or competitive protein binding assay [ 100, 142,754,821,9551. They have also been found useful in biosynthetic studies of plants for the convenient separation of carotenoids from sterols [ 10291. Various other aspects of chromatography on lipophilic Sephadex have been reviewed [978]. For instance, it is used in recycling and capillary column chromatography with automatic detection systems [804]. Marker dyes facilitate the location of steroid fractions in routine applications [248]. Lipophilic Sephadex also exhibits cation-exchange properties when electrolytes are present in the solvents or samples [981]. This effect can be exploited for the isolation of conjugated steroids from biological sources. For instance, from a column of methylated Sephadex, eluted with a 1:1 mixture of chloroform and a 0.02 M methanolic solution of some salt, free steroids emerge before steroid monosulfates, which are followed by disulfates. The mixed steroid sulfates in cu. 5 ml plasma can be separated on a 4-g column of Sephadex LH-20 by elution of the monosulfates with 30-60 ml chloroform-0.01 M NaCl in methanol (1 : 1) and then of the disulfates with 65- 115 ml methanol [ 5 141. The separation of individual steroid sulfates can be accomplished by liquid-liquid partition chromatography on Celite columns [138]. The use of polyamide columns for the isolation of steroid conjugates has also been reported [807].

8

LIQUID COLUMN CHROMATOGRAPHY

2.2. INSTRUMENTATION As in other fields of application, there has been a continuous trend toward automation in steroid analysis by liquid column chromatography. This section deals with instrumentation applied to gravity flow; high-pressure chromatography is treated in Section 2.3. One of the earliest solvent pumping systems for gradient elution in the Steroid Analyzer [19] consisted of a metal cam, shaped like a plot of solvent ratio us. time. The position of the arm of a linear potentiometer, following this cam, then regulated the rate of two reciprocating piston pumps, which delivered two solvents into a mixing chamber. This allowed elution programs to be designed for any particular fractionation [3 181. Fig. 2.2 illustrates this for the programmed gradient elution of corticosteroids. The solvent gradient system was subsequently modernized by substituting the more flexible punched tape for the metal cam to control the pumps [521] and an improved mixing chamber with minimum hold-up [522]. The Steroid Analyzer is suitable not only for multiple quantitative assays on collected fractions, but also for the isolation of minor components from large amounts of contaminants [520]. Another gradient system designed for LC of lipids produces concave gradients by the controlled addition of one solvent to the other in a mixing chamber with a cam-operated positive-displacement pump [772]. Equations permitting the calculation of any elution gradient produced in this manner have been derived by Cavina et al. [ 1581. Castellana and Kelly [ 1461 have calculated the shape of elution gradients that can be generated without the use of pumps by connecting two vessels of a certain shape, containing solvents of different densities. The application of such gradients to steroid chromatography was demonstrated.

Fig. 2.2. Programmed gradient elution of adrenocortical hormones. The tube number is plotted against elution composition, in % dichloromethane (DCM) in light petroleum ether, in the top portion and against UV absorbance of the fractions in the bottom portion. Q = Deoxycorticosterone; A = dehydrocorticosterone; S = deoxycortisol; B = corticosterone; E = cortisone; ALDO = aldosterone; F = cortisol. (Reproduced from Anal. Chem., 35 (1963) 2020, with permission; [318].)

INSTRUMENTATION

9

Vestergaard [ 11 16-1 1181 has made many contributions to the automation of steroid analysis by liquid column chromatography. He can now elute 25 columns simultaneously [ 1 1211 by pumping a solvent mixture with nitrogen at low pressure from a mixing bottle. The polarity of the eluent is gradually increased by metered addition of a more polar solvent to the mixing bottle. The chromatographic tubes are 6-ft.-long PTFE capillaries [ 1 1231. They are packed by either pumping a slurry from a wider tube into them or, preferably, by allowing the dry sorbent to enter the capillaries from funnels which are attached to a shaking machine [927]. Eluate fractions are analyzed with the aid of a computerized multichannel high-capacity read-out system [ 11 19,11201 . It has a capacity of 100 chromatograms per day when used as a three-channel instrument, but it can be expanded to 400 chromatograms per day by the use of twelve channels. In isocratic elution, a recording refractometer can be used for monitoring the column effluent. For RI determinations with changing eluent composition, the solvents must be evaporated and the residues must be redissolved in a single solvent. This is accomplished automatically in a device which collects the effluent on a moving Mylar or PTFE tape [ 11541. The tape passes from the column outlet first through an evaporating chamber and then past a solvent outlet, where the residue is redissolved, to an aspirator, which draws the solution into a recording refractometer. A universal detector for LC with great potential is the flame-ionization detector (FID). It requires a device for transferring an aliquot of the column effluent to the detector after the removal of the eluent. In the design of Haahti and Nikkari [382] the transfer is effected by an endless gold chain, which passes through a stream of hot air, where the solvent is evaporated, and then through the flame of a hydrogen FID. The combustion of the residue on the chain gives rise to an ionization current, which is amplified to give a detector response. Karmen’s [533] design differs from the one just described in the construction of the FID. Here, the chain passes through a T-tube, in which the volatile components of the residue are evaporated in a stream of hot nitrogen, and the nonvolatile components are then pyrolyzed. The resulting vapors are aspirated into the hydrogen-nitrogen line of the FID to ensure a quantitative transfer. The Barber-Colman Model 5400 Liquid Chromatography Detector, based on the design of Haahti and Nikkari [382], has been used in forerunners of HPLC, such as the microcolumn system of Stouffer and Oakes [ 10141. That system includes a miniature gradient mixer and permits rapid analysis of lipid mixtures in submicrogram quantities. Cavina et al. [ 1581 have made extensive use of the Barber-Colman FID. Combining elution with a concave gradient (see p. 8) of diethyl ether in petroleum ether (b.r. 65-75?, conventional silicic acid column chromatography, stream splitting, continuous detection, and gravimetric analysis of the eluate, they were able to fractionate milligram amounts of neutral lipids into classes, including sterols and steryl esters. In the Pye System 2 Liquid Chromatograph for HPLC, the effluent coats a steel wire, which passes through an evaporator oven and then through a pyrolyzer oven [ 1 1681. A stream of inert gas transports the vapors from the pyrolyzer into a hydrogen FID. The liquid spectroradiochromatograph is an apparatus for the automatic determination and radioassay of steroids and other nonsaponifiable lipids in the effluent from a chromatographic column [221]. In addition to an automatic gradient elution device, the machine features a special sample injector and three monitors: a recording spectrophoto-

10

LIQUID COLUMN CHROMATOGRAPHY

meter, a scintillation counter (both equipped with flow cells), and an argon ionization detector. The response of all three monitors is recorded, and the effluent is collected in a fraction collector. A device for forcing eluents through very fine silica gel consists of a centrifuge head with assemblies of short chromatographic columns and loading funnels in radial array [889, 8901. After these assemblies have been centrifuged, the adsorbent may be extruded and stained. Steroid mixtures applied to columns of microparticulate silica, ranging in particle size from 0.02-0.7 pm [ 11831, were sharply separated. The commercial instrument (Centri-Chrom) also permits the collection of eluents in attached cups, but steroids may also be eluted from sections of the extruded column.

2.3. HIGH-PRESSURE LIQUID COLUMN CHROMATOGRAPHY Siggia and Dishman [971] were among the first to apply HPLC to steroid analysis. Best results were obtained by dry-packing of glass tubes, 50 cm X 2 mm I.D., with a trifluoroethylene polymer (Plaskon CTFE-2300, <325 mesh) that had been coated with 18-28% (w/w) n-dodecenal(trialkylmethy1)amine (LA-1). Eluents forced through this column at room temperature under less than 600 p.s.i. pressure separated a variety of 4-en-3-ones or aromatic steroids that could be detected by their UV absorption. The elution order indicated that the separation was based on reversed-phase partition and that adsorption on the support occurred when the amount of stationary phase applied was too low. Corticosteroids and androgens were well separated by water, but progesterone and other less polar steroids required up to 33% methanol as eluents, and estrogens were best eluted with water or aqueous methanol containing NaOH (pH 1 1S). Karger and Berry [532] recommended loading the support heavily with a stationary phase in order to avoid adsorption effects. In this case, the support, a porous silica (Porasil C, 10-25 pm), was coated with 35% by weight of formamide and packed into a column, 50 cm X 2 mm, which was maintained at 2OoC. Under increasing pressure, a series of steroids was eluted in decreasing order of polarity with hexane-dioxane mixtures (e.g., 19: l), saturated with formamide. Both UV and RI detectors were used. Heavily loaded columns have other virtues, such as high load capacity, good reproducibility, and great versatility. Relatively inexpensive packing materials may be coated with a variety of stationary liquid phases by a very simple in situ method [282]. A column, 30 cm X 4 mm I.D., is packed with a nonsettling suspension of silica (SI-100, < 10 pm) in a tetrabromoethane-methanol mixture. The silica is coated by pumping the eluent, partially or totally saturated with the stationary liquid, through the column under pressure. A convenient method for saturating the eluent is to pass it through a forecolumn, packed with Chromosorb W, which has been coated with about 30% by weight of stationary liquid. In that way, silica gel will take up 20-80% of its weight in stationary liquid. For steroids, a coating of 50% (w/w) formamide and elution with dichloromethane, saturated with formamide, give satisfactory results. When the thiee-component two-phase solvent system water-ethanol-dichloromethane is used for silica gel chromatography, the polarity of the system may be adjusted in accordance with the polarity of the steroid class by varying the proportion of ethanol [432].

HIGH-PRESSURE LIQUID COLUMN CHROMATOGRAPHY

11

After phase equilibration, the less polar phase is pumped through the column, which has been packed with silica by the slurry method. For corticosteroids and estrogens, the solvent system is prepared by making a mixture of 94 ml ethanol and 34 ml water up to 2 1 with dichloromethane at 25°C. After phase equilibration, the less polar phase is pumped through a column, 300 mm X 3 mm, which has been packed with silica (Spherosil XOA-400, 4-8 pm), at a rate of 1.14 ml/min. The steroids can be determined in nanogram quantities by UV absorption. In order to increase the sensitivity of detection for ketosteroids without UV-absorbing chromophores (which is in the microgram range for UV and RI detectors) Henry et al. [427] converted them to the 2,4-dinitrophenylhydrazones.The strong W absorption of these derivatives enabled them t o determine ketosteroids at the nanogram level with the W detector. This in turn allowed them to use a highly efficient but low-capacity porous-layer support, Zipax, which was coated with various stationary phases before dry packing. The stainless-steel column, 1 m X 2.1 mm I.D., was maintained at room temperature, at 40°C or at 50°C and operated at 600, 1200, or 1500 p.s.i. For liquid-liquid partition chromatography of less polar steroids, such as progesterone, the stationary/mobile phase system 1% P,$-oxydipropionitrile/heptanewas used, but the more polar corticosteroids required either more polar mobile phases, such as heptane-tetrahydrofuran (4: l), or less polar phases. For reversed-phase partition chromatography, such systems as 1% ethylenepropylene copolymer (HCP)/I 5% aq. methanol or 1% cyanoethyl silicone polymer (ANH)/2.5%aq. methanol were used. Permaphase ODs, a commercially available packing consisting of octadecylsilane bonded to Zipax, produced a reversed-phase partition chromatogram when eluted with a linear gradient of water toward 50% aq. methanol at a rate of 5% per min and a flow-rate of 1 ml/min. The authors also demonstrated that pellicular ion-exchange packings are applicable to HPLC of steroids. A useful derivative for steroids that do not absorb UV but contain a hydroxyl group is the benzoate orp-nitrobenzoate [314]. As little as 10 ng of a benzoate or 1 ng of a p nitrobenzoate can be detected by UV absorption. These derivatives are easily prepared and are well separated on Corasil CI8 or Permaphase ODS columns by reversed-phase partition chromatography with aqueous methanol. If HPLC is to serve for preparative purposes, relatively wide columns may be used [47]. For example, the isolation of 1 g of cholesteryl phenylacetate was accomplished on a Spherosil XOA-400 column, 1 m X 23 mm I.D., which was eluted with 0.1% methanol in dichloromethane. It is estimated that about I g of porous adsorbent or 10 g of pellicular adsorbent is required per mg of any component to be isolated. Direct identification of steroids in mixtures by coupling of HPLC and mass spectrometry (MS) has now become possible. Field desorption MS, which permits the molecular weight of nanogram quantities of polar compounds t o be determined, has so far not been used “on line” [945]. However, the effluent from a liquid chromatograph has been vaporized through an atmospheric pressure ionization mass spectrometer, and the output of the quadrupole mass analyzer was fed into a computer [461].

This Page Intentionally Left Blank

Chapter 3

Paper and thin-layer chromatography 3.1. PAPER CHROMATOGRAPHY Except for some isolated cases of rather polar compounds, e.g. steroid conjugates [387], paper chromatography (PC) has now been almost completely replaced by TLC. However, a few publications on general techniques of PC of steroids that have not been dealt with in Neher's book [769] should be mentioned here. Bush, who has done outstanding work in this field [ 1291, has developed a Chromatogram Automatic Soaking, Scanning, And Digital Recording Apparatus (CASSANDRA), suitable for processing over 500 paper chromatograms, 5 cm X 20 cm, per day [ 130,13 I]. Passing through this machine, the paper strips are treated with a reagent, then heated and scanned, the data being processed by a digital computer [ 1321. Schneider [934] has compared TLC with PC in the separation of steroidal glucosides and glucosiduronic acids, and preferred the latter. Results of that work are summarized in Table 3.1. Evidently, separation was effected not only of glucosides from glucosiduronic acids, but also of axial/equatorial (ale) pairs, the axially oriented members being more TABLE 3.1 RANOSIDURONIC ACIDS AND PC OF STEROID p-DGLUCOPYRANOSIDES, ~-DCLUCOPY AGLYCONES [934] Solvent systems: 1 = ethyl acetate-toluene-methanol-water ( 5 : 15:7:13); 2 = ethyl acetate-toluenemethanol-water (5:35:16:24); 3 = ethyl acetate-toluene-acetic acid-water (5:15:6:14); 4 = ethyl acetate-toluene-acetic acid-water (3: 17:6: 14); 5 = 2,2,4-trimethylpentane-toluene-methanol-water (17:3:17:3); 6 = 2,2,4-trimethylpentane-methanol-water (10:9:1). Aglycone

30-Hydroxy-5-androsten-1 ?'-one 3a-Hydroxy-5a-androstan-17-0ne (a) 3p-Hydroxy-5a-androstan-17-0ne (e) 3a-Hydroxy-50-androstan-17-one ( e ) 3p-Hydroxy-Sp-androstan-17-one ( a ) 3p-Hydroxy-S-pregnen-20-one 3a-Hydroxy-Sa-pregnan-20-one (a) 3p-Hydroxy-5a-pregnan-20-one ( e ) 3a-Hydroxy-5p-pregnan-20-one ( e ) 3p-Hydroxy-Sp-pregnan-20-one (a) (22S,25S)-Spirost-S-en-3p-o1 (22S,25S)-5or-Spirostan-3p-ol 5Cholesten-3p-01 5aCholestan-3p-ol

Giucoside System No.

hRF

1 1 1 1 1 2 2 2 2 2 2 2 2 2

14 25 17 18 23 11 22 14 16 19 17 18 21 21

13

Glucosiduronic acid

Aglycone

System No.

hRF

System No.

hRF

12 21 15 17 21 11 22 12 14 17 19 21 24

5 5 5

21 38 29 33 40 25 40 26 26 31

5 5

6 6 6 6 6

14

PAPER AND THIN-LAYER CHROMATOGRAPHY

mobile in all cases. Whatman No. 1 paper was used. The conjugates were detected by dipping the paper into 10%phosphomolybdic acid in ethanol and then heating it at ca. 90°C. The yellow fluorescence, produced when a,/.?-unsaturatedketosteroids on filter paper are treated with a NaOH solution and then heated, is fairly sensitive (2 ,ug/cm2) and specific [129]. Edwards and Trafford [261] have made use of this test to detect 30hydroxysteroid-5-enesafter their conversion to 6-hydroxysteroid-4-en-3-ones by oxidation with rut.-butyl chromate. Another fairly sensitive (5 ,ug/cm2) and specific test for this very common feature in natural steroids, which, however, is also applicable to TLC, is the color produced after treatment with a reagent containing 50 mg of picric acid in 16 ml of glacial acetic acid and 4 ml of 70% HC104 [258], Glass-fiber paper, impregnated with adsorbents, has been used for steroid analysis, and it is available commercially under the name Instant Thin-Layer Chromatography (ITLC). It has the advantage over filter paper that aggressive detection reagents, such as sulfuric acid, can be used, but it is inferior to thin-layer plates in producing more diffuse zones and thus poorer resolution, owing to its coarse texture.

3.2. THIN-LAYER CHROMATOGRAPHY Several review articles [419,65 1,6601 and chapters [659,661,771,1079] on TLC of steroids have appeared in the last decade. The following pages represent a selection of recent publications on techniques that have been applied to steroids in general. Spreadlayer chromatography, i.e. chromatography on a loose layer of adsorbent, is a forerunner of TLC, i.e. chromatography on an adherent layer of adsorbent. Having no particular advantages but rather serious disadvantages, spread-layer chromatography is now obsolete. However, some of its applications will be mentioned, because the procedures are readily adaptable to modern TLC.

3.3. SORBENTS The most generally useful adsorbent for TLC of steroids is silica gel. In this book, silica gel may be assumed to be the adsorbent for all thin-layer chromatograms, unless otherwise specified. Both particle size and pore diameter of the silica play a role in the efficiency of the sorbent. Optimal resolution of steroids was obtained with pore diameters of 50-60 A [397]. Whereas highly active silica was originally recommended, most workers now prefer partially deactivated silica, at equilibrium with the laboratory atmosphere. Honegger I4571 prepared activity gradients by dipping activated silica gel plates into successively shallower layers of aqueous acetone. Commercially available silica gel plates usually contain plaster of Paris as a binder and may be ordered with or without an inorganic fluorescent indicator. The latter permits the detection not only of W-absorbing steroids or their derivatives but also of many other organic compounds. Workers who prepare plates in their own laboratory sometimes incorporate inorganic [ 1021 or organic fluorescent indicators in the adsorbent slurry.

SORBENTS

15

F‘yrene derivatives permit the detection of as little as 10 ng of steroids [ 10781. Rhodamine 6G is generally less sensitive [ 1 1361. Other organic phosphors are mentioned in Section 3.6. Other additives have been suggested. For instance, silica gel plates prepared with 1% (NH4)2S04 solution instead of water have unaltered chromatographic properties, but when they are heated at 200°C for 20 min, sterols and other lipids are revealed as charred zones [1141]. By laying a silica gel plate into a satd. solution of NH4HS04 in abs. ethanol for 30 sec the adsorbent is modified in such a way that steroids can be detected in daylight or long-wave UV light by heating the plate at 140°C for 15 min [ 10671. Moreover, the modified adsorbent permits the resolution of the two 1 6 , 1 7 4 s epimers of estriol, which are unresolved on ordinary silica gel. Thin-layer plates prepared by mixing 10 g Silica Gel G with 22 ml of 5% aqueous cupric sulfate solution are suitable for the detection of steroids by charring at 180°C for 1 h [593]. When 10% phosphomolybdic acid is incorporated in the silica gel slurry or when the finished plate is dipped in an alcoholic solution of phosphomolybdic acid, steroids may be detected by heating it at 110°C for 20 min [ 10591. However, solvent mixtures containing more than 15% ethanol in benzene cannot be used for developing such a chromatogram, unless they also contain phosphomolybdic acid. Addition of an equal weight of Celite to silica gel decreases the development time 2.5to 3.5-fold [ 10861. “Critical pairs” of steroids, i.e., steroids of closely similar constitution and therefore inseparable by ordinary TLC, can often be resolved on specifically treated adsorbents. Thus, addition of 10% digitonin to the silica gel causes 3P-hydroxysteroid-Senes to be specifically retarded in relation to other groups of steroids [ 10451. When 10% boric acid is used to prepare the silica gel slurry, the thin-layer plates will specifically retard cis-ol,P-diols [664]. Incorporation of silver nitrate (argentation chromatography) allows unsaturated steroids to be separated according to the degree of unsaturation. One method of preparing plates for argentation TLC is t o spray them with a saturated solution of AgN03 in 90% ethanol and to dry them at 100°C for 10-20 min [35]. Most commonly, however, AgN03 solution is incorporated in the slurry before it is spread (see Section 6.2). Instead of silica gel, porous glass (pore diameter 30-40 A) can be affixed t o glass plates, but this has no advantage, because steroids tend to streak and to give more diffuse zones on porous-glass layers [696]. However, by mixing one part of silica gel with two to five parts of glass powder of similar particle size, applying this mixture in a slurry of solvent to glass plates, and then heating them at 450--750”C for a few minutes, one can obtain thin layers with several advantages over plates prepared with other binders [820]. The silica gel-sintered glass plates not only resist heat, abrasion, and strong acids, but they are also reusable after soaking in cleaning solution and they imbibe sufficient amounts of polar and nonpolar stationary phases for partition chromatography. Alumina- sintered glass plates may be analogously prepared from a mixture of one part of alumina with one to six parts of glass powder [818]. In addition to the previously mentioned advantages, alumina-sintered glass plates require less time for solvent development than alumina-gypsum plates. Other metal oxides may be substituted for alumina [815], and inorganic fluorescent indicators may be incorporated in the sintered layers [817]. TLC of steroids on alumina, produced by electrolytic oxidation of the surface of aluminum plates, has also been reported [222]. An advantage of alumina over silica in TLC of A4-3-ketosteroidsis that they form fluorescent oxidation products when the chromatographic plate is heated to 150-180°C

16

PAPER A N D THIN-LAYER CHROMATOGRAPHY

[484]. The reaction appears to be specific and permits the detection of nanogram quantities of these steroids. Another sorbent used in TLC of steroids is polyamide [ 1 1691. Polyamide plastic sheets, developed with hexane-acetone (4: 1) for the less polar steroids or with chloroform-acetone (4: 1) for the more polar ones, produce satisfactory separations. The disadvantage of plastics is that corrosive reagents cannot be used, but one of the advantages is that, for elution, zones can be simply excised and dropped into the appropriate solvent. Layers of about 250 pm thickness of polyamide on glass plates may be prepared from a 20% suspension of polyamide powder in ethanol 19501. A number of steroid glucuronides were separated on these layers by solvent systems such as methanol-water-formic acid (1 2:7: 1 ) and ethyl formate-methanol-water-formic acid (1 0:4:5: 1). After spraying the chromatogram with 4% A1C13 in 75% aq. methanol and heating it at 120-1 50°C for 15 min, the glucuronides could be detected by their fluorescence in W light (365 nm) in amounts of 0.1 pg. Polyamide layers have also been used for the isolation of various steroid conjugates [807].

3.4. LAYERS Most analysts now prefer ready-made plates, because they are more convenient, uniform, and sanitary than those prepared in the laboratory [395]. However, the preparation of certain layers, which has been described in connection with steroid research in recent years, should be discussed here. Peifer’s method permits the preparation of excellent microchromatoplates in a matter of minutes without any equipment [843,1189]. Two clean microscope slides are pressed together and dipped in a suspension of, e.g., 30 g silica gel and 1.5 g gypsum in 160 ml chloroform, then separated and allowed to dry. Such plates are extremely useful in exploratory work, such as the selection of solvent systems or the periodic testing of reaction mixtures. Another simple and rapid method of preparing thin layers is to dip scrupulously clean glass rods or test tubes into a suspension of one part of Silica Gel G and two parts of water [500]. The test tubes are then inverted and allowed to dry. The uniform layer on the outside of the tubes is spotted with the sample near their closed end, and the chromatogram is developed by dipping the tube into a few milliliters of solvent in a larger test tube. Several advantages have been claimed for coating test tubes on the inside rather than on the outside [641]. Uniform layers are easily prepared by filling clean test tubes with a suspension of two parts of silica and five parts of chloroform and then slowly emptying them. The layers are protected and require no binder. When the sample is applied near the lip of the test tube, the latter can be inverted over the mobile phase and acts as its own chromatographic chamber. For the detection of lipids, the test tube is simply inverted over iodine crystals. Plates for the purification of weighable amounts of steroids may be prepared in the laboratory [594], but the commercial availability of preparative plates with layers up to 2 mm in thickness makes this tool more accessible to the occasional user [393,394]. Depending on a number of conditions, up to 1 g of mixture can be resolved by 40 g of adsorbent. Fluorescent layers and multiple development are often found helpful.

DEVELOPMENT

17

Flexible thin layers are manufactured by coating aluminum foil [396,596] or plastic sheets [ 185,3961 with alumina or silica containing some proprietary binder. They have several advantages over the conventional glass plates: They are easily stored and cut, elution of zones is simplified, they can be used for long-distance, descending, and continuous development, etc. In short, they have all the advantages of PC. However, they lack some of the advantages of TLC, such as load capacity and resistance to corrosive detection reagents (see Section 3.6).

3.5. DEVELOPMENT Thin-layer chromatograms are most frequently developed by a single solvent escent, but some pairs of closely related steroids have been resolved by multiple development of silica plates with a cyclohexene-cyclohexanone (9: 1 or 4: 1) mixture [ 1 1791. For mixtures of steroids with a wide range of polarities, gradient elution TLC is desirable but requires special apparatus [779]. Tarr [ 10441 has solved this problem by saturating the atmosphere in the chromatographic chamber with a less polar, volatile solvent (e.g., benzene) while the more polar developing solvent (e.g., ethyl acetate) rises on the thinlayer plate. This causes the developing solvent to become less polar as it rises and sharpens the zones. Vapor-programmed TLC depends on the adsorption of different solvent vapors on different parts of a thin-layer plate, which lies with the adsorbent facing downward over a series of transverse solvent troughs [1156]. Substances that are difficult to separate by conventional TLC (e.g., stenols and the corresponding stanols) may be separated by allowing a solvent mixture [e.g., benzene-diethyl ether (9: l)] to traverse a series of zones on the plate, which are saturated with different vapors [e.g., in the order from front to origin, benzene-diethyl ether (9: l ) , carbon tetrachloride-diethyl ether (4: I), carbon tetrachloride, carbon tetrachloride-diethyl ether (3: 2), carbon tetrachloride, carbon tetrachloride-diethyl ether (3:2), carbon tetrachloride, carbon tetrachloride-diethyl ether (2:3), carbon tetrachloride]. Difficult separations sometimes require continuous descending development with solvents in which the migration rates are low. In ascending TLC, continuous development may be achieved either by attaching an absorbent pad to the top edge of the plate [63], by allowing the top edge to protrude from the lid of the chamber [66,635], or by allowing a paper wick, in contact with the layer, to extend from the chamber [ 1541. The continuous withdrawal of solvent from the top causes continuous upward flow in each case. Continuous development in horizontal solvent flow is accomplished by using the thin-layer plate as a lid for a rectangular solvent tray of smaller dimensions [635]. The solvent is fed to one end of the layer on the underside of the lid by a rolled filter-paper wick, traverses the layer, and evaporates at the protruding other end. Solvents for specific applications are given in connection with that application. Here, only some solvent systems for general use will be mentioned. Among the partition systems for various steroid hormones adopted from PC are [1144] : cyclohexane-methanol-water (5:4:1), cyclohexane-toluene-methanol-water (89: 1 1:80:20 or 4: 1 :4: l), cyclohexaneacetic acid-water (20: 17:3), cyclohexane-toluene-acetic acid-water (67:33:80:20 or

18

PAPER AND THIN-LAYER CHROMATOGRAPHY

16:4: 17:3), and cyclohexane-toluene-90% formic acid -water (80:20:89: 1 1). The aqueous (bottom) layer is used to saturate the chamber atmosphere before the organic (top) layer is added to a solvent trough under the silica gel plate. The impregnation of silica gel layers with formamide [133,997,11003], ethylene glycol [ 1331, or propylene glycol [996] in preparation for partition chromatography or with paraffin oil [996] in reversed-phase partition chromatography is cumbersome and now rarely practiced. In thin-layer adsorption chromatography, the composition of solvent mixtures changes as they move along the plate, their more strongly adsorbed components acting as displacers [417]. Examples of binary solvent mixtures generally useful for adsorption chromatography of steroids are: cyclohexane-ethyl acetate (1 : 1) [ 1941 ;benzene-diethyl ether (4: l), benzene-acetone (9: l), chloroform-benzene (1 :I), chloroform-methanol (99: 1) [389] ;benzene-acetone (4: l), benzene-ethyl acetate (1 :l), benzene-methanol (49: l), diethyl ether-acetone (9: 1) [ 10381 ;chloroform-acetone (3: 1) I4081 ; chloroformethanol (9: l), benzene-ethanol (4: 1) 17831;chloroform-ethyl acetate (4: l), chlorofomacetic acid (9:l) [917]. Ternary solvent mixtures are rarely needed, but some examples follow: cyclohexanechloroform-acetic acid (8: 1 :I), toluene-petroleum ether (b.r. 40-6O0)-methanol (2:2: l), cyclohexane-acetone-chloroform (1 5 :5 :4), chloroform-cyclohexane-isopropanol (5:10:2) [917]; ethyl acetate-cyclohexane-ethanol(16:3:1 or 9:9:2 or 10:9:1), ethyl acetate-hexane-acetic acid (1 5:4: 1) [783]. Two-dimensional development increases resolution; it has been used for several individual classes of steroids. For a large variety of steroid hormones, a silica gel plate can be developed with 2% acetone in chloroform in the first dimension and with 20% acetone in chloroform in the second [931] or with chloroform-methanol-water (188: 12: 1) in the first dimension and two successive runs with cyclohexane-ethyl acetate (1 :1) in the second [ 10461.

3.6. DETECTION Most of the reagents used for detecting steroids by destructive methods contain sulfuric acid, but sulfuric acid without additives also produces characteristic color and fluorescence responses, as well as black zones of charred material [423]. Table 3.2 lists the response of 141 representatives of various classes of steroids to heating at 78°C after 4 pg of sample had been sprayed with 50% HZSO4. The time required for the initial color to appear in daylight, the color after heating has been continued for 10 min, and the color in UV light of 366 nm are presented. As little as 0.01 pg of steroids can be detected. The sulfuric acid reaction was studied in detail by Carstensen [ 1431, and further data on different ways of performing this reaction can be found in the literature [57,408,994, 11361. A spray reagent made by dissolving 20 g (NH4)?S04 in 100 ml water and adding 4 ml conc. H2S04 has been suggested as a less corrosive substitute for H2S04 [1191]. Many organic compounds, including steroids, on thin-layer plates give color responses to a 0.05%V205 solution in 5% H2S04 [694]. A reagent prepared by adding 70 ml98% H2SO4 to 30 ml satd. aq. K2Cr20, also gives

DETECTION

19

characteristic colors with steroids at 76°C [962]. Oxidation of steroids by heating the chromatogram after spraying it with a H2S04-KMn04 reagent gives products that stain in characteristic ways with Toluidine Blue or Alcian Blue [ 10881. A spray reagent containing 0.5 ml anisaldehyde and 1 ml conc. H2S04 in 50 ml glacial acetic acid produces a variety of colors with steroids after a 10-min heating period [599]. Another useful reagent, 20%p-toluenesulfonic acid in ethanol, gives a variety of colors after the ehromatograms have been heated at 120°C for 10 min [996,1126]. Both silicotungstic and phosphotungstic acid, used as 15% aq. solutions and followed by heating at 110°C for 15 min, are sensitive spray reagents for steroid [948]. In order to avoid spraying and heating of chromatograms in recovery experiments, a 10% phosphomolybdic acid solution in ethanol can be applied to the pilot lane of a chromatogram with a dropper [77]. When this is followed by a treatment with conc. H2S04,steroids immediately appear as blue spots. However, even without H2S04, steroids will be stained by phosphomolybdic acid if the plates are kept at room temperature for 12-1 5 h [ 1941. Silicomolybdic acid is more sensitive than phosphomolybdic acid when used as 3% methanolic spraying reagents [965]. Thin-layer plates of alumina or silica which have been thoroughly washed by continuous development with methanol exhibit fluorescent zones wherever organic compounds are concentrated when they are heated in a closed chamber containing dry NH4HC03 [949]. Steroids in amounts as low as 0.1 pg respond after 1-2 h at 110°C with a lasting greenish blue fluorescence, detectable at 365 nm. The response can be used for quantitative analysis. When 3-hydroxy-5-enesand 3-hydroxy-4-eneson layers of Silica Gel HFZj4+366 are exposed to hydrogen chloride vapors at 40°C for 10 min, a pink or violet color develops which gradually fades, but a yellow fluorescence remains visible for days under shortwave UV light [516]. The reaction is quite specific and sensitive, less than 2 pg/cm2 being detectable. When the plates are exposed to the vapors at 4"C, only the 3-hydroxy-4-enes give pink spots [5 171. 3-Hydroxy-5-enes give characteristic colors after the chromatogram has been sprayed with a solution of 100 mg picric acid in 36 ml glacial acetic acid plus 6 ml 70% HC104 and then gently heated [258]. Various steroids give color reactions on thinlayer chromatograms after having been sprayed with 10%K2[Hg14], K2[Hg(SCN)4], or K2[HgBr4], heated at 140°C for 20 min, and then exposed to HC1 vapors [863]. Steroid glucosiduronates may be detected in microgram amounts by spraying the plate with a freshly prepared mixture of one part of 0.4% 1-(2-pyridylazo)-2-naphtholin ethanol and four parts of dichloromethane and, after it has dried, with a mixture of 8 mlO.8% Co(NO&, 4 ml 2 M acetate buffer of pH 4.6, and 88 ml water [209]. Steroid sulfates give various colors after treatment with a solution of 25 mg Methylene Blue in 100 ml 0.05 N HZSO4. Other steroid reagents exhibiting some specificity are [ 10081: a freshly prepared mixture of one part of 1% 2,5-diphenyl-3-(4-styrylphenyl)tetrazoliumchloride in methanol and ten parts 3% NaOH solution, which is more sensitive than other tetrazolium salts in detecting reducing steroids; Komarowsky reagent (one part of 50% H2S04 plus ten parts of 2%hydroxybenzaldehyde in methanol), which reacts with ketones adjacent t o a methylene group in 10 min at 60°C; and 30% methanolic ZnClz, which produces fluorescent zones with 1 1-oxygenated steroids after the plate has been heated at 105°C for 1 h. The specificity of many reagents may be enhanced by subjecting the steroids to reactions on the thin-layer plates, e.g., borohydride reduction or periodate oxidation [401].

20

PAPER AND THIN-LAYER CHROMATOGRAPHY

TABLE 3.2 SULFURIC ACID REACTION OF STEROIDS [423] Abbreviations: BE = blue; BK = black; BN = brown; BT = bright; DK = dark; DL = dull; ED = emerald; GN = green; GY = grey; LC = lilac; LT = light; OE = orange; PE = pale; PK = pink; PU = purple; RD = red; VY = very; YW = yellow. Compound

Color after heating at 78°C In daylight

In UV -

Min

C , , steroids 1,3,5( 1O)-Estratrien-3-01 4-Estrene-3J7-dione 17P-Hydroxy-4estren-3-one 17p-Hydroxyd(l O)estren-3-one Estrone Equilin Equilenin Estradiol-170 Estradiol-l7a 1,3,5( 1O)-Estratriene-3,16adiol 1,3,5(10),6-Estratetraene-3,17pdiol 1,3,5( 10),6,8-Estrapentaene-3,17adiol 3,17p-Dihydroxy-1,3,5(10)estratrien-16-one Estriol 16-Epiestriol C , , steroids 5wAndrostan-17-one 5a-Androstan-l7p-01 5a-Androstan-3a-01 5a-Androstan-3p-01 17p-Hydroxy-Sp-androstan-3-0ne Androsterone

3p-Hydroxy-5a-androstan-16-0ne 17p-Hydroxy-5cu-androst-len-3-one 17P-Hydroxy-l,4-androstadien-3-one Testosterone Dehydroepiandrosterone

5a-Androstane-3,16-dione 5a-Androstane-3,17-dione 50-Androstane-3,17-dione l-Androstene-3,17-dione 4-Androstene-3,17-dione 4-Androstene-3,16dione

1,4-Androstadiene-3,17-dione Sa-Androstane-3p, 16p-diol 5-Androstene-3p,16pdiol 5a-Androstane-3a, 17p-diol 5a-Androstane-3p,l7p-diol 5-Androstene-3p,l7pdiol 4-Androstene-3p,li’p-diol

Initial

Final

Yw

BE LC BE BT-BE GN-BE GN-BE DL-RD GN-BE BTGN BE BTGN DL-RD GN DL-RD DL-LC

VY -PE-BE PE-BE VY -PE-BE VY -PE-BE PE-BE BE BE BT-BE DL-OE BE BT-BE BT-BE DL-BE DLGN DLGN PE-BE BE YW PE-BE BE DL-RD DLGY DL-BE BT-BE

1.00 1.50 1.75 1.00 1.25 0.50 0.50 1.00 0.50 1.00 0.25 1.50 1.50 1.75 1.50

OE YW PE-YW PE-QE PK PE-PK BTGN-YW PE-OE OE PE-PK PE-OE

DL-YW GY-BN BN QE BT-OE RD-QE BN OE BT-PK OE-PK OE RD OE PK QE

3.00 1.00 0.75 1.50 1.75 2.50 1.50 1.00 2.00 1.00 0.50 3.00 2.25 1.50 1.00 1.50 1.25 1.75 1.50 0.50 1.50 2.00 0.50 0.50

PEGY LT-BN BEGY GYGN BT-YW PE-YW GY-YW GYGN PE-OE BEGN BT-PK PEGY PEGN GN GN BEGN PEGN PK GY LT-PK GYGN GY GY-PK PK

GY DLGY PEGY BN DK-\W GY GY GYGN QE EDGN PU GY GN GN GYGN EDGN GY OE OE BE-PU PU GY-RD PU GY-PU

GN GYGN

Yw

DETECTION

21

TABLE 3.2 (continued) Compound

Color after heating at 78°C

In daylight

In UV

3p,l7p-Dihydroxy-5-androsten-16-one 5a-Androstane-3,11,17-trione 4-Androstene-3,11,17-trione

Min Initial 0.50 PK 0.75 BEGN 1.50 BEGN 0.50 PK 2.75 GY 1.00 PEGY

Final BE-PU BE GN DK-PK GY PEGY

BE BEGN GN GN-BE VY-PE-BE VY -PE-BE

C , , steroids 5a-Pregnan-3p-01 5p-Pregnan-3a-01 3p-Hydroxy-Sa-pregnan-2O-one 20p-Hydroxy-5a-pregnan-3-one 3p-Hydroxy-5p-pregnan-20-one 3p-Hydroxy-5-pregnen-2O-one 3p-Hydroxy-Sp-pregn-16en-20-one 3p-Hydroxy-5,16-pregnadien-2O-one 20a-Hydroxy-4-pregnen-3-one 20p-Hydroxy4-pregnen-3-one 5 a-Pregnane-3,20-dione 5 p-Pregnane-3,20-dione Progesterone 4,16-Pregnadiene-3,20-dione 5p-Pregnane-3a,20a-diol 5p-Pregnane-3p,20a-diol 5a-Pregnane-3p,20@-diol 5p-Pregnane-3p,20p-diol 4-Pregnene-3p,20p-diol 5-Pregnene-3p,20p-diol 12a-Hydroxy-Sp-pregnane-3,2O-dione 6p-Hydroxy-1 -pregnene-3,20-dione 1 la-Hydroxy-4-pregnene-3,20-dione 15a-Hydroxy+pregnene-3,2O-dione 1 7a-Hydroxy-4-pregnene-3,20-dione Deoxycorticosterone 3p,16a-Dihydroxy-5a-pregnan-20-one 3p, 16a-Dihydroxy-Sp-pregnan-20-one 3p,17a-Dih ydroxy-5 p-pregnan-20-one Sp-Pregnane-3,11,20-trione 4-Pregnene-S,11,20-trione Corticosterone 11-Dehydrocorticosterone 11-Deoxycortisol Cortisone Cortisol 17a,2 1-Dihydroxy-Sp-pregnane-3,20-dione 3a,l7a,20a,2l-Tetr~hydroxy-5p-pregnan-ll-one 3a,l7a,20p,2 1-Tetrahydroxy-5p-pregnan-l l-one

1.00 1.50 1.25 2.00 2.50 0.75 1.50 0.75 1.25 1.00 1.75 2.50 1.50 1.75 1.25 1.50 1.00 1.75 0.50 0.75 0.75 2.50 1.50 1.50 1.50 0.75 1.50 1.50 2.25 1.25 2.00 1.00 2.00 1.00 2.50 0.75 1.00 1.50 2.00

BN PKGY GY-PK GY GY-PK PK-PU PK BN OE YW YWGY GY GY LT-BN GY-PK GY -PK RD-BN RD GY BE-PU GY YW BN-RD OE BN DK-BE OE BN OE YW GY DKGN OE PU OE BN BN OE GY

YW-BE VY-PE-BE BE LT-BE PE-BE BE YW YWCN BT-BEGN BT-BE BT-BE PE-BE BE LC VY -PE-BE VY -PE-BE DL-BE PE-BE PE-BE BT-BE OE BT-BE BE BT-BE BE DL-BE DL-OE YWGN BT-LC BT-BE BE BTGN BE RD BT-BE BTGN BE BT-BE BT-BE

5-Androstene-3p,l7a-diol 1lp-Hydroxy-4-androstene-3,17-dione 1la,l7a-Dihydroxy-4-androsten-3-one

GY-BN GY-PK GY-PK PE-YW PE-PK PK PE-PK OE YW-PK PE-YW GY-YW PE-YWCY GY-PK YW GY-PK PK PK PK PE-PK GY-PK GN PE-YE YW YW GY GN PK YW LT-PK PE-YW GY GN PE-YW PK YW OE-PK YW GY-OE PEGN-YW

(Continued on p . 22)

22

PAPER AND THIN-LAYER CHROMATOGRAPHY

TABLE 3.2 (continued) Compound

Color after heating at 78°C

Min

Initial

_____-

In U V

In daylight Final BN BN GYGN

BTGN-BE DL-BE PE-BE

YW YW YW GN PE-YW

GN OE OE GN RD-BN

GN GN BT-BE BE BT-BE

2.00 2.00 1.50 1.25 1.00 1.50

LTGY PE-PK GN-YW PK GY-PK PEGN-YW

GY GY-PK YW PE-PK GY YW

DLGN BE GN-BE BE BE BE

0.25 3.00 2.25 2.25 0.75 2.00 2.00 1.00 1.25 2.00 1.75 1.75 1.00 1.00 1.00 0.00 0.50 2.25 1.00 0.50 1.25 0.00 0.75 1.25 0.50 0.50 1.75

PK PE-YW LT-BN PE-YW PE-YW GY-PK PK PE-YW PK GY PEGY-PK GY-PK DL-YW GY-PK YW GYGN PK PE-YWGN GY PE4E GN YW GY GY-PK PK YW GY-PK

DK-PK LT-BN LT-BN GY PK BN PK-OE YW BN-RD BN PKGY LT-BN RD PU GY GY GY YWGN GY OE-RD BN BK BN

GN BE PE-BE BE BE DL-RD BE PE-BE OE BK W-PE-BE BK GN-BE DL-BE YW-BE BE DK-BN BE DLGN BE DLGN GN DL-BE DL-RD DK-BN GN-BE PE-DL-RD

1lp,l7a,21-Trihydroxy-4-pregnene-3,20-dione 17a,2 l-Dihydroxy-1,4-pregnadiene-3,11,20-trione Sp-Pregnane-3a,l lp,l7~,2Oa,2lpentol

0.50 YW 1.50 YW 1.00 GY

Cardenolides Uzarigenin Strophanthidin Digoxigenin Digitoxigenin Gitoxigenin

1.50 0.50 1.00 1.00 1.00

Bile acids Cholanic acid Lithocholic acid Deoxycholic acid Chenodeoxycholic acid Hyodeoxycholic acid Cholic acid Sterols 3,5Cholestadiene 5 a C h olestan-3-one 5pCholestan-3-one 4Cholesten-3-one 5Cholesten-3-one 1,4€holestadien-3-one 4,6Cholestadien-3-one 3,5Cyclocholestan-6-one 3,5-Cyclocholestan-6p-ol SaCholestan-3p-01 5pCholest an-3a-01 5pCholestan-3p-01 5a,6a-Epoxycholestan-3p-ol Cholesterol Sa-Cholest-7-en-30-01 7-Dehydrocholesterol Desmosterol 3p-Hydroxy-5crcholestand-one 3p-Hydroxy-5-cholesten-7-one 4Cholestene-3,6-dione 5Cholestene-3p,4p-diol Ergosterol 22-Stigmasten-30-01 Sitost er ol Stigmasterol Lanosterol Dihydrolanosterol

PU GY BN PK-BN

DETECTION

23

TABLE 3.2 (continued) -.

Compound

-

Color after heating at 78°C In UV

In daylight Min Initial ~. Sapogenins Srnilagenin Sarsasapogenin Tigogenin Neotigogenin Y arnogenin Diosgenin Hecogenin Gentrogenin Kryp togenin

1.25 0.75 1.00 1.25 0.75 1.50 1.00 0.75 0.50

PE-PK PK PK OE PE-YW PK DK-PK

Alkaloids Holaphyllamine hydrochloride Holamine Holaphylline Solasodine Toma tidine 5-Tomatiden-3p-01 Solanidine

2.00 1.75 1.50 0.75 0.50 0.50 0.50

PE-YW PE-PK PE-OE PK PK PK DK-PK

Yw Yw

Final OE OE OE GY GY Yw RD DK-BN

GN-BE GN-BE BE PE-BE BE BT-BE BT-BE BE BT-BE

YWGY LT-BN LT-BN PU GN PU RD

W-PE-BE DL-RD BE BE BT-BE BE BT-BE

OE

A number of nondestructive detection methods are available in case recovery of unaltered steroids from thin-layer plates is desired. For instance, the sample can be mixed with marker dyes selected in such a way that certain groups of steroids will migrate between certain dyes [759,1093]. In an example given by Vandenheuvel [ 10931, the following migration distances (in mm) were obtained on a silica gel plate developed (over a distance of 150 mm) with chloroform-methanol-water (800:36:3): Sudan Blue, 136; progesterone, 130; cholesterol, 1 13;p-aminoazobenzene, 112; androsterone, 106; etiocholanolone, 95 ;2,4-diaminoazobenzene, 89 ; 1 1-ketoandrosterone, 84; pregnanediol, 62; 4,4’-dihydroxyazobenzene, 26; pregnanetriol, 20; estriol, 10. Layers containing fluorescent indicators are used t o detect steroids that absorb W or show fluorescence or quenching (see Section 3.3). Ordinary thin-layer plates can be made fluorescent by spraying or predevelopment with fluorescent organic compounds. A spray reagent suitable for photography at 366 nm is prepared from 100 mg Rhodamine B, 35 mg 2’,7’-dichlorofluorescein, 150 ml diethyl ether, 70 ml95% ethanol, and 16 ml water [524]. Dimethyl-POPOP [stock solution: 0.2% 2,2’-p-phenylene-bis(4-methyl-5phenyloxazole) in methanol, kept in the dark] can be used either as a spray (stock solution diluted ten times with a volatile solvent) or by predeveloping the plate with a dilute solution ( 5 ml of stock solution made to 100 ml with chloroform-methanol (1 : l ) ] to detect microgram quantities of most steroids under a short-wave W lamp [55]. To make Silica Gel G plates suitable for the detection of UV-absorbing as well as UVtransparent steroids, they may be pretreated by dipping them in a solution, prepared by

24

PAPER AND THIN-LAYER CHROMATOGRAPHY

mixing 1 ml 0.1% primuline with 100 ml aq. acetone [ 11801. Greater sensitivity (< 5 pg) is achieved by spraying the finished chromatograms with that solution and observing them under a 365-nm source of radiation while they are still damp. Exposure of thin-layer chromatograms to iodine vapors is a widely practiced method of detecting lipids. Most steroids are recovered unchanged after exposure for half an hour, but estrogens and presumably some unsaturated steroids undergo chemical changes [ 10 101 (cf. also p. 83). The sensitivity of the iodine test can be greatly increased by the use of layers containing Rhodamine 6G [1136]. As little as 50 ng of some steroids can thus be detected in UV light.

3.7. QUANTITATIVE AND RADIOCHEMICALMETHODS Quantitative analysis of steroids after separation by TLC is generally more accurate if they are eluted than if they are assayed in situ,provided the elution is quantitative.

After a steroid zone has been located on the layer by one of the nondestructive tests described above or by tests on pilot chromatograms, the adsorbent in that area is removed and extracted. Attal et al. [34] have packed the adsorbent in a small column, which was eluted with a series of solvent mixtures of increasing polarity. A syringe, equipped with a suitable filter (Alpha-8 Metricel), served as the column [697]. By recovery studies on I4Clabeled steroids Idler et al. [496] have determined that destruction of microgram quantities of material occurs not so much during TLC as during elution and concentration of the eluate. The problem was traced to the presence of silica gel in the eluate and remedied by the use of nonpolar eluents containing a minimum of polar solvents for the inactivation of the adsorbent, e.g., dichloromethane--methanol(9: 1) [495]. A number of extractors for the collection and subsequent elution of steroid zones have been described [488,910, 947,10921. For the quantitative analysis of steroids without elution from the plate, visual estimation or, preferably, densitometry is used. Of the various densitometers designed in connection with steroid analysis, reference should be made to the two-dimensional scanning densitometer for transmitted [410] or reflected 19321 light, the double-beam spectrodensitometer [ 10611, and the scanning fluorometer [574], w h c h can be used to determine the quenching of fluorescence produced by, e.g., 4-en-3-ones on Silica Gel GFZM.Densitometry is more rapid and convenient than elution, but requires uniformity of layers, sample application, and staining for reproducible results [ 10631. Amounts of 0.1-1 .O pg of UVabsorbing or stained steroids can be determined in this manner. The densitometer output can be integrated manually or by instrumentation, and the peak areas can be related to amounts of steroids by calibration curves [ 1471. Chromatograms developed in a quartz glass tube that has been coated on the inside with a thin layer of silica or a 9: 1 mixture of silica and CuO can be scanned by slowly passing the tube through a ring furnace at 600-800°C while blowing a gas through it [75 1, 7521. The gas carries the pyrolysis or combustion products - either directly or after catalytic reduction of the C02 formed to methane - into a gas chromatograph and through a FID (if the carrier gas is nitrogen) or through a thermal conductivity detector (if it is helium). When air is used as the carrier gas, combustion takes place even in the absence of

25

STEROID DERIVATIVES

CuO, and the chromatographic tubes may be reused [750]. A variant of this method employs a quartz rod, coated with silica gel by mixing it with a borosilicate binder and sintering the mixture [816,819]. A chromatogram developed on such a rod can be scanned by analogous techniques. Steroids labeled with I 4 C or 3H may be detected by radioautography [891]. This simple but time-consuming method has now been largely replaced by scanning techniques. A methane flow counter for 3H-labeled steroids has been described [88], but the counting efficiency for 14C is much higher. The determination of the specific activity of labeied steroids may be based on a combination of densitometry and scintillation counting of the eluted compounds [ 1 1711. When TLC is performed on silica-coated plastic sheets, the steroids can be located by exposure to iodine vapors, and the zones can be excised for scintillation counting by use of a manual punch [625]. In tubular TLC (see above) the combustion or pyrolysis products may be assayed either continuously in a proportional flow counter or serially after being trapped in a hyamine solution. TLC is also suitable for the isotope derivative method of analyzing steroid hormones [1158].

3.8. STEROID DERIVATIVES Conjugated steroids are quite polar and require special chromatographic systems. The use of PC and partition systems has persisted in this application (see p. 13). Separations obtained on thin layers of ion-exchange cellulose by Oertel et al. [809] are detailed in Tables 3.3 and 3.4. Mobilities of steroidal glucosides and glucosiduronates (glucuronosides) [934] and of their methyl and acetyl derivatives [935] on silica layers (Silica Gel IB-F sheets) hzve been reported. Steroidal sulfates have been chromatographed on Silica Gel G [1181], Silica Gel HF254+366and ECTEOLA-cellulose layers [852]. Mono- and diphosphates of steroids are best separated by TLC after conversion to their methyl esters [893]. This is

TABLE 3.3 h R p VALUES OF STEROID CONJUGATES IN TLC ON DEAECELLULOSE [ 8091 Solvent systems: 1 = 0.5 M acetate buffer, pH 4.25; 2 = 0.5 M acetate buffer, pH 4.75; 3 = 1.0 M acetate buffer, pH 4.75; 4 = 1.5 Macetate buffer, pH 5.00; 5 = isopropanol-water-formic acid (65:33:2); 6 = ethanol-water-acetic acid (80:15:3); 7 = methanol-water-acetic acid (15:3:2). Conjugate

Estrone sulfate Dehydroepiandrosterone sulfate Androsterone sulfate Pregnenolone sulfate 17-Hydroxypregnenolone sulfate Dehydroepiandrosterone glucosiduronate Androsterone glucosiduronate Etiocholanolone glucosiduronate

Solvent system

1

2

3

4

5

6

7

8 19 23 -

3 9 15

-

38 45 40

1 14 22 21 9 41 48 45

8 20 24 56 62 58

8 22 33 72 I1 75

2 7 8 29 35 31

3 6 I 36 46 39

54 59 56

-

26

PAPER AND THIN-LAYER CHROMATOGRAPHY

TABLE 3.4

~ R VALUES F OF STEROID CONJUGATES IN TLC ON ECTEOLACELLULOSE [ 8091 For solvent systems, see Table 3.3. ~

Conjugate

Estrone sulfate Dehydroepiandrosterone sulfate Androsterone sulfate Pregnenolone sulfate 17-Hydroxypregnenolone sulfate Dehydroepiandrosteroneglucosiduronate Androsterone glucosiduronate Etiocholanolone glucosiduronate

Solvent system 1

2

3

4

5

6

8 16 23 65 72 61

5 14 18 45 49 41

8 23 29 34 12 61 61 64

7 15 21

54 79 85

5 11 13

4 8 9

64 70 65

90 91 91

64 I1 71

48 59 55

7

accomplished on the silica gel layer by treating the initial zone with a solution of diazomethane in diethyl ether prior to development with cyclohexane-ethyl acetate (1 : 1). Other derivatives may be prepared directly on the thn-layer plate. For instance, fungal spores entrapped in Silica Gel G will transform steroids during incubation prior t o TLC [388]. The “spore plate” is prepared by substituting 0.1%glucose solution for the water normally used for slurrying the silica gel. Chromatography involving derivatization at the origin (elatography) has been advocated for steroids with various functional groups. Thus, ketosteroids are specifically retarded by impregnating the starting line with 0.1% Girard’s Reagent T [654] or 0.5% isonicotinic hydrazide [664] in 10%acetic acid and spotting the steroids on the moist area some time before the chromatogram is developed. The order of migration is: uncombined steroids > mono- > di- > trihydrazones. To convert ketosteroids to the 2,4-dinitrophenylhydrazoneson thin-layer plates, they may be sprayed with a solution of 0.1% 2,4-dinitrophenylhydrazinein ethanol, containing 0.1%conc. HC1, and then heated at 100°C for 5 min [695]. To reconvert the hydrazones to ketones, the plates are sprayed with a solution, prepared by dissolving 1.3 g SnC12-H20in 16 ml conc. HCl and 24 ml water, and then heated at 100°C for 15 min. Kent and Rawitch [553] have separated various ketosteroid dinitrophenylhydrazones by TLC with hexane-ethyl acetate (17:3) for quantitative analysis after elution. It should be kept in mind that a diketone, such as progesterone, may not only give rise to three derivatives, i.e., two monohydrazones and one dihydrazone, but that the hydrazones themselves may exist in two isomeric forms. In some solvent systems, such as chloroform-benzene (3: l ) , the 3-keto derivative of progesterone separates into two zones, corresponding to the two geometric isomers [ 11451. The chromatographic properties of several steroid derivatives, such as the formates, acetates, dinitrophenylhydrazones,and bromides, have been recorded [300]. For GC, hydroxylic steroids are often converted to trimethylsilyl (TMS) ethers (see p. 31). These ethers are easily purified by TLC, and even nanogram quantities of steroids with one or more alcohol groups may be recovered quantitatively [ 1 13,1241. Suitable developing solvents are: cyclohexane-ethyl acetate (9: l), benzene, chloroform, and cyclohexanechloroform (7:3 or 4: 1). A spray reagent of 1% Ce(S04)2 in 10%H2SO4 may be used for detection, and diethyl ether is a good eluent for recovery. TMS derivatives of phenols readily undergo hydrolysis and are therefore unsuitable for TLC.

STEROID DERIVATIVES

27

TLC may be used not only for the purification of TMS derivatives prior to GC, but also for the analysis of the effluent from a GC column. This may be accomplished automatically by slowly passing a thin-layer plate across the exit of a gas chromatograph in such a way that individual TMS ethers are adsorbed sequentially along the starting line of the plate [215]. After spraying the plate with 1% HC1 in methanol to hydrolyze the ethers, the individual steroids are identified by TLC.

This Page Intentionally Left Blank

Chapter 4

Gas chromatography 4.1. INTRODUCTION The application of GC to steroids is a very unlikely success story. Steroids are generally thermolabile and insufficiently volatile to be good candidates for vapor phase migration, and gas chromatographs are still among the most expensive pieces of apparatus in the routine laboratory. That GC has nevertheless achieved the most prominent place in the routine analysis of steroids is mainly due to a major research effort in developing suitable steroid derivatives, stationary phases, and instrumentation. The large volume of information developed in this field of analysis has been reviewed in numerous books [266,371,646, 841,859,939,11771,chapters [122,187,214,466,468,535,1103,1110,1178],and articles [301,460,464,467,559,615,711,795,1108,1109,1176].

4.2. STEROID DERIVATIVES There are several reasons for derivatizing steroids prior to GC: the derivatives may be more stable or volatile, less adsorbed, or easier t o separate, detect, and identify. For instance, oxidation of 3P-hydroxy-Sene-steroids and 3/3-hydroxy-Sa-steroids, such as sterols, by cholesterol oxidase produces ketones which are easier to separate by GC [988]. Certain derivatives can be produced on the column by injecting first the sample and then, e.g., acetic or propionic anhydride (“peak-shift technique”) [ 181, but esters are usually prepared prior to sample injection. The steroid acetates are stable derivatives, having the desirable property that the electron-capture detector (ECD) selectively responds t o acetyl derivatives of a-ketols [958]. Steroid formates are easily prepared and, in some cases, they separate better than other esters [690]. Sulfonate esters are unstable under the conditions of GC and are known to undergo elimination reactions [ 1100,1104]. Halogenated esters permit the detection of picogram quantities of steroids with the ECD [ 1861. The heptafluorobutyrates (HFB) are prepared by heating the steroids in a 2: 1 mixture of heptafluorobutyric anhydride (HFBA) and benzene at 70°C for 30 min [29 1 1. Under these conditions, 4-en-3-ones are converted to the corresponding 3,5-enols and will also form electron-capturing derivatives [ 1701. Closer examination of the reaction showed that monosubstitution of nonphenolic hydroxyl groups requires a lower proportion of HFBA/benzene (1 : 1 or 1:SO) than that needed for the formation of enol monoheptafluorobutyrates, di-, or triesters [ 1641. The diheptafluorobutyrates, unlike the monoesters, are relatively unstable, but they may be purified on small columns of Sephadex LH-20 by elution with hexane, ethyl acetate, or mixtures of these. Conversion of steroids to HFB esters with minimal contamination by byproducts is achieved by adding 0.1 ml of heptafluorobutyryl chloride to a solution of 1 mg of steroids in 0.2 ml pyridine, 0.5 ml benzene, and 0.5 ml hexane, and keeping the reaction mixture at room temperature for 30 min [854]. Chlorodifluoroacetates may be prepared in

29

W

TABLE 4.1

0

GROUPS SILYLATED BY DIFFERENT REAGENTS AFTER 24 HOURS AT ROOM TEMPERATURE [359] t = positive

reactions; - = negative reactions; * = semi-quantitative reactions. -

Silylating agent*

__

--_________. -

c=o

OH

Side chain -~ ~-

3

Ilp

1601 17p

20

21

3

17

20

YH3

CH, I

CH, I

COH

I\

HMDS TMCS HMDS t TMCS BSA BSA + TMCS TMSDEA TMSDEA t TMCS BSTFA BSTFA -+ TMCS MSTFA MSTFA t TMCS MSTFA t KOAc** TMSI TMSI t MSTFA

f

-

f

-

-

t

-

t

t

-

4

t

+

t

-

t

i

t

f

t

-

t

t

t t

i

i

t t

-

-

i

t

+

+ t

-

-

t

t t

-

-

t

t

+

-

t

t

t

t

-

t

t

t

i

t

t

-

t

-

t

i

i

t

-

t

t

t

t

i

t

-

+

-

t

-

+ t

+ t

+ t

i t

f -

t

-

+

+

i

*For abbreviations, see List of Abbreviations (p. 125). **KOAC = potassium acetate.

t

-

CH,OH I

CHOH

C=O

EOH

SH

I

I

CH, I

CH,OH I

C=O

C=O

I ,,COH

fiOH

I

STEROID DERIVATIVES

31

analogous manner by replacing the heptafluorobutyryl chloride by chlorodifluoroacetyl chloride. Some HFB derivatives are unstable in GC, e.g., the cholesteryl HFB is pyrolyzed to 3,s-cholestadiene [861]. If steroids are converted t o methoximes (MO, see p. 33) before perfluoroacylation, stable MO-HFB derivatives are obtained and, moreover, side reactions due to enolization of carbonyl groups are prevented [708]. The ECD is not as sensitive to the HFB esters of saturated steroids as it is to unsaturation in the steroid molecule. Derivatives of saturated steroidal secondary alcohols with superior affinity for thermal electrons may be prepared in high yields by dissolving ca. 2 mmoles of the steroid in 0.1 ml dry acetone and 0.1 ml trimethylchlorosilane (TMCS). When 0.05 ml HFBA is added and the mixture is allowed to stand at room temperature for 1 h, an exocyclic conjugated heptafluorobutanoyl group [C3F7COCH:C(Me)O-] is introduced [225]. The reaction does not work well with equatorial hydroxyl groups, which tend to form the HFB esters instead. The phosphinic esters of monohydroxy steroids have good GC properties and can be precisely assayed in the range of 10 pg [ 11341. They are prepared by heating the steroid with dimethylaminodimethylphosphine in the presence of oxygen and absence of water at 50-70°C for 0.5-2 h and are determined with a Rb2S04 alkali flame-ionization detector (AFID). Silyl ethers have been used most extensively in GC of steroids, because they can be easily and selectively prepared, they separate well, and they are very suitable for quantitative analysis and MS [359]. The TMS derivatives may be prepared by treating 0.1 1.O mg of the dry steroids with 50 p1 of hexamethyldisilazane [HMDS, (Me3Si)2NH] and 50 pl of 10%TMCS (Me3SiC1) in chloroform at room temperature for 3 h [ 10991. Alternatively, 0.4 mg of steroids, dissolved in 200 pl dry pyridine, may be left overnight in a mixture of 200 p1 HMDS and 40 p1 TMCS [ 1 131. A third version of this method requires heating the steroids in 0.5 ml tetrahydrofuran with twenty drops of HMDS and one drop of TMCS at 56°C for 3 h [630]. Most commonly, however, 0.5-1 .O mg of a steroid is kept in 0.51.O ml of N,O-bis(trimethy1silyl)acetamide [BSA, MeC(OSiMe,):NSiMe,] in a tightly stoppered vessel at room temperature overnight [462]. Pyridine (0.1-0.2 ml) may be added to promote solution. In any case, the excess reagents are later evaporated below 50°C. The silylation reaction was studied in detail by Yasuda [ 11881,by Chambaz and Horning [ 1671, and, more recently, by Gleispach [359]. Neither pyridine nor tetrahydrofuran were found necessary. Incubation at room temperature for 24 h gave more reproducible results and less byproducts than heating at 60°C. Table 4.1 shows the results of incubating 100 pg of various steroids with 200 pl of various reagents (500 p1 in the case of HMDS) at room temperature for 24 h. Phenolic hydroxyl groups were readily silylated by all reagents. HMDS or TMCS alone may be used for the partial silylation of 3- and 21 -hydroxyl groups, and other unhindered hydroxyls are silylated by BSA, BSTFA [N, 0-bis(trimethylsilyl) trifluoroacetamide, F3CC (OSiMe3):NSiMe3] , TMSDEA [ trimethylsilyldiethylamine, Et,NSiMe3], and MSTFA [N-methyl-N-trimethylsilyltrifluoroacetamide, F3CC:ON (Me)SiMe,] . Silylation of the sterically hindered 17a-hydroxyl group requires TMSI [trimethylsilylimidazole,N:CHNSiMe,C:CH], but the 1lp-hydroxyl group is attacked by various reagents in the presence of TMCS. A mixture of BSTFA and TMCS

32

GAS CHROMATOGRAPHY

(99: 1) is commercially available under the name Regisil. The steroids, dissolved in 1 ml acetonitrile, are heated with 0.1 ml of Regisil at 100°C for 1 h [854]. When trimethylbromosilane is used instead of TMCS, slowly reacting hydroxy- and ketosteroids are converted to TMS ethers more rapidly, ketosteroids forming enol ethers [30].Only MSTFA reacts with keto groups without a catalyst, but potassium acetate and other basic catalysts promote enolization (Table 4.1) [ 166,7781.In order to avoid reaction of the silylating agent with the carbonyl group of steroids containing both hydroxyl and carbonyl groups, the latter can be masked with methoxylamine [342]. Prior to GC, the TMS ethers may be purified by TLC (p. 26) or by eluting the reaction products from a silicic acid column with 5% and 60% ethyl acetate in hexane, containing 2% HMDS [ 1 131.Comparison of the behavior in GC of TMS derivatives of steroids with that of the corresponding trifluoroacetyl derivatives has shown that the FID is generally more sensitive to the TMS ethers [379],but that the trifluoroacetates have much shorter retention times on OV-17and OV-1columns [ 11321. Steroid glucuronosides may be analyzed by GC after conversion to TMS ether methyl esters [508,1102].First, the carboxyl group is esterified with diazomethane in methanol-. diethyl ether and, after purification of the ester by TLC, the hydroxyl groups of the sugar are trimethylsilylated with HMDS and TMCS in pyridine. Another possibility is the conversion of the glucuronosides to the tri-0-acetylated methyl esters 12301. The dimethylsilyl (DMS) ethers, which generally give more intense molecular peaks in GC-MS than the corresponding TMS ethers [487],may be made by treating 0.5-2.0 mg of steroids with 50 p1 of tetramethyldisilazane and 50 p1 dimethylchlorosilane reagent for 3 h at room temperature [ 11011 or by warming a mixture of < 2 mg of steroids in 1 ml ethyl acetate with 20 p1 bis(dimethylsily1)acetamide (BDSA) at 50°C for 1 h [487],or by simply leaving a solution of 1 mg of steroids in 1 ml BDSA at room temperature overnight [462]. The preparation of chloromethyldimethylsilyl (CMDMS) ethers, which are electroncapturing derivatives, requires only a 3-h incubation at room temperature of < 2 mg of steroidal alcohols with 50 p1 each of 1,3-bis(chloromethyldimethyl)-l,1,3,3-tetramethyldisilazane and chloromethyldimethylchlorosilane.The response of the 63Ni ECD to different (halogenomethy1)dimethylsilyl steroid ethers depends on the halogen atom [255].For the corresponding iodo, bromo, and chloro compounds, it is in the ratio of 50: 18:1. The CMDMS ethers are even more useful derivatives for combined GC-MS than the TMS derivatives [ 1211.The chlorine atom increases the separation between mono-, di-, and trihydroxysteroids and facilitates the interpretation of spectra by virtue of the abundant 35Cl and 37Cl isotopes. The relatively intense peaks at high mass, particularly the M+ - CH2C1 peak, permit the highly specific and sensitive determination of steroids by single and multiple peak monitoring techniques [ 1791.The pentafluorophenyldimethylsilyl (flophemesyl) ethers of steroids are stable, volatile, detectable at picogram levels with the ECD, and yield characteristic mass spectra with a strong molecular ion [738,739]. Another type of electron-capturing derivatives proposed for the GC of steroidal alcohols is the methyl haloacetone ketal [925].The ketals are made in two steps from the hemiketals. First, the steroids are kept overnight at room temperature in a 20-60% (vlv) benzene solution of 1,1,3-trichlorotrifluoroacetoneor sym-dichlorotetrafluoroacetone. After the reagents have been evaporated at 60"C,the hemiketals are converted to the cor-

PACKED COLUMNS

33

responding 0-methyl ethers by dissolving them in ethereal diazomethane at -10°C. After 6-8 h, the products are ready for GC. They have desirable properties, not only in GC, but also in MS. The most widely used ketosteroid derivatives are the 0-methyloximes (methoximes, MO), which may be prepared simply by heating 2 mg of steroids and 8 mg of 0-methylhydroxylamine hydrochloride in 0.5 ml of pyridine at 60" for 3 h [469]. Steroids containing several hydroxyl groups in the molecule are not heated, but kept at room temperature overnight [294]. The methoximes are stable derivatives which may be converted to methoxime acetates or to methoxime trimethylsilyl ethers (MO-TMS derivatives) by treating 1-mg samples of MO steroids with 0.2 ml BSA in the presence of 50-100 p1 of TMCS at room temperature for 3-5 h , if silylation of hindered groups is desired (see p. 31) 12811. In many cases, the syn and anti isomers of the methoximes are separated by GC as well as TLC [247]. The benzyl oximes (BO) are prepared by using 0-benzylhydroxylamine hydrochloride instead of 0-methylhydroxylamine, in analogy to the MO derivatives [231]. BO and BO-TMS derivatives have a higher molecular weight and greater effect on retention behavior of ketosteroids than the corresponding MO derivatives. As little as 5 pg of ketosteroids can be detected with the ECD, if they are chromatographed in the form of their 0-(2,3,4,5,6)-pentafluorobenzyloximes(0-PFBO) [595]. The derivatives are readily prepared from 5 ng of the steroid by heating it with 5 pg of a reagent, containing 50 mg 0-pentafluorobenzylhydroxylamine/mlpyridine and 10 pl pyridine at 65°C for 30 min. Again, syn and anti forms may be separated. Steroid ketones have also been chromatographed in the form of N,N-dimethylhydrazones (DMH derivatives) [1107]. These derivatives are easily prepared, but are much less stable than the methoximes on exposure to light and air. The ethylene thioketals, prepared by incubating 10 pg of ketosteroids with 100 pl of a reagent containing 7% (v/v) ethanedithiol and 1.7%(w/v) p-toluenesulfonic acid in glacial acetic acid overnight at room temperature, behave very well in GC, but are not often used [ 1 1921.

4.3. PACKED COLUMNS

One of the first stationary phases used in GC of steroids was the methylpolysiloxane SE-30, supported on silanized Gas-Chrom P. In a typical procedure [3 11, Gas-Chrom P (100-200 mesh) was soaked in conc. HCl, then washed with water and dried at 100°C for 4 h. Next, 100 g of this support was suspended in 700 ml of a 1%solution of dichlorodimethylsilane in toluene. After degassing and standing for 20 min, the suspension was filtered under suction and the solid was washed on the filter with ca. 500 ml of toluene, followed by ca. 500 ml of methanol. The support was dried at 100°C for 2 h, and then 60 g of this was added to 250 ml of a 1% (w/v) solution of SE-30 polymer in toluene. The degassed suspension was allowed to stand for 30 min and then filtered under suction until the foaming had diminished. The solid was then dried at 100°C for 2-4 h and finally packed into the chromatographic tube. Except for copper, metal tubing is as acceptable as glass tubing, but PTFE connections must be kept as short as possible. SE-30 is a nonselective stationary phase which, nevertheless, separates a variety of unmodified steroid hormones at 230°C on a column 6 ft. X 4 mm I.D. [367]. A nonpolar

34

GAS CHROMATOGRAPHY

stationary phase of great stability and capable of high reproducibility is the dimethylpolysiloxane JXR [1097]. Columns, 18 ft. X 1/8 in. O.D., packed with 3% JXR on 100-200 mesh Gas-Chrom Q, gave 6000 theoretical plates when TMS ethers of androstane, pregnane, and cholestane derivatives were chromatographed at 21 5", 230", and 240°C. The temperature stability of Epon Resin 1001, a product of the reaction of bisphenol A and epichlorohydrin, is about the same as that of SE-30, but it is selective for ketosteroids and requires no inactivation of the support [ 11641. A column, 2 ft. X 4 rnm I.D., packed with 2% Epon Resin 1001 on 80-100 mesh Diatoport S and operated at 235"C, exhibited greater retention of ketosteroids than of hydroxylic steroids. Another stationary phase exhibiting high temperature stability, low adsorption on the support, and selectivity for ketosteroids is the silicone nitrile elastomer XE-60 [414]. On a column, 6 ft. X 1/8 in. O.D., packed with 3% of this phase on 80-100 mesh siliconized Diatoport S and operated at 262"C, acetates as well as TMS ethers of ketosteroids had greater retention times than the corresponding derivatives of nonketonic steroids. When the same column was used with 1% of the polyester HI-EFF-8BP instead of the silicone polymer, the derivatives of C19 and Czl steroids were also well separated at 242°C. Creech [208] reported quantitative resolution of such mixtures with the aid of 2% neopentyl glycol succinate (NGS) in 6-ft. X 4-mm columns, operated at 210-215°C. Treatment of the silanized support with a small amount of a polar stationary phase, such as Versamide 900 (a long-chain linear polyamide), Ucon 50-HB-2000 (a polypropylene glycol), or a polyester, before coating it with a silicone gum not only blocks remaining adsorption sites on the support, but also imparts a slightly polar character to the column [527]. By coating 25 g silanized Gas-Chrom S, 100-120 mesh, with a solution of 2 g SE-30 and 1 g NGS in 150 ml toluene and 50 ml chloroform, Nair et al. (7601 obtained a packing material which, in an 8-ft. X 4-mm I.D. column at 205"C, served to separate the

TABLE 4.2 RELATIVE RETENTION TIMES OF STEROIDS AND THEIR CORRESPONDING TRIMETHYLSILYL ETHERS, CHLOROMETHYLDIMETHYLSILYL ETHERS, HEPTAFLUOROBUTY RATES, AND CHLOROFLUOROACETATES ON SE-30 TMCBA* 18541 Abbreviations: PC = parent compound; TMS = trimethylsilyl ethers; CMDS = chloromethyldimethylsilyl ethers; HFB = heptafluorobutyrates; CDFA = chlorodifluoroacetates. Compound

PC**

Diketones 4-Androstene-3,17-dione 1,4-Androstadiene-3,17-dione 4-Pregnene-3,20-dione

1.44 1.72 2.17

-

2.85 2.85 D§% 2.54 3.80 4.72

2.60 2.55 3.05 1.67 3.38 4.56

Monohydroxy compounds SorCholestan-3p-d SCholesten-3p-01 5,7Cholestadien-3p-o1

24p-Methyl-5,7,22(9p,lOor)-cholestatrien-3p-01 24p-Methyl-5,7,22-cholestatrien-3p~l 24p-Ethyl-5-cholesten-3p-ol

TMS***

CMDS**

HFB§

CDFA§§

1.98 1.87 D D D 3.30

3.79 3.62 D D D 6.21

-

5.81 5.18 6.54 3.72 7.27 9.27

PACKED COLUMNS

35

TABLE 4.2 (continued) Compound

PC**

TMS***

Monohydroxymonoketones 3a-Hydroxy-5a-androstan-17-0ne 3P-Hydroxy-5a-andro st an-17-one 17p-Hydroxy-5a-androstan-3-0ne 30-Hydroxy-5P-androstan-1 7-one 3p-Hydroxy-5-androsten-17-one 17a-Hydroxy-4-androsten-3-0ne 17p-Hydroxy-4-androsten-3~ne 3p-Hydroxy-Sa-pregnan-2O-one 3a-Hydroxy-SP-pregnan-20-one 20a-Hydroxy4-pregnen-3-one 2Op-Hydroxy-4-pregnen-3-one 3P-Hydroxy-5-pregnen-20-one 3-Hydroxy-l,3,5( lO)-estatrien-l7-one

0.97 1.00 1.19 0.88 1.01 1.57 1.64 1.49 1.33 2.65 2.33 1.54 2.05

Monohydroxydiketones 3a-Hydroxy-5a-androstane-ll,17-dione 3or-Hydroxy-5p-androstane-ll,17-dione 17a-Hydroxy4-pregnene-3,20-dione

CMDS**

HFB§

CDFA§§

0.46 0.6 1 0.60 0.5 3 0.6 1 0.77 0.96 1.03 0.88 1.95 1.75 1.02 0.89

1.43 1.94 2.22 1.61 1.89 2.41 3.01 2.82 2.40 5.33 4.79 2.84 2.48

0.36' 0.5 1 D 0.4 1 0.47 0.6 1 0.83 0.80 0.65 1.61 1.37 0.78 0.52

0.84 1.16 1.28 0.98 1.05 1.44 1.78 1.74 1.51 3.22 2.85 1.67 1.22

1.59 1.51 -

0.78 0.93 2.29

2.27 2.40 5.90

0.58 0.69 2.03

1.33 1.70 1.98

1.01 1.02 1.10 1.58 1.60

2.91 3.31 3.93 6.98 6.28 7.22 4.90

0.25 0.26 0.34

0.58 0.53 0.65 0.36

1.03 1.09 1.33 2.33 2.18 2.46 2.58

Dihydroxy compounds Sa-Androstane-3~~, 17pdioI 5-Androstene-3p,17adiol 5-Androstene-3p,l7pdiol 5a-Pregnane-3p,20p-diol 5p-Pregnane-3~1,20a-diol 5-Pregnene-3p,20a-diol 1,3,5(10)-Estratriene-3,17p-diol

2.34

0.43 0.48 0.53 1.oo 0.87 1.09 0.70

Dihydroxymono ketones 3p,l Ip-Dihydroxy-5a-androstan-17-one 3a,llp-Dihydroxy-5p-androstan-l7~,ne 3,17p-Dihydroxy-l,3,5( lO)-estratrien-l6-one

2.90 2.42 4.38

1.95 1.48 1.52

8.76 6.84 9.84

0.77 0.57 1.06

3.71 2.95 4.48

-

1.06 1.52 1.26

15.22 12.10 19.79

0.42 D 0.46

3.06 D 3.66

Trihydroxy compounds 5-Androstene-3&16a,17a-trio1 5p-Pregnane-3~~,17a,2Oa-triol 1,3,5( 1O)-Estratriene-3,16~~,17p-trioI

-

-

*Mean of three determinations; data presented here are relative to cholestane = 1.00. **Cholestane retention time: 5.2 min; column temperature: 228°C. ***Cholestane retention time: 14.6 min; column temperature: 205°C. kholestane retention time: 21.5 min; column temperature: 195°C. S§Cholestane retention time: 11.2 min; column temperature: 210°C. §@D means that the compound decomposes.

36

GAS CHROMATOGRAPHY

TMS and TFA derivatives of Cla and CI9 steroids. The combination of stationary phases combines the fractionation according to molecular weight by the nonselective, nonpolar silicone rubber with the selectivity of the polar ester phase. An improved version of this binary mixture, in which tetramethylcyclobutanediol adipate (TMCBA) replaces NGS, is stable at temperatures up to 25OoC for four days or 240°C for seven days [854]. Table 4.2 shows the relative retention times of a number of steroid derivatives, as determined with both argon-ionization and electron-capture detectors on a 6-ft. X 5-mm I.D. column at the temperatures given in the footnotes. The derivatives are not only better separated, but also easier to detect than the parent compounds. Several other combinations of stationary phases were tried by Touchstone et aE. [ 10691. Best results were obtained with the following procedure: A solution of 10 g QF-I and 3 g SE-54 in 100 ml dichloromethane was mixed with 100 g Gas-Chrom Z, 80-100 mesh, the solvent was evaporated, and the packing was dried in a vacuum oven at 100°C for 18-20 h.

I

OF 1-5 L 45-0

3.75 1.25

2.5 2.5

1.25 3.75

I

0

5

* I . Combination

Fig. 4.1. Retention behavior of steroids on combination columns. (Reproduced from J. Chromatogr., 29 (1967) 237, with permission; [1072].)

COATED CAPILLARIES

31

Columns, 6 ft. X 4 mm I.D., were packed with this material and operated at 255°C. In another series of experiments [ 1068,10721, 5% stationary phase on Gas-Chrom Q was tested on 75 steroids belonging to four classes. The selective substrate QF-1 was combined with the nonselective methylsilicone L-45 in various proportions to make a total of 5 g of stationary phase per 100 g of support. At a column temperature of 250"C, the rete.ntion times relative to estrone were determined and then plotted, as shown in Fig. 4.1 [ 10721. The separation, R 3, of two steroids can be calculated from the expression R3

= PlR1 +P2R2)I(P, +PZ)

where R and R z are the retention times of the two steroids, and P I and Pz are the percentages of the two stationary liquids. When the more thermostable OV substrates became available, OV-210 was substituted for QF-1, and OV-1 was used instead of L-45, giving similar results with 5% mixed stationary phases on Supelcoport, 80-100 mesh, at a column temperature of 260°C [ 10621. Mueller et al. [749] have evaluated six liquid phases of the OV series, coated in 3% concentration on Chromosorb W-HP, 100 mesh, for optimal retention times and molar responses in the chromatography of steroid hormones. They concluded that the cyanopropylmethyl-phenylmethylsilicone OV-225, used at 24OoC, is best for the uncombined steroids, whereas OV-101, used at 21OoC, is the preferred stationary phase for steroid heptafluorobutyrates and perfluorooctanoates. An interesting suggestion is the use of steroid derivatives as stationary phases for the GC of steroids [558]. Liquid-crystalline esters of cholesterol, such as 1%cholesteryl benzoate or cholesteryl p-phenylbenzoate, produced promising separations on the basis of molecular shape. Another interesting possibility is the so-called dense-gas chromatography [686]. Gases under high pressure may have solvent properties useful for chromatography. Thus, COz at 500 atm and 40°C separated various sterols on a 15% Ucon column. Finally, the use of steam as the mobile phase in GC should be mentioned [52]. Steroids that tend to tail in ordinary GC showed better peak shapes and shorter retention times on columns such as 1% XE-60, a polar silicone elastomer, at 230°C when steam was used as the carrier gas instead of a permanent gas. The FID is suitable for such work.

4.4. COATED CAPILLARIES The high separating efficiency of wall-coated open tubular columns makes them very desirable for GC of the complex steroid mixtures in biological samples. However, several problems have prevented this technique from being exploited for steroids until about 1970. The coating must be continuous and thermostable, because irreversible adsorption and catalytic activity on the capillary walls is detrimental to the analysis of nanogram amounts of steroids. Also, the sample must be in concentrated form to prevent stripping of the coating, detector noise, loss of sensitivity, and other problems associated with the detection of minute amounts of steroids in the presence of large amounts of solvents. The most effective way of concentrating dilute steroid samples for capillary column chromatography is to introduce them through a forecolumn packed with Apiezon L grease on a diatomaceous earth support [794].

38

GAS CHROMATOGRAPHY

Glass capillaries are sufficiently inert for steroid chromatography and may be coated uniformly with thin films of appropriate stationary liquids. Their inner walls are first etched and then treated with silylating agents possessing selective groups that match the stationary phase: a mixture of HMDS and TMCS for nonpolar silicone phases, phenyltrichlorosilane for methylphenyl silicone liquids, and allyltrichlorosilane - which is subsequently oxidized - for more polar phases. NovotnL and Zlatkis [796] coated glass capillaries, 25-70 m X 0.2-0.5 mm I.D., pretreated this way, with 1-5% toluene solutions of SE-30, LC-l,OV-lOl, OV-17, and OV-225. Such columns are claimed to have efficiencies varying from 2500 to 4300 theoretical plates per meter, depending on the stationary phase, and are suitable for TMS, MO-TMS, and HFB derivatives of steroids at programmed temperatures up to 270°C. In an evaluation of stationary phases with adequate temperature stability for capillary column chromatography, based on the resolution of model mixtures of steroids, SE-30, SP-400,0V-101,0V-17,0V-210,Dexsil-300 GC, and Polyimide received high scores [797]. Vollmin [1128] has demonstrated the resolution attainable by the use of glass capillary columns, 20 m X 0.3 mm, coated with SF-96 or OV-101, in GC with programmed temperatures. Fig. 4.2 shows a chromatogram of nineteen TMS derivatives, obtained with a SF-96 column, which was heated to 180°C in 10 min, then by 3"/min up to 230°C in ca 0.5 h. Rutten and Luyten [914] have made a detailed study of the pretreatment of glass capillary columns by etching, silanization, and wetting with surfactants. Etching is not only unnecessary for the uniform spreading of apolar phases, such as SE-30 or OV-101, but actually interferes with surface deactivation by silanization. For the latter, a 5: 1 mixture of HMDS and TMCS, filling 25% of the glass capillary, was passed through the column. The most durable columns were obtained by sealing both ends and keeping them at 200°C for a few days. The most convenient pretreatment consists of quickly passing enough of a 1% solution of surfactant to fill 15% of the capillary through the column, which is then dried with a stream of nitrogen, rinsed with the solvent, and then dried again. Suitable surfactant solutions are Gas-Quat L (trioctadecylmethylammonium

Fig. 4.2. GC separation of TMS derivatives of steroid reference compounds on a glass capillary column, coated with SF-96. 1 = Androsterone; 2 = etiocholanolone; 3 = dehydroepiandrosterone; 4 = 1 l-ketoandrosterone; 5 = 1 I-ketoetiocholanolone; 6 = epitestosterune; 7 = testosterone; 8 = pregnanolone (internal standard); 9 = 1I-hydroxyandrosterone; 10 = 1I-hydroxyetiocholanolone; 11 = 16-hydroxydehydroepiandrosterone; 12 = allopregnanediol; 13 = pregnanediol; 14 = pregnenediol; 15 = 16-hydroxypregnenolone; 16 = estriol; 17 = pregnanetriol; 18 = pregnenetriol; 19 = pregnanetriolone. (Reproduced from Chromutogruphiu, 3 (1970) 235, with permission; [ 11281.)

COATED CAPILLARIES

39

bromide) or benzyltriphenylphosphonium chloride (BTPPC) in dichloromethane and Kalignost (KGn, sodium tetraphenylborate) in acetone. After repeating the pretreatment, if necessary, a static or dynamic coating procedure (see below) can be used. In contrast to the nonpolar stationary phases, the polar liquids do not spread on the glass surface unless it has been suitably prepared. The deactivation of the surface is apparently less important when polar stationary phases are used than in the case of nonpolar phases. A capillary tube made of soda-lime glass is readily etched by filling it with dry hydrogen chloride, sealing it at both ends, and then heating it at 350°C for 1-2 h. Such tubes may then be coated with 0.25% solutions of OV-225,OV-210, or OV-17 in dichloromethane by the static method [ 141. The effects of various treatments on the surface properties of glass capillaries have been studied in detail [15]. Instead of etching the glass capillaries, German and Horning [353] and German et al. [354] have conditioned the walls by adding very fine silanized silica particles to the stationary liquid. This produces a thermostable film that does not break up into micro droplets when the column is repeatedly heated and cooled. The chromatographic apparatus of these authors provides for the evaporation of the sample in a precolumn, maintained at 250"C, which vaporizes the sample before it is split, traps nonvolatile substances, and prevents adsorption and decomposition of sensitive steroids. The inlet system, which is shown in Fig. 4.5, keeps split ratio and column flow-rate constant [353]. To reduce adsorption, a glass capillary, ca. 70 m X 0.3 mm I.D., was silanized by forcing a plug of 5% dimethyldichlorosilane in toluene through the column at room temperature. The column was rinsed, first with toluene and then with methanol, and dried with a stream of nitrogen. Immediately before the dynamic coating, the capillary was wetted by passing chloroform through it. The coating suspension, prepared from 2 g Silanox (Grade 101, particle size 6-10 pm) in 100 ml chloroform containing 0.2 g BTPPC and 0.5 g SE-30, was then passed through the column at a rate of 5 cm/sec in the form of a plug occupying cu. 25% of the column. Subsequently, nitrogen was blown through the column at room temperature for 3 h. Then, an additional plug - again occupying ca. 25% of the column - of 2 g SE-30 in 100 ml isooctane was passed through the column at a rate of 2 cmlsec, and this was followed by another 3-h period of drying with a stream of nitrogen. The column was conditioned with a flow of nitrogen at the rate of 10 ml/min by raising the temperature at a rate of l"/min to 200°C. After at least 6 h at 200"C, the flow-rate was lowered to 2 ml/min and the temperature was increased at a rate of l"/min to 300°C. After 2 h at 300"C, the column was ready for use. For the separation of TMS, MO-TMS, and BO-TMS derivatives of steroids, 30-m columns were operated at an initial temperature of 200°C, rising at the rate of l"/min, with gas flow-rates as shown in Fig. 4.3. Such columns have efficiencies of 42,000-48,000 theoretical plates (1400- 1600/m) with capacity ratios of 1.2-1.5, and give reproducible results over periods of several months, if maintained at 200°C when not in use. Silanox 101 is also suitable for coating capillary columns with polar stationary phases, but the dynamic two-step technique is not satisfactory [ 11 111. Durable, thermostable columns with efficiencies of 1700-2400 theoretical plates per meter were prepared by coating glass capillaries, 20 m X 0.3 mm I.D., with either PZ-176, HI-EFF-8BP, SILAR-SCP, OV-17, or SP-2401 by the following method. First, 1 ml of a sonicate of 0.1 g of polar phase and 1.O g Silanox 101 in chloroform was drawn through the tube at the rate of

GAS CHROMATOGRAPHY

40 PRECOLUMN - INLET SPLITTER inject ion I

Pyrex tube 3,4 mm I

precolumn flow control

14

10% SE-30 on Gas-Chrom P 100-120 mesh, A W S 1 % SE-30 on Gas-Chrom P 100-120 mesh, A W S

glass Wool

capillary column 0 3 mm I D 'vent

W diluent flow

I

column inlet pressure control

I

cappillory column

Fig.4.3. Capillary column inlet system. The precolumn flow is flow controlled. PC = pressure controller; FC = flow controller; FI = flow indicator (mass flow meter). The flow-rate determines the split ratio when the pressure at the head of the capillary column is held constant. The capillary inlet is under pressure control.(Reproduced from J. Chromatogr. Sci., 11 (1973) 78, with permission; [353].)

6

I

I

10

20

I

30

40

50

Time ( m i n )

Fig. 4.4. Separation of the TMS derivatives of four isomeric cholestanols and stigmasterol plus an internal reference compound on a 20-m PZ-176open-tubular glass capillary column, temperatureprogrammed 1"C/min from 200°C. 1 = SpCholestan-3p-ol; 2 = Spcholestan-3a~I; 3 = Sacholestan-3cu-ol; 4 = Sasholestan-3P~l; 5 = stigmasterol; 6 = cholesteryl butyl ether. (Reproduced from J. Chromatogr., 99 (1974) 108,with permission; [ I l l l ] . )

INSTRUMENTATION

41

5-8 cmlsec and then nitrogen was blown through it for 2 h. Then a static method was used to apply a second coat of stationary phase. For this, 72 mg of the polar liquid was dissolved in 40 ml of acetone in each case, except HI-EFF-8BP, where dichloromethane was used instead of acetone. After the removal of dissolved gases by distilling off 10 ml of solvent, the capillaries were filled with this solution under reduced pressure. One end was then sealed with water glass and the other end was attached to a vacuum pump. Slow removal of the solvent by evaporation at room temperature took 36-48 h. The columns were conditioned by raising the temperature under nitrogen flow at the rate of l"/min up t o the maximum temperature of 250°C in all cases, except HI-EFF-8BP (230°C) and PZ-176 (300°C). A sample chromatogram is shown in Fig. 4.4.

4.5. INSTRUMENTATION At the present time, the most frequently used detectors for steroid chromatography are the FID and the ECD [708]. The FID has the advantage that it is relatively insensitive to temperature changes during analysis and to minor structural differences in the steroids and that it has a large linear range. When oxygen is used instead of air to support combustion of hydrogen, the sensitivity of the FID is increased 2.7-3.5 times, and as little as 1 ng of steroids can be accurately determined [ 11861. When used with capillary columns, where the flow-rate is inadequate for the FID, the detector is modified t o allow for the introduction of nitrogen to make up for the deficit [l 11 I ] . The ECD is more sensitive than the FID, but more difficult to use [729]. The use of 63Ni as a source of thermal electrons makes the ECD usable at temperatures above 300°C. This not only obviates contamination but also allows operation at optimum selectivity for certain steroids, especially in the pulsed mode. When it is necessary to collect effluent fractions from a gas chromatograph, a stream splitter may be used to divert an aliquot to the detector. The rest of the effluent is quantitatively condensed by passing it through a U-tube, which is immersed in liquid nitrogen [125]. Good recoveries have also been obtained by connecting a silanized glass tube, 10 mm X 2 mm I.D., plugged with silanized fiber glass, filled with KBr, and kept at 130"C, to the outlet [372]. The KBr can be used for infrared (IR) spectrophotometry, but for qualitative analysis even glass capillary pipettes, attached to the outlet at room temperature, are adequate for collecting steroids [lOOl]. For the preparation of up to 1 mg of pure sterols, a chromatographic column, 1.8 m X 6 mm I.D., packed with 3% QF-1 on Gas-Chrom Q and operated at 230°C was used [642]. The desired fractions were collected in capillary melting point tubes, which were attached to the effluent end of the stream splitter with a PTFE sleeve.

4.6. GAS CHROMATOGRAPHY-MASS SPECTROMETRY COMBINATION The combination of gas chromatography with mass spectrometry (GC-MS) has provided the steroid chemist with one of the most powerful tools for the qualitative analysis of biological extracts. Because the structural features affecting Chromatographic

42

GAS CHROMATOGRAPHY

behavior and fragmentation patterns are quite different, the likelihood of two different steroids showing the same propefties in the two tests is very small, and the association of a particular retention time with a particular mass spectrum is convincing evidence of identity. Applications of the GC-MS combination to steroids have been reviewed by Brooks and Middleditch [ 1221. The mass spectrometer may be used to identify unknown steroids in the column effluent, to detect inhomogeneity in chromatographic peaks, to determine the abundance and distribution of stable isotopes in metabolites, and to act as a highly sensitive and specific detector by single-ion monitoring [687]. Multiple ion detection, i.e., the monitoring of several selected fragment ions, makes the analysis even more specific and accurate. Under the name mass fragmentography this mode of detection has greatly enhanced the value of capillary columns [706,707]. A diagram of such a combination is shown in Fig. 4.5 [707]. The capillary column is connected to an all-glass solid injector on one end, and to a special adapter, shown in detail in the upper right corner of Fig. 4.5, at the other. An extra supply of helium maintains the flow-rate required by the two-stage jet separator. The low background permits mass spectra of as little as 10 ng of a steroid to be recorded. The amount of information generated by the GC-MS combination in a very short time is so large that the data are best processed by on-line or off-line computers. A computer

Fig. 4.5. Scheme of the combination between solid injector, glass capillary column, and separator of the mass spectrometer. Inset: Enlargement of the adapter between column and separator. A = Adapter between column and separator; C = capillary column, 20 m X 0.25 mm I.D., coated with SE-30; ES = extra helium supply; injection = septum for sample injection; F = flow meter; IS = ion source of the mass spectrometer; L = top leak by the use of a metal capillary tube; R = flow regulator; Sep = two-stage jet separator; T = thermo-shrinkable PTFE tube; V1 = needle valve on the GC Line; V2 = needle valve for extra flow supply (about 30 ml/min). (Reproduced from J. Chromatogr. Sci., 11 (1973) 608, with permission; [ 7071.)

QUANTITATIVE AND RADIOCHEMICAL METHODS

43

can be programmed to produce a fragment ion chromatogram, i.e., a plot or matrix list of the intensities of ion current for a few selected ions vs. time [881]. From the tape recording the computer can also locate certain peaks in the chromatogram, suggest structures for unknown steroids, and integrate peak areas for quantitative analysis, even if the chromatographic separation is incomplete [ S 1,8821. Derivatization plays an important role in GC-MS, not only because most steroids are best chromatographed in the form of derivatives, but also because the various types of adducts influence the mode of fragmentation and provide additional mass spectrometric information [ 11l o ] . Methyl ethers are particularly useful for quantitative analysis by single-ion monitoring, because their base peak in the mass spectrum is usually the molecular ion [ 1221. The most commonly used derivatives are the TMS ethers [30], but the DMS ethers give more intense molecular peaks [487]. The CMDMS ethers not only separate better than the TMS ethers, because they have longer retention times, but they also facilitate the interpretation of mass spectra because ions containing the CMDMS group are easily recognized by the two abundant natural isotopes of chlorine [ 1211.

4.7. QUANTITATIVE AND RADIOCHEMICAL METHODS Yasuda [ 1 1871 has made a very thorough study of the problems in quantitative analysis of steroid hormones by GC. Various factors were evaluated by calculating a relative peak area ratio (peak area of any steroid/peak area of an internal standard). Although internal standards can be used to eliminate some of the experimental errors [ 10301, some difficulties - mainly due to adsorption remain. Of the derivatives studied, the TMS ethers gave the most satisfactory recoveries. Huck [483] has suggested that steroids can be accurately determined without integration of the peak areas by calculating the product of peak height, breakthrough time, and a correction factor (ratio of molecular weight to carbon number). Most radiochemical methods involve the determination of the specific activity, i.e., the ratio of radioactivity to mass. This was obtained by splitting the effluent stream from a gas chromatograph so that 10%passed through the mass detector and 90% through a transfer line, which was heated to 300"C, leading to a combustion tube, which contained CuO wire and Mg(C104)2 (for I4C) or CuO wire and steel wool (for 3H) and was heated to 630"C, and thence to a proportional counter [426,103 1,10321. In another approach, the effluent was split in such a way that one part passed through a detector and twenty parts through a tube at 220°C into a series of glass cartridges, loosely packed with glass wool, which were standing in a fraction collector [ 1971. The individually collected steroids were then eluted with toluene containing scintillation phosphor. Good recoveries were also obtained by filling the cartridges, made from 35-mm X 7-mm I.D. glass tubing plugged with glass wool, with 3% DC-200 silicone on Florisil [245]. The best recoveries were obtained with a two-stage trapping system [777]. The effluent, 10%of which was diverted to the FID, was first passed through 2 ml toluene in a glass U-tube and then through a 50-mm X 6-mm glass tube packed with 1% OV-1 on Gas-Chrom Q, which was plugged with glass wool on both ends and had its contents moistened with toluene. The traps were attached to the outlet of the chromatograph prior to the appearance of carrier steroids in the chromatogram. Both traps were washed with toluene and the washings were collected ~

44

GAS CHROMATOGRAPHY

and counted in scintillation vials. Accurate counting of samples with low 14C activity requires trapping of 14C02 from combustion (see above) in ethanolamine and scintillation counting [427]. Using earlier versions of the trapping technique and of the combustion and continuous flow method, Karmen er al. [534] devised a method for measuring steroids by a combination of radioassay and GC, which they called derivative ratio analysis. It is based on the addition to the substance to be determined, e.g., testosterone, of a known amount of an internal standard having a common functional group, e.g., the hydroxyl group in methyl' ricinoleate, and the preparation of a radioactive derivative, e.g., by acetylating the mixture with [14C]-acetic anhydride. From the ratio of the total radioactivity, C,of the unknown, u , and the standard, s, the amount of the unknown, U, can be calculated by substituting the known amount of standard, S , in the equation

C,

M.W.,

n,

C,

M.W.,

n,

u = s - x -x -

where M. W. is the molecular weight and n the number of derivative groups per molecule in the unknown and the standard. Another interesting isotope method is the twin ion technique [ 1061. It is a method for tracing the metabolism of a compound that requires neither carrier steroids nor purification to constant specific activity. It does require starting compounds that are labeled with both 14C and D. The D-labeled substrate is diluted with unlabeled substrate until the molecular ions of both show equal intensity in the mass spectrum. The metabolites will then be characterized by twin ions and can be assayed by liquid scintillation counting of the l4Clabeled species.

Chapter 5

Relations between structure and chromatographic mobility 5.1. RM VALUES IN LIQUID CHROMATOGRAPHY Correlations between the structure and mobility of steroids are necessary for describing and explaining their chromatographic behavior and useful for selecting appropriate experimental conditions. However, the complexity of the constitution of steroids and the rudimentary understanding of the physico-chemical factors involved in chromatographic processes, together with all the possible interactions in chromatographic systems and the problems of reproducing experimental conditions, have largely prevented earlier hopes of using chromatography for structure elucidation [ 1291 from being realized. It will be recalled that RM = log(l/RF - 1) and that RM is a function of the free energy required to transport a solute molecule from one phase to another. Inasmuch as RM is made up of the free energy contributions of the individual groups of which that solute molecule is composed, it is assumed that the potential differences of the individual groups contribute additively to the chromatographic mobility of the solute. The value represents the change in RM resulting from the substitution of a group,g, for H; the AR~M, value is the change resulting from some other reaction (e.g., reduction or acylation); and the ARM^ value is the result of changing the solvent system. The literature after 1964 contains only a few references to the use OfRM values in liquid-liquid partition chromatography. Cathro et al. [ 1501 have determined a large number of R F and R M values for CIS,CI9,and C21 steroids by PC with various solvent systems containing aqueous methanol. The ARM^ values for functional groups at positions C-17, (2-20, and C-21 of the pregnane side chain were similarly recorded by Schneider and Lewbart [936]. An interesting method for determining the partition coefficients for a series of testosterone esters is based on the determination of their R M values in reversed-phase partition TLC [78]. Similarly, the partition coefficients for a number of steroids were determined from their retention time, t R , in a HPLC column [479]. The more polar liquid phase was held stationary on porous particles of diatomaceous earth, while the less polar phase was pumped through the column. The partition coefficient, K , was calculated from tR = t ( I + K q )

where t is the retention time of the mobile phase and q is the volume ratio of stationary/ mobile phase. In liquid-solid chromatography, too many irregularities in the correlation between steroid structure and RM are observed to make this index useful, even for descriptive purposes [ 175,7801. The deviation from “typical” behavior depends on the nature of the liquid and the solid. The most “atypical” solvents are alcohols and ethers, and the most “atypical” adsorbents are silica and silicates. “Atypical” chromatographic behavior has been demonstrated in the pregnane series, where it was exploited for the separation of certain members [781]. More “typical” behavior of several steroid classes has been observed by avoiding alcoholic solvents [406]. 45

46

RELATIONS BETWEEN STRUCTURE AND CHROMATOGRAPHIC MOBILITY

5.2. RM VALUES IN GAS CHROMATOGRAPHY

Knights and Thomas [ 172,220,582,583,1054] have introduced the RM concept into the gas-liquid partition chromatography of steroids. The RM value for GC can be calculated from the retention time, fR , by determining t o , the time interval between injection and the appearance of the air peak, and by using the expressions fR = to(l/RF - 1)

and RM = log(l/RF - 1) Since the relative retention time, r , is the ratio of the retention times of two substances, log r is the difference between them. The logarithm of the relative retention time of a steroid is made up of the additive contributions of the individual substituent groups, g, and the logarithm of the retention time contribution of the nucleus,N log r =

AR,ng

+ log rN

The values for ARM^ and ARMr depend on various experimental conditions, such as the amount and nature of the stationary phase and the column temperature. The ARM, value for any functional group depends not only on its nature, position, and orientation, but also on the configuration of the nucleus and the presence of interacting groups in the same molecule. The relative retention times of 3-hydroxysteroids and their derivatives were found to be in the following order: 3-hydroxysteroids on QF-1: 3P(5a) > 3p(A5) = 3a(5a) = 3 4 5 0 ) > 3P(5P); 3-acetates on QF-1: 30(5a) > 3p(A5) = 3a(5a) > 3p(50) > 3a(5P); TMS ethers of 3-hydroxysteroids on QF-1: 3/3(5a) = 3/3(A5) > 3a(Sa) = 3/3(5/3) = 3a(5p); and TMS ethers on NPGA: 3/3(5a) = 3/3(A5) > 3a(5/3) > 3a(5a) > 3@(5P). The ARMr value is the change in log r resulting from a change in the nature of a functional group as a result of performing a chemical reaction. For instance, ketosteroids [ 1741 have been characterized by ARMr(redn.), obtained by reduction with LiAlH4 in diethyl ether [ 1731, or by ARM,.(l7/3 +. 17a), obtained by isomerization of 17P-acetoxy20-ketosteroids with methanolic KOH [ 1761. Knights [576] has used ARMr(hydroborafion) values to distinguish double bonds in sterols. The adducts are prepared by dissolving 1-5 mg of the sterol or its acetate in 1 ml diethyl ether and adding 5-10 mg LiAlH4. After dropwise addition of BF3 etherate until the vigorous reaction has ceased, the mixture is allowed to stand for 30-60 min. Then, 0.5-1 .O ml30% H202 is cautiously added, the mixture is shaken, and allowed to stand for 1 h. The adducts are isolated by extraction with chloroform and converted to TFA or TMS derivatives prior to GC. Trisubstituted double bonds give rise to a single product, but di- and tetrasubstituted double bonds and steroids containing more than one double bond produce several derivatives. Knights [577] has also made use of ARM^, the difference between log r for a sterol chromatographed in two different columns, containing either siliconized or polyvinylpyrrolinidone-coated Gas-Chrom P with HI-EFF-8BP as stationary phase. A list of RM values for forty-seven C L Band CI9 steroids, chromatographed on 1% SE-30 and on 1.5%

GROUP RETENTION FACTORS

47

QF-1has been published by Hara et al. [412], and some values for CI9 and CZl steroids appear in the paper by Heitzman and Thomas [425].

5.3. GROUP RETENTION FACTORS To correlate the structure of steroids with their behavior in GC, Brooks and Hanaineh [ 1 141 have made use of the group retention factor, k . The latter is simply the change in retention accompanying the introduction of a certain group into the steroid nucleus and, therefore,

logk

=

ARM^

To calculate k for that group, the relative retention time of a steroid containing it is divided by the relative retention time of the analog lacking it. Retention factors due to the introduction of hydroxyl and 0x0 groups into CI9-C2, steroids, obtained by chromatographing steroids on two silicone stationary phases, SE-30 and QF-I, at 200°C, are shown in Table 5.1. TABLE 5.1 RETENTION 1:ACTORS DUE TO THE INTRODUCTION OF HYDROXYL AND OX0 GROUPS INTO C,,-C,, STEROIDS [ 1141 Group

3u-Hydroxyl 3p-Hydroxyl

1la-Hydroxyl 1lp-Hydroxyl 17a-Hydroxyl 170-Hydroxyl

Other relevant groups

Sa-Hydrogen 5p-Hydrogen 5u-Hydrogen 50-Hydrogen AS -

3-0xO 20a-Hydroxyl 2Op-Hydroxyl 3-0x0

-

17- or 2 0 4 x 0 ll-oxo

-

204x0 17 - 0 ~ 0 174x0

-

3-0X0 20-Ox0

-

3-0xO A4-3-0xo

-

SE-30 No. of examples

3 4 8 3 2 2 2 2 4 2 3 4 6 3 1 5 4 4 2 5 2 6

QF-1 Retention factor Range

Mean

2.06--2.12 2.08-2.12 2.09-2.14 2.09-2.10 2.11-2.12 1.78-1.91 1.82-1.86 2.09-2.16 2.13-2.19 2.19-2.20 2.06-2.10 1.91-1.93 2.24-2.32 2.27-2.29 1.36 1.35-1.46 1.23-1.30 2.04-2.08 2.05 -2.09 1.87-1.91 1.88-1.90 2.82-2.90

2.09 2.11 2.12 2.09 2.12 1.84 1.84 2.13 2.16 2.20 2.08 1.92 2.28 2.28 1.36 1.41 1.27 2.06 2.07 1.88 1.89 2.85

No. of examples

Retention factor Range

3 3 7 3 2 2 2 1 3 2 3 4 5 3 1 5 4

3 2 5 2 5

Mean

3.14-3.24 3.18 3.60-3.62 3.61 3.46-3.66 3.58 3.15-3.19 3.17 3.32-3.44 3.38 2.47 2.47 1.95-2.16 2.06 3.02 3.02 3.10-3.15 3.13 3.22-3.27 3.24 3.10-3.17 3.13 2.74-2.78 2.76 6.7 1-7.00 6.84 7.31-7.47 7.39 2.60 2.60 2.33-2.67 2.53 1.75--1.94 1.88 5.02-5.29 5.19 5.63-5.78 5.70 4.18-4.33 4.28 4.54-4.56 4.55 10.64-11.65 11.2

RELATIONS BETWEEN STRUCTURE AND CHROMATOGRAPHIC MOBILITY

48 TABLE 5.2

RETENTION FACTORS DUE TO THE INTRODUCTION OF THE 1 1 4 x 0 GROUP INTO STEROIDS OF THE ANDROSTANE AND PREGNANE SERIES [ 1141 ~~~

Parent compound

Sa-Androstane-3,17-dione Sp-Androstane-3,lII-dione 3a-Hydroxy-Sp-androstan-l 'I-one

4-Androstene-3,17-dione Sa-Pregnane 5a-Pregnane-3,20-dione 3p-Hydroxyd a-pregnan-20-one 5p-Pregnane-3,20-dione 5a-Pregn-2-en-20-one 16a-Methyl-Sa-pregn-2-en-2O-one

Retention factor SE-30

QF-1

1.30 1.25 1.28 1.23 1.36 1.39 1.44 1.35 1.46 1.41

1.94 1.87 1.94 1.75 2.60 2.44 2.67 2.33 2.66 2.56

Although the k values for hydroxyl substitution at position 3a or 3p in both 5a-and 50-steroids are similar on SE-30, the equatorial hydroxyl steroids (3a,50 and 30,5a) are retained much longer than their axial epimers on QF-1 and are thus separable. The sterically hindered 1 1-hydroxyl groups give lower retention factors. On both phases, the k values for ketones decrease in the order A4-3> 3 > 17 > 20 > 11, but the differences between them are much larger on the more polar stationary phase, QF-1, evidently through a combination of polar and steric effects. Vicinal effects are also more pronounced on QF-1 than on SE-30. Hydroxy- and ketosteroids have generally similar retentions on SE-30, but on QF-1 the ketosteroids are more strongly retained than the hydroxysteroids, with the exception of the 1 1-ketones. The latter exhibit different k values in the androstane and pregnane series (Table 5.2). Brooks and Hanaineh [ 1141 also determined k values for the acetylation of 3-hydroxysteroids. Whereas the axial and equatorial hydroxyl groups gave constant values of 1.45 f 0.03 and 1.58 k 0.04, respectively, on SE-30, the situation was more complex on QF-1, where axial (3/3,50) and equatorial (3p,5a) compounds gave the same value of 1.75. Although the separation factors, i.e., the ratios of the retention of Sa-steroids/5fl-steroids, for a series of epimers are constant and equal on QF-1 and SE-30, there is a great difference in the character between the two stationary phases, SE-30 responding essentially to molecular skeletal size and shape, and QF-1 interacting selectively with substituent groups.

5.4. STEROID NUMBERS

Another way in which the relation between steroid structure and retention behavior in GC can be expressed is the steroid number, SN [ 11061. SN = S + F ,

+ . . . .F,

where S is the number of C atoms in the steroid nucleus and F1.. ..Fn are values charac-

STEROID NUMBERS

49

TABLE 5.3 STEROID NUMBERS AND RELATIVE RETENTION TIMES FOR REPRESENTATIVE STEROIDS DETERMINED AT 211°C WITH AN SE-30 PHASE [ 11061 Conditions: 6 ft. X 4-mm glass U-tube; 1% SE-30 on 100-120 mesh GasChrom P at 211°C; 20 p s i ; cholestane retention time, 13.5 min. ______ Steroid

RRT*

Steroid number ( S N

5a-Androstan-17-one Sa-Androstane-3,17-dione

0.18 0.39 0.48 0.49 0.40 0.48 0.57 0.85 0.81 0.38 0.38 0.37 0.37 0.36 0.37 0.36 0.4 1 0.37 0.46 0.48 0.6 1 0.60 1.10 0.66 0.89 0.66 0.62 0.66 0.62 0.86 0.78 2.00 1.98 1.80 2.14 2.64 3.12 1.88 1.66 2.6 1 2.71 2.24 2.21 2.12 1.95

21.3 23.9 24.6 24.7 23.9 24.6 25.1 26.5 26.3 23.8 23.8 23.7 23.7 23.6 23.7 23.6 24.1 23.7 24.4 24.6 25.4 25.3 27.4 25.6 26.7 25.6 25.4 25.6 25.4 26.6 26.2 29.4 29.4 29.1 29.6 30.3 30.9 29.2 28.8 30.3 30.7 29.8 29.8 29.6 29.3

4-Androstene-3,l’I-dione 17p-Hydroxy-4-androsten-3-one Sa-Androstane-3,16-dione 4-Androstene-3,16-dione 4-Androstene-3,11,17-trione 11a-Hydroxy-4-androstene-3,17-dione 1lp-Hydroxy-4-androstene-3,17-dione 19-Nor-4-androstene-3,17-dione 5a-Androstane-3p,l7pdiol Sa-Androstane-3a,l7p-diol 3p-Hydroxy-Sa-androstan-17-one 3a-Hydroxy-5a-androstan-1 ?’-one 5-Androstene-30,178-diol 30-Hydroxy-5-androsten-1 7-one 17a-Methyl-5-androstene-3P,17p-diol 5a-Androstane-3p,l60-diol Estrone Estradiol Equilenin Sp-Pregnane-3,2Odione 2l-Hydroxy-Sp-pregnane-3,20-dione 5a-Pregnane-3,20-dione 5a-Pregnane-3,11,20-trione 5a-Pregnane-3p,2Oa-diol 5a-Pregnane-30,200-diol 20p-Hydroxy-Sa-pregnan-3-one 3P-Hydroxy-Sol-pregnan-20-one 3~-Hydroxy-5a-pregnane-1lI 20-dione 4-Pregnene-3,20-dione Cholestdnol Epicholestanol Coprostanol Cholestan-3-one 4Cholesten-3-one Cholestanyl acetate Cholestanyl methyl ether Cholestanyl trifluoroacetate Cholestanyl trimethylsilyl ether Tigogenin Solanidan-30-01 7Cholesten-30-01 Desmosterol Cholesterol *Retention time relative to cholestane

so

RELATIONS BETWEEN STRUCTURE AND CHROMATOGRAPHIC MOBlLlTY

teristics for the functional groups present. The steroid numbers of cholestane and androstane being 27 and 19, respectively, a linear relationship between the steroid numbers, SN, and the logarithm of the retention times relative to cholestane, log r , is postulated. Using this relationship, all log r values determined under a given set of chromatographic conditions can be converted to SN values, either graphically or by substituting in an equation [ 10991. TABLE 5.4 STEROID-NUMBER CONTRlBUTlONS FOR REPRESENTATIVE FUNCTIONAL GROUPS DETERMINED WITH A NONSELECTIVE PHASE (SE-30) [ 11061 Functional group

Parent steroid*

Sene Sene 7-ene 24ene 3-one 3-one 3-one 3-one-l-ene 3-one-4-ene 11-one 11-one 16-one 1%one 20-one 3a-01 (a) 3p-01 ( e ) 3p-01 ( e ) 3p-01 ( e ) lla-ol ( e ) 11p-01 (a) 160-01 170-01 (sec.) 170-01 ( t e r f . ) 20a-01 2op-01 21-01 Aromatic B ring N Spiroketal 30-Trifluoroacetoxy ( e ) 3p-Methoxy ( e ) 3a-Trimethylsilyloxy (a) 38-Trimethylsilyloxy ( e ) 3p-Acetoxy ( e ) A/B cis A/B cis

(Cholestanol/cholesterol) (Cholestane/S-cholestene) (Cholestanol/7-cholesten-3p-ol) (Cholesterol/desmosterol) (Androstane) (Sa-Pregnane) (Cholestane) (Androstane) (Cholestane) (4-Androstene) (So-Pregnane) (Androstane) (Androstane) (5a-Pregnane) (Androstane) (Androstane) (Sa-Pregnane) (Cholestane) (Androstane) (Androstane) (Androstane) (Andros tane) (17a-Methylandrostane) (5a-Pregnane) (5a-Pregnane) (Sa-Pregnane) (Estrone/equilenin) (Solanidan-3p-01) (Tigogenin) (Cholestane) (Cholestane) (Cholestane) (Cholestane) (Cholestane) (Coprostane/choles tane) (Coprostanol/cholestanol)

Steroid number (F)** 0.1

0.0 0.4 0.3 2.6 2.6 2.6 3.3 3.3 0.5 1.1 2.3 2.3 2.0 2.3 2.4 2.4 2.4 1.9 1.7 2.3 2.4 1.7 2.2 2.0 2.1 1.o 0.4 1.3 1.8 2.2 2.5 3.3 3.9 4.2 -0.3

of the steroid nucleus for the reference compound used in the determination of the SN value. ***Structure Determined with the column and conditions described in Table 5.3.

STEROID NUMBERS

51

TABLE 5.5 STEROID-NUMBER CONTRIBUTIONS FOR REPRESENTATIVE FUNCTIONAL GROUPS DETERMINED WITH NGS AND QF-1 LIQUID PHASES [398] Functional group

Parent steroid

Sene Cholestanol/cholesterol 5-ene Cholestanyl methyl ether/cholesteryl methyl ether Sene Cholestanyl TFA/cholesteryl TFA Sene Cholestanyl TMS/cholesteryl TMS 3-one Cholestane 3-one DMH Cholestane 3-one-4-ene Cholestane 3-one4-ene DMH Cholestane Androstene 11-one (4-ene) Androstane 1%one SaPregnane 20-one Cholestane 301-01 (0) Coprostane 36-01 ( 0 ) 301-01 ( e ) Coprostane Cholestane 3p-01 (el 3a+l(u) TMS Cholestane 36-01 (u) TMS Coprostane 301-01 (e) TMS Coprostane Cholestane 3p-01 (e) TMS 3&trifluoroacetoxy (e) Cholestane Cholestane 3p-methoxy (el Cholestane 3p-acetoxy (e) 1lp-ol(u)-(4-ene) Androstene 17ct-o1(sec.) Androstane 17p-01(sec. ) Androstane 2001-01 So-Pregnane 200-01 SwPregnane 20p-01TMS Sa-Pregnane 5a-PregnanelSp-pregnane A/B cis Cholestane/coprostane A/B cis

NGS

0.4 0.2 -0.2 4.1 8.0 6.6 9.9 1.9 3.2 6.9 6.3 7.2 1.2 7.5 1.7 1.3 1.6 2.6 3.1 3.4 4.2 1.3 5.9 1.0 1.3 6.9 6.3 2.2 -0.3 -0.3

QF-1

-0.3 4.3 -0.5

-0.1 8.1 5.2 10.0 5.9 2.0 6.1 5.9 4.1 4.6 5.1 5.2 2.1 2.6 2.4 3.4 5.0 3.5 7.1 2.5 4.2 4.3 4.4 4.0 2.9 -0.3 -0.3

In contrast to ARM^ and k values, the SN values determined with a nonselective stationary phase, e.g., SE-30, are independent of small changes in experimental variables, such as flow-rate, amount of liquid phase, and temperature. Table 5.3 is a listing of some representative steroids with their relative retentions and steroid numbers. The F values derived from these SN values are presented in Table 5.4. Some variation in these values with temperature is observed when selective phases, e.g., NGS or QF-1, are used [398]. Table 5.5 shows the steroid-number contributions, F , for a series of functional groups, observed when representative steroids were chromatographed on 6-ft. X 4-mm columns of 1% NGS or 2% QF-1 at 222°C with an argon flow-rate of 40 ml/min. Although selective stationary phases give more structural clues, the contribution of the parent hydrocarbon to the SN value is more difficult to assess than when nonselective phases are used. Steroid numbers are useful in defining relationships based primarily on molecular weight

RELATIONS BETWEEN STRUCTURE AND CHROMATOGRAPHIC MOBILITY

52

and shape. For an index reflecting the number, nature, position, and stereochemistry of the functional groups, VandenHeuvel and Horning [ 11061 recommended the T value. It is defined as

T =

(tsf-tnf)/tnf

where ts’ is the relative retention time observed with a selective phase and f n f that observed with a nonselective phase. It is thus related to the ARM^ value mentioned earlier (Section 5.2). The T values in Table 5.6 were calculated from rn for SE-30 and tsffor NGS and TABLE 5.6 VALUES OF T DETERMINED AT 21 1”FOR REPRESENTATIVE FUNCTIONAL GROUPS WITH NGS AND OF-1 PHASES I1 1061 Functional group

3-one 3-one-4ene 17-one 3a-0l(u) 30-01( e ) 3p-ol-S-ene-(e) 3,17-dione 3,17-dione-4ene 3,20-dione(A/B cis) 3,20-dione(A/B trans) 3p,l7pdiol 3a,l7p-diol 3p,l7p-diol-Sene 3p,2Opdiol 3p,2Oadiol 3p-01-1%one 3a-01-17-one 3p-ol-17-one-5-ene 3p-ol-20-one 17p-ol-3-one-4-ene 2Op-ol-3-one 3,17-dione-4-ene-l lp-01 3,11,20-trione(A/B trans) 3-01-17-one, A aromatic 3-01-170-01, aromatic 3a-Trimethylsilyloxy (a) 30-Trimethylsilyloxy( e ) 30-Trifluoroacetoxy ( e ) 3p-Trifluoroacetoxy-Sene (e) 3p-Methoxy(e) 3p-Methoxy(e) 3p-Acetoxy(e) A/B cis *ME = Methyl ether.

Steroid

Cholestan-3-one 4-Cholesten-3-one Androstan-l7-one Cholestandcr-01 Cholestan-3p-01 Cholesterol Androstane-3,lIl-dione 4-Androstene-3,lIJ-dione Pregnane-3,20-dione 5a-Pregnane-3,20-dione Androstane-3p,l7pdiol Androstane-34,l 7pdiol 5-Androstene-3p,l7p-diol 5a-Pregnane-3@,20pdiol Sa-Pregnane-3a, 20ordiol Androstan-3p-ol-l’hne Androstan-3a-ol-17-one 5-Androsten-3p-ol-17-0ne 5a-Pregnan-3p-ol-2O-one 4-Androsten-17p-Ol-3-one Sa-Pregnan-20p-ol-3-0ne 4-Androsten-1 lp-ol-3,17-dione Sa-Pregnane-3,11,20-trione Estrone Estradiol Epicholestanyl TMS Cholestanyl TMS Cholestanyl TFA Cholesteryl TFA Cholestanyl ME* Cholestanyl ME Cholestanyl acetate 5a- and SpPregnane-3,20-dione

T value NGS

QF-1

2.1 2.9 2.5 1.9 2.2 2.5 12.1 15.6 9.7 9.9 11.8 10.7 12.5 9.4 10.6 11.0 10.0 12.1 9.5 16.5 10.6 42.0 34 .O 37.2 4.4 4.2 0.3 0.3 0.4 0.4 0.8 4.2

2.0 2.9 2.6 0.4 0.7 0.6 10.7 13.5 8.2 7.8 2.5 2.3 2.3 2.1 2.5 5.1 4.4 4.5 3.8 7.1 4.6 15.0 15.1 4.9 2.5 4.2 -0.1 0.8 0.7 0.2 0.2 0.7 0.4

__

OTHER INDICES

53

QF-I, respectively, all at 21 1°C. Distinct differences can be seen in the NGS column between T values of steroids, depending on the number of functional groups and the presence or absence of an aromatic ring or conjugated double bond. In the QF-1 column, differences are seen between steroids with hydroxyl and carbonyl groups and diastereoisomers . The utility of SN values was demonstrated by Nambara et al. in applications to a series of androstanones and their oximes [765] and to the SN contribution of the C/D ring fusion [767]. Fehir and Bodrogi [303] have applied the SN treatment to a number of hydroxy- and ketoandrostane derivatives and have distinguished between contributions to SN by addition (ASNg)and substitution (ASNr)of a group, both being called group numbers (ASN). An empirical correlation between the structure and retention index of sterol acetates has been published by Gassiot et al. [343]. 5.5. OTHER INDICES A parameter closely related to SN is the methylene unit (MU). In analogy to SN, it is a linear function of log r , which is the sum of the M U values for the steroid nucleus and the functional groups, and there is a linear relationship between SN and MU. Using either unbranched alkanes or C19, Czl, and C2, Sa-steranes as reference compounds and GC with a temperature program of l"/min, Horning e l al. [462] have characterized a large number of TMS and MC-TMS derivatives of steroids by their MUvalues on OV-1 and OV-17. The retention index, which for practical purposes is equal to M U X 100, is sometimes used for steroid correlations [ 10361, but Mori and Sat0 [742] have found that SN values show a smaller and more reliable temperature dependence. Vandenheuvel and Court [ 1097,10981 have calculated retention constants (RC)in order to make retention times independent of temperature. When nonpolar JXR columns (see p. 34) were used, remarkable agreement between most calculated and observedRC values was observed, but it was impossible to predict retention times on polar stationary phases accurately, owing to interactions between steroids and polar phases. Retention constants 2 and B for any steroid are obtained by determining its retention times, R l and R 2 , relative to Sa-cholestane at two absolute temperatures, T I and T2 (at least 20°C apart). These values are substuted into the expressions

lo3 X logR1 = A + lo3 X BT1-' and

A + lo3 X BT2-' which are solved for A and B . A and B are structure-specificconstants, independent of lo3 X logR2 =

both temperature and carrier gas flow-rate, but they must be obtained under standard conditions, and the retention times of steroids - which vary with the quantity injected require a correction. However, the lack of constancy in incremental factors, e.g., for the 1 la- and 1 10hydroxyl groups, has now forced Vandenheuvel [ 10941 to elaborate new correlations, based on two distinct sets of structural features of the steroid molecules: M-features,

-

54

RELATIONS BETWEEN STRUCTURE AND CHROMATOGRAPHIC MOBILITY

which affect the structure of Ring A, and G-features, which affect the remainder of the molecule. The sum of these two features, LR ,

is lo3 X the logarithm of a retention time that has been corrected for various deviations from “ideal” normal conditions. In a plot of LR vs. MR for steroids of the same group, some members were found to give points lying on a straight line (GR-normal) and others were found to have GR values considerably below those of the group (GR-odd). In addition to M, G , and L values, Vandenheuvel has presented a number of very useful micro procedures for reactions of members of the androstane [ 10941 and pregnane [ 10951 series and their derivatives [ 10961.

Chapter 6

Sterols If the vitamins D and the molting hormones, which are dealt with in Chapters 13 and 14, respectively, are disregarded, the sterols emerge as the most lipophilic and least reactive group among the steroids. This group is made up of homologous series with, usually, 27-30 carbon atoms and has many members differing from each other in the number and location of double bonds and in the juncture between Rings A and B. All natural sterols have an oxygen function at C-3 and a side chain of at least seven carbons at (2-17. Aside from articles in which the applications of specific chromatographic techniques to sterols are reviewed, there are no surveys of the analysis of sterols by chromatography, with possibly two exceptions. Subbiah’s brief paper [ 10191 deals with applications of chromatography to sterol balance studies, and an obsolete article [543] deals specifically with cholesterol and its esters.

6.1. LIQUID COLUMN CHROMATOGRAPHY

The most frequently used adsorbent in the conventional column chromatography of sterols and their esters is silica. For example, a mixture of up to 12 mg of free sterols is separated on a column, 1 m X 1 cm, prepared under 10 p.s.i. pressure from a slurry of 40 g silicic acid (Mallinckrodt, 100 mesh, “suitable for chromatographic analysis by the method of Ramsey and Patterson”) and 20 g Hyflo Super-Cel in 275 ml benzene and eluted with benzene at the rate of ca. 5 ml/h/fraction [319]. Klein et aZ. [570] have made a very thorough study of the behavior of sterol acetates on silica surfaces (see p. 3). For a given type of silica gel, elution orders of nuclear double bond isomers and side-chain homologs can be established. However, reversals in the order are frequently observed when the elution orders on silica gels with different pore diameters are compared [572]. Some destruction of 5,7-dienes occurs on silica columns. In the analysis of doubly labeled compounds with silica gel columns, isotope effects have been observed [571]. For instance, elution with 16% benzene in pentane effected some fractionation of a mixture of cholesterol [2-14C]-acetate and cholesterol [2-3H]acetate. Silica impregnated with a solution of AgN03 and dried at 120°C displays highly selective adsorption properties with respect to the number and the geometry of double bonds in unsaturated lipids. The separation of cholesterol from 5a-cholestan-30-01by argentation chromatography on a Si02-AgN03 column was first demonstrated by De Vries [1135] in 1962. This method was used to fractionate esters of cholesterol according to the degree of unsaturation of the fatty acids [383,836]. The separation of steryl acetates according to the number of double bonds in the nucleus or side chain by stepwise elution of a Si02-AgN03 column was reported by Vroman and Cohen [ 1 1371. The adsorbent was prepared by mixing a solution of 25 g AgNOB in 500 ml water with 100 g

55

56

STEROLS

Si02. After removal of water in a rotary evaporator, the adsorbent was dried overnight at 110°C. The column, 105 mm X 1Qmm, was packed with a slurry of 6 g of this adsorbent in hexane. Mixtures of 15 mg of each of two steryl acetates were resolved by elution with 50-ml portions of 0-35% benzene in hexane in 5% increments. Similarly, cholesteryl acetate was eluted by lo%, desmosteryl acetate by 20%, and 7-dehydrocholesteryl acetate by 40% benzene in hexane from a column prepared by mixing 10 ml40% AgN03, 10 g Silica Gel G, and 10 g Celite [340,341]. Paliokas et al. [827] have succeeded in separating 7-cholesten-30-01 from 8-cholesten-30-01 and 7,24cholestadien-30-01 from 8,24-cholestadien-30-01 by elution with chloroform-acetone (97:3) from a column of A1203-Hyflo Super-Cel, impregnated with AgN03. AdsorbosilCABN, a commercially available 25% AgN03-Si02 adsorbent, was used for the preliminary separation of plant sterols [789]. Florisil, an activated natural magnesium silicate, is a useful adsorbent for the preliminary fractionation of plant sterols, their esters, glycosides, and acylated glycosides [ 539,5571. For example, ca. 300 mg of a sample, obtained from soybeans by acetone extraction, was applied to a Florisil column, 40 cm X 1 cm, in hexane solution. A stepwise increase in the concentration of diethyl ether in hexane up to 50% yielded steryl esters, followed by free steiols. Then, increasing concentrations of acetone in diethyl ether up to 30% eluted the acyl glycosides, followed by glycosides. Factice 31-B, Coarsely Ground, a hydrophobic polymer produced from soybean oil, has been used as the stationary phase for reversed-phase partition chromatography of nonpolar lipids, including steryl esters [442], A slurry of Factice, 60-140 mesh, in acetone was packed under pressure into a jacketed column, measuring 150 cm X 1 cm I.D. and kept at 18-20°C. The column was eluted with 98% aq. acetone at the rate of 40-90 ml/h, and the effluent was automatically monitored with a differential refractometer. Fig. 6.1 shows a RI recording of the separation of the esters of cholesterol with a series of saturated fatty acids and some glycerides. The order of elution (from right to left) is that of increasing chain length of the fatty acids, the logarithm of the elution time being a linear function of the number of C atoms. Using another gel, LH-20 with a 49% hydroxyalkyl content [268] (cf. Section 2.1), Hyde and Elliott [492] succeeded in the separation of sitosterol from campesterol. Mixtures of 100-mg quantity were completely resolved by pumping three cycles of methanol-hexane (19:l) through a column, 88 cm X 2.5 cm. Products of the autoxidation of cholesterol, including several hydroperoxides, were readily separated on Sephadex LH-20 with dichloromethane [643]. A simple and rapid method for separating free cholesterol from esterified .. .

5, .en1 2% H20/ote10ne $1\;

: 2425x10 “ t i

li,,,li

1

. limn \

Fig. 6.1. Separation of the saturated series of cholesteryl esters from triolein, cholesterol, and partial glycerides on Factice. Solvent system: acetone-water (49:l). At the beginning of the run on the far right, a sharp peak of solvent front disturbance is seen. The asymmetry of the dipalmitin peak is most likely due to the presence of the 1,2- and 1,3-isomers. (Reproduced from J. Lipid Res., 4 (1963) 6, with permission; [442].)

THIN-LAYER CHROMATOGRAPHY

57

cholesterol in the analysis of blood is based on fractionating a lipid extract equivalent t o 0.5 ml of plasma on a column of 25 g Sephadex LH-20 in a tube of 15 mm I.D. [ 1651. A 13:7 mixture of chloroform and hexane elutes cholesterol in the first 39 ml, and then esterified cholesterol in 49-70 ml. Sterol digitonides, in benzene-methanol (1 :1) solution can be decomposed by chromatography on a Sephadex LH-20 column [271J .

6.2. THIN-LAYER CHROMATOGRAPHY A discussion of the TLC of sterols and its application to the study of biological and pharmacological problems was published in 1973, unfortunately without bibliography [ 1901. Earlier methods of TLC were adaptations of PC methods, and therefore reversedphase partition systems, which are fairly successful in the PC of sterols [734], were widely used. One of these systems requires the impregnation of the silica gel with undecane and development with a mixture of acetonitrile and acetic acid [ 199,11661. Addition of 0.5% bromine to the mobile phase improves the separation of several “critical pairs” [ 1991. Other reversed-phase systems consist of paraffin-impregnated kieselguhr plates and mobile phases of ethyl methyl ketone-acetonitrile (7:3) [542] or 80% aq. acetone [998]. More recently, silanized silica gel plates, which are commercially available (Silica Gel HS) but are also easily prepared in the laboratory, have been tried for the separation of sterols and other lipids [ 1 1251. These plates showed reversed-phase partition behavior when polar water-containing nobile phases were used, but with less polar solvents they behaved as in adsorption TLC. TABLE 6.1 RcVALUES OF CHOLESTEROL ANALOGS [ 1891 Compound

SCholestene-3p,4p-dioI SCholestene-3p, 7adiol S€holestene-3p, 7p-diol 5Cholesten-3-one 3p-Hydroxy-5-cholesten-7-one 3,5Cholestadien-7-one 4Cholestene-3,6-dione 1,4Cholestadien-3-one 4,6Cholestadien-3-one SaCholestan-3p-01 5aCholestan-3a-ol 5pCholestan-30-01 3~-Hydroxy-5or-cholestan-6-one SaCholestane-3p, 501,6p-triol 3P,Sor-Dihydroxy-Sa-cholestan-6-one 3,SCholestadiene 5,7Cholestadien-3p-ol 7-Cholesten-3p-ol

Benzene-ethyl acetate mixture 9:l

2:l

1:2

0.23 0.10 0.11 1.60 2.32 2.16 1.60 1.38 1.51 0.93 1.46 1.31 0.25 0 0 2.58 1.00 1.00

0.42 0.28 0.33 1.36 1.53 1.46 1.35 1.25 1.32 1.00 1.26 1.20 0.54 0.0s 0.0s 1.53 1.00 1.00

0.69 0.46 0.55 1.09 1.10 1.09 1.09 1.07 1.08 1.02 1.08 1.04 0.73 0.17 0.17 1.10 1.oo 1.oo

STEROLS

58

Cargill [ 1411 described both adsorption TLC with benzene-ethyl acetate (2: 1 or 19: 1) as the mobile phase and reversed partition TLC with either 2-phenoxyethanol or 2-methoxyethanol as the stationary phase and methanol as the mobile phase, or with undecane as the stationary phase and either methanol or methanol-diethyl ether (49: 1) as the mobile phase. Cholesterol analogs with additional double bonds or functional groups in Rings A or B can be analyzed by adsorption TLC on silica gel with mixtures of benzene and ethyl acetate (9:1,2:1, or 1:2) [189]. Table 6.1 shows the resulting& values (mobilities relative to cholesterol). Other TLC methods for sterols and their esters involve single development with benzene-petroleum ether (b.r. 35-45') (7:4) or triple development with either benzenepetroleum ether (17:3), petroleum ether-diethyl ether (99: l), isooctane-diisopropyl ether (49: l), or isooctane-diethyl ether (99: 1) [542]. A series of sitosteryl esters has been prepared and their mobilities on silica gel plates, developed with various solvent systems, were recorded [67]. Silica or alumina plates have been used with benzene-ethyl acetate (2:l or 4:l) or benzene-ethanol (95: 1 or 95:2) for separating stanols from the corresponding brominated stenols [499]. Alumina also served as the adsorbent in the fractionation of steryl esters [381] and of the oxidation products of ergosterol [332]. The use of a 15:5: 1 mixture of MgO, Alz03, and CaS04 in a wedge-shaped layer was recommended for TLC of steryl acetates [95 11. TABLE 6.2 ~RFVALUESOF STEROLS AND STERYL ESTERS [ 5 3 8 ] Solvent systems: 1 = cyclohexane-diethyl ether-glacial acetic acid (40: 159: 1); 2 = cyclohexanediethyl ether-glacial acetic acid (100:99: 1); 3 = petroleum ether (b.1. 40-6O0)-glacial acetic acid (999: 1); 4 = Detroleum ether-acetone (49: 1): 5 = carbon tetrachloride. Compound

Cholesterol Stigmasterol Sitosterol Ergosterol Campesterol Lumisterol Lanosterol Cholesteryl acetate Stigmasteryl acetate Sitosteryl acetate Ergosteryl acetate Cholesteryl palmitate Stigmasteryl palmitate Sitosteryl palmitate Ergosteryl palmitate Cholesteryl stearate Stigmasteryl stearate Sitosteryl stearate Ergosteryl stearate

Solvent system 1

2

86 80 94 61 90 10 100 -

51 41 60 27

3 -

-

55

-

15 75

51 45 68 21

-

-

5

4

100 83 100 53 86 66 95 43 16 55

93 38

-

-

80 I0 96 55 18 60 92 48

THIN-LAYER CHROMATOGRAPHY

59

Whereas the separation of plant sterols and their esters on silica plates ordinarily requires derivatization, impregnation, or special development techniques, good resolution is obtained on MgO plates by simple development with the solvents listed in Table 6.2 [538]. Spraying of the chromatograms with water permits the detection of ca. 20 pg of sterols without destruction. Argentation TLC was introduced into sterol analysis by Morris [746]. He prepared 5% AgN03 plates by spreading a slurry of 23.75 g Silica Gel G in 50 ml water, containing 1.25 g AgN03, and then activating the resulting layers at 1 10°C for 30 min. Development with diethyl ether or diethyl ether-hexane (1 :4) separated the cholesteryl esters according to the degree of unsaturation of the fatty acids. A 9: 1 mixture of hexane and benzene may also be used [383]. The optimal AgN03 concentration for this method is below 15% and may be around 5% 15701. The resolution of “critical pairs”, e.g., of stanols and the corresponding stenols, is readily accomplished on AgN03-impregnated silica gel plates, developed once with chloroform [498], chloroform-acetone (19: 1) 12361 or (49: 1) [ 10771, chloroform-diethyl ether-acetic acid (970:23:5) [ 1991, or continuously for 6 h with benzene-ethyl acetate (19: 1) [635]. Alumina, impregnated with AgN03, appeared to be equally suitable for TLC with a mobile phase of chloroform-acetone (19: 1) [530], and has served to prepare pure sitosteryl propionate [ 161. In many cases, sterol derivatives separate better than the sterols themselves. Thus, desmosteryl acetate and cholesteryl acetate separate on ordinary silica gel plates, continuously developed for 1 h with benzene-hexane (1:3) [635]. The 2401- and 24fl-hydroxycholesterols cannot be separated by TLC, unless they are converted to the dibenzoates [644]. Continuous development with benzene-hexane (1 :1) for 15 h resolves these TABLE 6.3

RC (C = CHOLESTEROL) VALUES OF STERYL ACETATES ON SILVER NITRATE-IMPREGNATED SILICA GEL HF254+36L IN HEXANE-BENZENE (5:2) AFTER TWO DEVELOPMENTS [497] Steryl acetate RC value 5cuCholestan-30-01 (cholestanol) 5pCholestan-3p-01 (coprostanoU 5pCholestan-70-01 SpCholestan-3cu-ol (epicoprostanol) (24R)-24-Ethylcholest-5en-3p-ol (sitosterol) (24R)-24-Methylcholest-S-en-3p-ol (campesterol) (24S)-24-Ethylcholesta-5,22-dien-3p-o1 (stigmasterol) Cholesterol* 4-Cholesten-3p-01 (24R)-24-Methylcholesta-5,22-dien-3p-o1(brassicasterol) 5,22Cholestadien-3f3-1 (cis and fruns) 22-fruns-24-Norcholesta-5,22-dien-3p-ol (C16 sterol) 5,24Cholestadien-3p-ol (desmosterol) 24-Ethylidenecholest-5-en-3p-01 (fucosterol) 24-Methylenecholest-Sen-36-01 5,2S€holestadien-3p-ol 5,7Cholestadien-3@-01 *The RF value of cholesteryl acetate is 0.65.

1.20 1.20 1.18 1.15 1.09 1.02 1.02 1 .oo 1 .oo 0.82 0.76 0.69 0.67 0.6 1 0.44 0.33 0.32

STEROLS

60

epimers. Other sterols known to pose difficult separation problems were converted to 3,5-dinitrobenzoates and separated on wedge-shaped layers of Mg0-A1203-CaS04 (15:5:1) with a solvent mixture of hexane-ethyl acetate (9:l) [952]. On AgN03-impregnated silica gel plates, steryl acetates have been chromatographed by a single development with benzene-hexane (3:5) [ 11371. Table 6.3 shows the R c values (mobilities relative to cholesteryl acetate) of some steryl acetates, observed on silica gel HF294+366-AgN0 (5: 1) plates, developed twice with hexane-benzene ( 5 : 2 ) [497]. The R c value decreases as the number of carbon atoms following the double bond in the side chain increases. The propyl esters of 7-dehydrocholesterol, cholesterol, desmosterol, and Sol-cholestan3/3-01 were separated on SiOZ-AgNO3 (1 5:4) by a single development with hexane-benzene (5:1) [ 1881. Gibbons et al. [356] have used ca. 10%AgN03-Silica Gel H with benzenehexane (1 :1) and ca. 20% AgN03-A1203 G with hexane-benzene (3: 1) at 5°C for the separation of 4,4-dimethylsteryl acetates differing in the number of double bonds. Most of the detection methods used for the TLC of sterols are nonspecific and have already been mentioned in Chapter 3. Additional tests, which have been reported in connection with TLC of sterols and other lipids but also have a low order of specificity, will now be reviewed. Observation and photography of thin-layer plates in UV light have been greatly improved in connection with sterol research [633]. Lipid material transparent to W, such as sitosterol, can be detected in amounts as low as 1 pg under the UV lamp on a kieselguhr layer impregnated with paraffin [ 11551. After having been sprayed with 0.01% fluorescein in ethanol and then having been exposed to steam, the chromatogram shows green fluorescent zones on a purple background. Malachite Green, in the form of a 0.5% aqueous spray reagent, stains cholesterol and other lipids with long aliphatic chains [ 10471. Background color can be bleached by spraying the dry chromatogram with 2% NaHS03 in 2 N HCl. A detection reagent for cholesterol and its esters, advocated as a replacement for the toxic and messy Carr-Price reagent, is prepared by dissolving 50 mg FeC13 - 6 HzO in 90 ml water and adding 5 ml glacial acetic acid and 5 ml conc. H2S04 [669]. The red or violet color which appears in 2-3 min when the plate is heated at 100°C is unstable. A highly sensitive test claimed to be specific for 5-en-3P-01~ and 5,7-dien-3/3-olshas been described by Richter [892]. The spray reagent is made by dissolving 100 mg 1,2-napthoquinone-2-sulfonic acid in 100 ml of a mixture of ethanol-60% HC1O4-40% formaldehyde-water (20: 10: 1:9). Sterols with the requisite structure in amounts as low as 30 ng give a characteristic blue color when the sprayed chromatogram is heated at 70-80°C. Oxidation products of cholesterol give blue zones when the thin-layer chromatogram is sprayed with a mixture of 5 ml 5% KI and 20 ml glacial acetic acid, followed, after 5 min, by a spray of starch solution [775]. A more recent method of detecting sterol hydroperoxides involves spraying the chromatogram with 1% solutions of Wurster dyes (bromine-treated N,N-dimethyl-p-phenylenediamine2HC1 or N,N,N',N'-tetramethyl-pphenylenediamine -2HC1) in 50% aq. methanol containing 1 ml glacial acetic acid per 100 ml of reagent [990]. For the detection of 7-ketocholesterol in oxidized sterol preparations, the keto group is first reduced by spraying the chromatogram with 1% NaBH4 in methanol and, after 5 min, the 7P-hydroxycholesterol which is formed is detected as a blue zone by spraying with 50% HzSO4 [992]. TLC is not ideal for the isolation of sterols from lipid extracts, but in metabolic

-

GAS CHROMATOGRAPHY

61

research, sterol analysis often involves a fractionation into sterols, steryl esters, and other classes of lipids. Fractionation into six classes of compounds can be accomplished by sequential development of a single Silica Gel G plate, activated at 110°C for 3 0 min, with two solvent systems 14541. The extract is applied 2 cm from the bottom edge of the plate, and diethyl ether-glacial acetic acid-petroleum ether (b.r. 38.5-40.0') (100:3:97) is allowed to rise on the plate to a height of 9 cm from the bottom. The plate is allowed t o dry and then the second solvent, diethyl ether-petroleum ether (3:97), is allowed to ascend to 15 cm above the bottom. The six separate zones contain: phospholipids (at the origin), cholesterol, fatty acids, triglycerides, cholesteryl esters, and squalene (near the solvent front). Fractionation of sterol mixtures by TLC permits the determination of 5a-cholestan-3cu01 in the presence of 5a-cholestan-3/3-ol and cholesterol [764], of cholesterol in autoxidation mixtures [473], of the individual products of cholesterol autoxidation [991], of 7-dehydrocholesterol in skin extracts [237], and the qualitative analysis of serum sterols [ 1911. However, even without separating them, the sterols in dietary and fecal lipids can be qualitatively and quantitatively analyzed by the application of MS to the eluates from thin-layer chromatograms [444]. Under carefully controlled conditions, the fluorescence produced by cholesterol on an ammonium sulfate-impregnated silica gel layer (see p. 15) when the chromatogram is heated to 150°C can be used for fluorometric analysis [727]. Analysis of eluted cholesterol and its esters by use of the sulfophosphovanillin reaction is more accurate and can be performed after the zones have been located by spraying the chromatogram with 50% H2S04and then heating it at 150°C for 7.5 min [728] or, better yet, by spraying with 0.001%Rhodamine 6G in methanol [ 10431. One of the main applications of the quantitative analysis by TLC t o the sterols is the determination of free and esterified cholesterol in blood [726]. Silica gel plates that have been freshly activated by heating at 150°C for 6 h and developed with carbon tetrachloride separate the cholesteryl esters in blood according to the number of double bonds [584]. Reproducible densitometric assays are obtained after spraying the chromatograms with SbCIS and heating them at 120°C for 5-10 min. Radioactive sterols and their esters are usually assayed following their elution from the thin-layer plates [639,919,1087].

6.3. GAS CHROMATOGRAPHY The analysis of sterols by GC has been reviewed earlier [334,345], in particular, the application of GC to plant sterols [581] and the GC-MS combination [122]. Sterols are usually chromatographed in the form of derivatives, but useful separations have also been obtained without derivatization. Thus, cholesterol, desmosterol, stigmasterol, and sitosterol are separately eluted, in that order, from 3%XE-60 or JXR on Gas-Chrom Q in a 6-ft. X 4-mm-I.D. column 13071. The dimethylpolysiloxane, JXR, is more thermostable and can be used at 240°C but is not as effective in separating cholesterol from its 24dehydroanalog, desmosterol, as the nitrile silicone, XE-60, at 2 15" or 225°C. Retention times of various C2, sterols relative to cholesterol, determined with 3% XE-60 on GasChrom Z in a column, 10 ft. X 2 mm, at 22OoC and with 10%SE-52 on Chromosorb W in a column, 5 ft. X 2 mm, at 260"C, are shown in Table 6.4 [ 1891.

STEROLS

62

TABLE 6.4 RETENTION TIMES OF VARIOUS C,, STEROLS RELATIVE TO CHOLESTEROL [ 1891 Compound Column ~.

5Cholestene-3p,4p-diol SCholestene-3p,7adiol 5-Cholestene-3p,7p-diol SCholesten-3-one 3p-Hydroxy-5-cholesten-7-one 3,5-Cholestadien-7-one 4Cholestene-3,6-dione 1,4Cholestadien-3-one 5aCholestan-30-01 5aCholestan-3a-01 5pCholestan-3p-01 3p-Hydroxydarcholestan-6-one SaCholestane-3p,5a,6p-triol 3p, Sa-Dihydroxy-5a-cholestand-one 3JCholestadiene 5,7-Cholestadien-3p-o1 7Cholesten-3p-01

XE-60

SE-52

1.87 0.39 0.39 1.73 1.28 1.45 4.18 2.37 1 .oo 0.94 0.84 4.38*

1.43 0.58 0.5 8 1.36 1.37 1.28 2.07 1.58 1.00 1.00 0.89 1.86 2.91 2.66 0.62

-

0.40 1.15 1.15

w

1.14

*Incipient decomposition. **Decomposition.

Ikekawa et al. [SO21 have determined the retention times of C2,, &, and CZssterols relative to cholestane on six different liquid phases (see Table 6.5). These were coated, in the concentrations shown, on Shimalite W and used in columns, 180 cm X 4 mm I.D., at the temperatures shown in Table 6.5. Generally, the polar phases gave better separations than the nonpolar phases. Sterols having a double bond at C-5, (2.14, or C-25 were eluted before the corresponding saturated sterols on nonpolar phases, but after the saturated analogs on polar phases. The 5-unsaturated sterols separated from the saturated analogs only on NGS. .The 22-unsaturated sterols were less retained than the saturated analogs on all liquid phases. On SE-30 and NGS, the elution order of sterols with unsaturated side chains was dependent on the position of the double bond: 22 < 25 < 24(28) < 24. The best separation of cholestanol from coprostanol was obtained on XE-61. Fumagalli et al. [335] have evaluated a series of stationary phases for their ability to separate cholesterol from desmosterol. Rest results were obtained with PhSi 19 1-43 (XE-6 1). In no case was it necessary to convert the sterols to TMS derivatives. More recently, Grunwald [377] found that OV-101, a liquid dimethylsiloxane polymer, gives considerably less tailing than SE-30 when free phytosterols are chromatographed. Comparing the performance of OV-17,OV-22, and OV-101 with 3% SE-30 (all on Anakrom ABS) in identical columns at 25OoC, he observed the best resolution of free phytosterols with 5% OV-101.No improvement was possible by chromatographing the sterols in the form of their TMS ethers. The procedure is applicable to the quantitative analysis of free sterols in plant extracts [378].

GAS CHROMATOGRAPHY

63

TABLE 6.5 RELATIVE RETENTION TIMES OF STEROLS (502) Sterol

C,,Sterols Cholestanol Coprostanol Cholesterol 7Cholesten-3p-ol 8 (14)Cholesten-3p-ol 14Cholesten-3psl 5,22Cholestadien-3p-o1 Desmosterol 7,22Cholestadien-3pl C,,-Sterols Campestanol Campesterol (240) 8 (14)-Ergosten-30-01 5,7Campestadien-3p-oI Brassicasterol (240) 5,24-Ergostadien-3p-oI 24-Meth ylenecholesterol

7,22-Ergostadien-3p-ol Ergosterol (240) C,,-Sterols Stigmastanol Sitosterol(24a) 7-Stigmasten-3p-ol 5,7-Stigmastadien-3p-ol Stigmasterol (24a) Fucosterol Chondrillasterol (24p) crspinasterol (240) 5,25-Stigmastadien-3p-ol 7,25-Stigmastadien-3p-ol 7,22,25-Stigmastatrien-30-01

Position of double bond

0 0 5

7 8(14) 14

5,22 5,24 7,22

0 5

8(14) 5,7

5,22 5,24 5,24(28) 7,22 5,7,22 0 5

7 5J 5,22 5,24(28) 7,22 7,22 5,25 7,25 7,22,25

Retention time relative to that of cholestane (= 1.00) SE-30*

SE-52**

XE61***

QF-15

XE-60§§

1.96 1.79 1.93 2.21 1.88 1.93 1.73 2.10 1.94

2.17 1.94 2.13 2.47 2.13 2.13 1.96 2.34 2.17

3.28 2.69 3.13 3.74 3.25 3.10 3.03 3.72 3.40

3.63 3.27 3.34 3.93 3.27 3.27 2.90 3.86 3.43

4.74 4.32 4.78 5.47 4.55 4.74 4.37 5.80 4.99

6.35 5.65 7.14 8.07 6.50 6.93 6.85 9.93 7.85

2.60 2.54 2.56 2.66 2.3 1 2.92 2.50 2.46 2.42

2.90 3.04 2.94

4.22 4.22 4.22 4.97 3.66 5.12 4.25 4.22 4.34

4.57 4.23 4.23 5.03 3.57 4.70 4.30 4.17 4.23

5.98 5.98 5.84 1.86 5.17 7.31 6.37 5.94 6.28

8.35 9.43 8.50 13.4 8.22 13.1 10.9 9.00 11.7

5.30 5.21 6.25 6.28 4.59 5.50 5.40 5.31 6.06 6.34 5.60

5.61 5.24 6.26 6.14 4.46 5.30 5.20 5.16 5.30 6.00 5.36

7.56 7.65 8.32 9.56 6.32 7.91 7.64 7.68 7.64 8.80 7.56

10.0 11.3 12.9 16.1 9.18 12.8 11.4 11.0 12.4 14.5 13.6

3.33 3.23 3.66 3.46 2.81 3.23 3.21 3.23 3.10 3.60 3.04

-

2.44 3.24 2.89 2.17 2.74 3.83 3.60 4.14

-

3.08 3.57 3.53 3.60 -

-

NGS§§§

9 . 5 % SE-30; column temperature, 210°C; nitrogen llow-rate, 80 mllmin; retention time of cholestane, 8.6 min. **1.5% SE-52; column temperature, 215°C; nitrogen flow-rate, 70 ml/min; retention time of cholestane, 8.0 min. ***1.0% XE-61 (PhSi); column temperature, 218°C; nitrogen flow-rate, 60 ml/min; retention time of cholestane, 5.7 min. § 1.5% QF-1; column temperature, 193°C; nitrogen flow-rate, 100 ml/min; retention time of cholestane, 5J min. 551.5% XE-60; column temperature, 221'C; nitrogen flow-rate, 100 ml/min; retention time of cholestane, 4.0 min. §§§2.0% NGS; column temperature, 220°C; nitrogen flow-rate. 100 mllmin; retention time of cholestane, 2.2 min.

Free sterols and their 4-methyl and 4p-dimethyl homologs have been chromatographed on 1.5% OV-17 columns at 25OoC [506],the dimethylsterols also on 1.5% SE-30 and 1.5% QF-1 at 22OoC [356]. For comparison of the results of chromatographing free sterols with those obtained by chromatography of acetyl derivatives, Itoh etal. [506] made use of R h values. These are the ratios of the relative retention times of the acetates to those of the corresponding free sterols. It was found that the R A values ~ for sterols range from 1.32 to 1.36, those for 4-monomethylsterols from 1.26 to 1.32, and those for 4,4-dimethylsterols from 1.I8 t o 1.23. Therefore, they may be of some diagnostic value. An earlier solution for the problem of separating cholesterol from Scu-cholestan-3~-01 was the oxidation of the former to cholestane-3/3,5a,6/3-triol,which can be isolated by solvent partition prior to GC [904]. The resolution of the cholestanol/cholesterol pair

STEROLS

64 TABLE 6.6 RELATIVE RETENTION TIMES OF STERYL ACETATES* [838] Steryl acetate

7Coprostenol Coprostanol 8 (14)Coprostenol cis-22-Dehydrocholesterol Epicholestanol tra ns-22-Dehydrocholesterol 5,22,24-Cholestatrienol 5,7,22Cholestatrienol Cholesterol Cholestanol 8,14Cholestadienol 14a-Methyl-8-cholestenol 8(9)Cholestenol 5 ,25Cholestadienol Desmosterol 5,7Cholestadienol Brassicasterol 7-Cholestenol 7,14,22-Ergostatrienol Zymosterol 140-Methyl-8,22-ergostadienol Pollinastanol 140-Methyl-7-cholestenol 1,9(1 1),22-Ergostatrienol Ergosterol 7,22-Ergostadienol 24-Methylenecholesterol Lophenol 8 (14)-Ergostenol 14a-Methyl-7,22ergostadienol Campesterol 5-Ergostenol 14a-Methyl-8,24(28)ergostadienol 8,24 (28)-Ergostadienol 8 (9),14-Ergostadienol Campestanol 14c~-Methyl-8ergostenol 31-norCycloartanol 8 (9)-Ergostenol 24-Dihydrolanosterol 5 ,I-Ergostadienol Stigmasterol Poriferasterol 7,24 (28)-Ergostadienol 24-Methylenepollinastanol 7-Ergostenol Obtusifoliol 24-Dihydroobtusifoliol

Gas-chromatographic system SE-30**

QF-1***

HI-EFF-gBS

PMPESS

0.86 0.86 0.86 0.87 0.91 0.91 0.94 0.99

0.88 0.90 0.91 0.88 0.93 0.89 0.90 1.00 1.oo 1.05 0.97 1.08 1.01 1.12 1.09 1.12 1.09 1.11 1.07 1.09 1.17 1.23 1.21 1.11 1.22 1.20 1.28 1.25 1.23 1.30 1.29 1.29 1.37 1.29 1.24 1.35 1.38 1.40 1.29 1.50 1.45 1.32 1.32 1.40 1.58 1.42 1.53 1.54

0.76 0.76 0.77 0.88 0.80 0.91 1.35 1.21 1.00 1.00 1.09 0.94 1.01 1.31 1.29 1.29 1.10 1.21 1.18 1.30 1.04 1.16 1.17 1.24 1.44 1.32 1.43 1.32 1.26 1.26 1.32 1.32 1.34 1.46 1.44 1.30 1.23 1.27 1.33 1.13 1.69 1.34 1.34 1.74 1.68 1.59 1.46 1.33

0.77 0.74 0.75 0.87 0.82 0.92 1.37 1.14 1.00 1.02 1.07 0.94 1.07 1.27 1.29 1.23 1.09 1.26 1.18 1.39 1.01 1.17 1.16 1.28 1.33 1.37 1.39 1.32 1.27 1.25 1.29 1.29 1.29 1.47 1.38 1.32 1.20 1.22 1.38 1.08 1.60 1.32 1.32 1.76 1.63 1.63 1.31 1.23

(0.92) (0.93) (0.93) (0.99)

1.oo

1.03 1.03 1.04 1.05 1.07 1.09 1.09 1.12 1.12 1.13 1.13 1.16 1.16 1.17 1.18 1.22 1.25 1.26 1.27 1.28 1.29 1.30 1.30 1.30 1.33 1.33 1.34 1.34 1.35 1.36 1.41 1.42 1.42 1.42 1.42 1.46 1.46 1.47 1.50

(1.32)

(1.39) (1.52)

(1.52) (1.54)

GAS CHROMATOGRAPHY

65

TABLE 6.6 (continued) Steryl acetate

Gas-chromatographic system SE-30**

4wMethyl-8(9),14-ergostadienol 24-Methylpollinastanol 14a-Methyl-7ergostenol Lanosterol 5,7,22-Stigrnastatrienol 40-Methyl-8ergostenol 5,25-Stigmastadienol 7,22,25-Stigrnastatrienol Spinasterol Chondrillasterol 24-Methylenelophenol Cycloartanol 8 (14)-Stigmastenol Sitosterol Clionasterol Fucosterol 8 (9),14-Stigmastadienol Stigmastanol 23-Demethylgorgosterol 140-Methyl-24S-8-stigmastenol 28-Isofucosterol Cycloeucalenol 8 (9)-Stigmastenol 8 (9),14,24 (28)-Stigmastatrienol 7,25-Stigmastadienol Cycloartenol 5,7-Stigmastadienol 7-Stigmastenol 7Chondrillastenol Macdougallin 4a-Methyl-8(9),14-~tigmastadienol

1.50 1.51 1.52 1.54 1.54 1.55 1.55 1.57 1.58 1.58 1.60 1.61 1.61 1.63 1.63 1.63 1.66 1.67 1.67 1.67 1.69 1.70 1.70 1.71 1.73 1.75 1.78 1.83 1.83 1.84 1.87 4a,14a-Dimethyl-24S-8-stigmastenol 1.88 28-1so-7,24(28)-stigmastadienol 1.88 1.92 4a-Methyl-8(9)-stigmastenol Peniocerol 1.92 1.94 Cy clolaudenol 24-Methylenecycloartanol 1.98 24-Methylcycloartanol 2.06 Citrostadienol 2.13 Gorgosterol 2.22 Saringosterol 2.50

(1.54) (1.66) (1.60)

(1.65) (1.71)

(1.75)

(1.87)

(1.92) (1.94) (1.99) (2.08) (2.12) (2.21) (2.22)

QF-1***

HI-EFF-8B5

PMPEg5

1.38 1.61 1.53 1.62 1.47 1.46 1.47 1.43 1.46 1.46 1.57 1.73 1.50 1.56 1.56 1.50 1.48 1.62 1.62 1.66 1.55 1.82 1.56 1.53 1.61 1.85 1.75 1.71 1.71 3.09 1.67 1.84 1.70 1.74 3.24 2.12 2.22 2.24 1.90 2.18 2.89

1.54 1.53 1.52 1.47 1.73 1.44 1.68 1.84 1.61 1.61 1.89 1.45 1.51 1.60 1.60 1.76 1.74 1.58 1.82 1.49 1.85 1.84 1.59 1.99 2.05 1.88 2.05 1.93 1.93 2.49 1.88 1.59 2.22 1.69 2.93 2.02 2.08 1.89 2.43 2.49 6.93

1.42 1.52 1.49 1.41 1.61 1.41 1.59 1.85 1.65 1.65 1.79 1.40 1.52 1.54 1.54

1.68 1.65 1.57 1.69 1.43 1.79 1.70 1.65 1.90 1.99 1.81 1.88 1.93 1.93 2.12 1.69 1.46 2.24 1.67 2.55 1.87 1.90 1.79 2.31 2.5 1 4.24

*Relative to cholesterol acetate; values in parentheses are for free sterols relative to free cholesterol. When no value is given, it is the same as the acetate value. **3% SE-30 on GasChrom Q, 244OC. ***I% QF-1 on GasChrom P, 231°C. §3% HI-EFF-8B on Gas-Chrom Q, 238°C. 592% PMPE on GasChrom Q, 250°C.

o\

TABLE 6.7

m

RETENTION TIMES OF STERYL ACETATES RELATIVE TO CHOLESTERYL ACETATE [788] Steryl acetate

Stationary phase GE-F-50

Dexsil-300

OV-17

ov-25

AN400

SP-525

OV-225

CTpA

SP-1000

Cholesterol Cholestanol Desmosterol 25-Dehydrocholesterol

1.oo 1.01 1.11

1.oo 1.05 1.12 1.15

1.oo 1.oo 1.22

1.00 0.99 1.25

1.oo 1.oo 1.18

1.00 1.03 1.28

1.00 1.03 1.28

1.00 0.96 1.35

1.00 0.95 1.37

Campesterol Ergostanol Brassicasterol Ergosterol

1.31 1.33 1.11 1.10

1.33 1.39 1.11 1.10

1.31 1.31 1.14 1.29

1.33 1.31 1.14 1.28

1.32 1.32 1.08 1.17

1.31 1.34 1.11 1.19

1.31 1.34 1.12 1.27

1.30 1.24 1.13 1.34

1.30 1.23 1.12 1.48

Sitosterol Stigmastanol Stigmasterol Fucosterol Isofucosterol

1.65 1.69 1.42

1.71 1.78 1.41

1.65 1.65 1.45

1.65 1.63 1.44

1.65 1.65 1.36

1.71

1.76

1.84

1.75

1.75

1.60 1.63 1.36 1.72 1.86

1.59 1.62 1.41 1.79 1.80

1.57 1.52 1.36 1.77 1.86

1.60 1.53 1.39 1.74 1.92

GAS CHROMATOGRAPHY

67

was subsequently improved by conversion to the TFA esters and by the use of 10% SP2401 at 220°C [565]. Another stationary phase with superior properties - high thermal stability, in particular - is PZ-176, a polyphenyl ether sulfone [700]. Temperature programs of up to 400°C at 2"/min can be used to chromatograph sterols in 4-m X 3.4-mm columns of 3% PZ- 176 on Gas-Chrom P, either in the free form or in the form of TMS ethers. For the GC of various cholestanols and cholestanones, Herz and Gonzilez [431] employed a 7-ft. X 1/4-in. 3% HI-EFF-8BP column. The free alcohols, chromatographed a t 225°C with a carrier gas flow-rate of 68 ml/min, gave broad peaks. Sharper peaks and better resolution were obtained when TMS and TFA derivatives were chromatographed at 200°C with a flow-rate of 9 1 ml/min. The ketosteroids and their methoximes were well resolved at 225°C with a flow-rate of 68 ml/min. Among the most frequently used esters of sterols are the acetates. The retention times relative to cholesteryl acetate of 92 acetates of natural sterols and their precursors on some commonly used stationary phases are listed in Table 6.6 [838]. Some of the generalizations made for the GC of free sterols (see p. 62) also apply to the steryl acetates, but improved separations may be noted in many cases. The 24(28)-unsaturated sterols separate better from their saturated analogs as acetates on HI-EFF-8BP and PMPE than on QF-1 or SE-30. The sterols with alkyl substituents at C-24 are easily separated from the lower homologs on both polar and nonpolar phases. The resolution of the commonly encountered triad campesterol, stigmasterol, and sitosterol is best accomplished on SE-30. Alkylation at C-4 produces greater increases in the retention times of sterols on the nonpolar than on the polar phases. The 14a-methylsterols are specifically retained by QF-1, whereas HI-EFF-8BP and PMPE have greater affinity for double bonds at C-24(25), C-24(28), and C-5,7 than the other stationary phases. Additional retention times for the acetates of commonly encountered sterols on more recently introduced phases are shown in Table 6.7 [788]. The stationary phases were applied as 1% coatings on Gas-Chrom Q and packed into 6-ft. X 4-mm columns, operated at oven temperatures between 200" and 250°C. Chemically, GE-F-50 is a methyl chlorophenyl silicone, Dexsil-300 is a polycarboranesiloxane, OV-17 is a SO% phenyl methyl silicone, OV-25 is a 75% phenyl methyl silicone, AN-600 is a 50% cyanoethyl methyl silicone, SP-525 is an aromatic hydrocarbon, OV-225 is a 25% cyanopropyl2S% phenylmethyl silicone, CTpA is Carbowax 20M-terephthalic acid, and SP-1000 is a modified Carbowax 20M. The greatest overall resolving power for "critical pairs" was shown by SP-1000. Synthetic and naturally occurring esters of sterols have been analyzed by GC [610,1018] Temperature programs have been found suitable for the separation of esters differing in molecular weight by at least one methylene unit, but isothermal conditions were required for the resolution of mixtures of esters differing only in the unsaturation of the fatty acid moiety. The glycosides of sterols must be chromatographed in the form of TMS, TFA, or HFB derivatives [580]. For GC-MS, the TFA derivatives are most satisfactory when a 1.5-m column, packed with 3% OV-17 on Gas-Chrom Q, is used at 270°C. Extensive studies on the characterization of sterols by GC-MS of their TMS ethers have been reported by Brooks et al. [ 1 16,1171. Although the separation on 1% SE-30 or OV-17 columns was inadequate, the combination of GC data with MS information allowed positive identification of most sterols. Knights [ 5781 has analyzed fifteen free sterols

68

STEROLS

and their acetyl, TFA, and TMS derivatives by GC-MS, using a 9-ft. column of 1% SE-30 at 250°C. The TFA and TMS derivatives proved to be most suitable for this type of analysis. Brooks et al. [ 1 191 have extended these studies to the 24-ethylidene sterols and their acetyl and TMS derivatives. From a 3-m X 3-mm column, packed with 3% OV-17 and operated at 260°C, the E-isomers were eluted consistently before the corresponding Z-isomers. Column bleed, which interferes with MS, was minimized by the use of some of the newer thermostable liquid phases [48]. The best resolution of sterol TMS ethers with the least substrate bleed was observed with 1% coatings of Dexsil-300, SE-30 "ultraphase", and Silar-SCP on Diatomite CQ at 220-260°C. Most of the sterols in cockles could be identified by a combination of one of these stationary phases with MS. Some of the technically interesting applications of GC to sterols will now be mentioned. The preferred method of determining the purity of cholesterol standards is based on GC [ 11591. In the analysis of the pyrolysis products of sterols, a pyrolyzer was coupled to a gas chromatograph [344]. Although steroidal peroxides decompose on GC columns, much information about their chemical nature can be obtained from the resulting chromatographic patterns [89,1049]. For several years, the standard procedure for determining plant sterols involved their conversion to TMS ethers, followed by GC at 230°C on a column, 16 ft X 1/4 in. O.D., packed with 0.75-1 .O% SE-52 on Gas-Chrom Z, 45-50 mesh [907]. Keeping the flowrates adjusted to 85 ml/min for helium, 57 ml/min for hydrogen, and 450 ml/min for air, the hydrogen FID was kept at 260°C. Injection of a sample of mixed steryl TMS ethers (including a known quantity of TMS stigmasterol), containing a maximum of 6 pg of any single component, into the injection port at 320°C produced a response in the hydrogen FID. The internal standard method gives results of high accuracy and precision. More recently, the quantitative analysis of free phytosterols has been described by Grunwald [378] (see p. 62). His method is also based on an internal standard and the determination of peak area ratios. The analysis of plant sterols in feces presents an extremely difficult problem because the mixtures are exceedingly complex [579]. It is best solved by preliminary fractionation of the lipid extracts on adsorption columns and thin-layer plates and conversion of individual fractions to suitable derivatives before analysis by GC [719]. The determination of coprostanol and coprostanone in water as an index of fecal pollution is an interesting application, which makes use of the specificity and sensitivity of GC [1041]. A U-shaped column, 180 cm X 2 mm I.D., was packed with 3% OV-1 from one end and with 3% OV210 (both on Gas-Chrom Q) from the other [244]. After solid injection, this column, at 21OoC, produced separate peaks of (in the order of emergence): Sa-cholestane, 50-cholestan3/3-01, 5-cholesten-3~-01,5/3-cholestan-3-one, 4-cholesten-3-one. Coprostanone (50-cholestan3-one), detectable in the 400 pg/ml range, was proposed as the most suitable index of pollution. One of the most important applications - at least as measured by the number of publications - is the analysis of plasma cholesterol by GC. Only two of the most recent methods can be mentioned here. A convenient and rapid procedure for determining cholesterol in 20 pl of plasma has been developed by Ishikawa et al. [SO51.The plasma is added to 100 pl of a solution, prepared by diluting 20 mg of 50-cholestane in 2 ml diethyl ether and 25 ml of 24% methanolic tetramethylammonium hydroxide to 100 ml with

GAS CHROMATOGRAPHY

69

isopropanol, in a 3-ml glass-stoppered centrifuge tube. The contents of the tube are briefly mixed and then heated at 80°C for 15 min. After a 30-sec cooling period, 50 pl of a methyl butyrate-l,l,2,2-tetrachloroethane (3: 1) mixture is added. This is followed by mixing, addition of 200 pl water, vigorous shaking, and then centrifugation at 2000-2500 rpm for 10 min. Finally, 0.5-1 pl of the 1,1,2,2-tetrachloroethanephase is injected into one of two columns, which have been silanized with Sylon-CT before packing. The first is packed with 3% OV-17 on Gas-Chrom Q and the second, column, 1.8 m X 2 mm I.D., 1,2 m X 2 mm I.D., with 3% SP-2250 on Supelcoport. Either one of the two columns gives excellent results when operated at 260°C with a nitrogen flow-rate of 50 ml/min. To calculate the cholesterol concentration in the sample, the peak-height ratio of cholesterol/ cholestane is multiplied by a correction factor, F , which is obtained by dividing the known cholesterol concentration of a reference serum by its peak-height ratio. A highly sensitive and accurate method for the determination of cholesterol in 10 p1 of serum by mass fragmentography (see p. 42) has been described by Bjorkhem et al. [86]. It is not a routine method, but it is so specific that it can serve as a reference standard. After the addition of [2,2,3 ,4?H4] -cholesterol to the serum sample, saponification by KOH, and extraction of the nonsaponifiable material with hexane, the extract is analyzed by GC-MS. A 4-m X 2-mm column, packed with 1.5% SE-30 on Chromosorb W, is used at 240°C. The amount of unlabeled cholesterol is obtained from the ratio between the recordings at m/e 386 (cholesterol) and m/e 389 (trideuterated cholesterol), which is ascertained by means of a multiple-ion detector. As little as 4 ng of cholesterol can be determined in t h s manner. This elegant method is, of course, also applicable to many other analyses. For instance, the amount of 5-cholestene-3P,7ar-diolformed by liver microsomal cholesterol 7a-hydroxylase can be used as an enzyme assay by adding a known prior to extraction, TLC, conversion amount of 5- [24,25,7p-' HJ]-cholestene-3~,7a-diol, to the TMS ether, and GC-MS [87].

This Page Intentionally Left Blank

Chapter 7

Bile acids and alcohols In the animal organism, cholesterol is reduced and hydroxylated to C2, bile alcohols. In the higher animals the side chain is degraded, and CZ4 acids, the cholanoic acids, are found in the bile and excreta, usually in conjugation with taurine and glycine. In most cases, the bile acids are 50-steroids with a 3a-hydroxyl group and additional oxygen atoms at C-6, (2-7, or C-12. General methods of isolating and chromatographing bile acids have been reviewed by Sjovall [976], Eneroth and Sjovall [279,280], and Subbiah [ 10201.

7.1. LIQUID COLUMN CHROMATOGRAPHY In spite of the ionic character of this class of steroids, the application of ion-exchange chromatography has been only moderately successful [614]. This is partly due to the great success of TLC and GC. However, ion-exchange and partition columns still find use in preliminary purification. The most useful column materials are the gels, such as Sephadex G-25 [790] ,DEAESephadex [ 11841, methylated Sephadex G-25 [Sol, 8021, and particularly carboxymethylated Sephadex LH-20 [799]. For the isolation of bile acids from urine, a 30-ml aliquot is percolated through 10 g Amberlite XAD-2 in a column, 20 cm X 1 cm, at a rate of about 1 drop/:! sec [691]. The column is washed with 60 ml water, and the bile acids are eluted with 60 ml methanol. After evaporation of the methanol, the bile acids are dissolved in 2 mlO.01 M NaCl in chloroform-methanol (1 :l), and this solution is applied to a column, 25 cm X 1 cm, containing 4 g Sephadex LH-20 in the same solvent. The glycine and taurine conjugates can now be eluted with 70 ml of this solvent, and the sulfated conjugates can be obtained by subsequent elution with 50 ml methanol. For the extraction from serum, a similar procedure is used [692]. A 2-ml aliquot, diluted with 18 mlO.1 MNaOH in saline is percolated through a column, 20 cm X 0.5 cm, of 1 g Amberlite XAD-2, and the column is washed with water until the effluent is neutral. The bile acids are then eluted with 10 ml ethanol.

7.2. THIN-LAYER CHROMATOGRAPHY The methods of TLC, as applied to the bile acids, their conjugates, and the bile alcohols, have been reviewed by Hofmann [452] and by Eneroth [274]. Layers of Kieselguhr G [ 11383,polyamide [322], and methylated Sephadex G-25 [803] have been used for these procedures, but most investigators prefer Silica Gel G [273,329,407,1084]. The RF values of free and conjugated bile acids obtained by use of some of the more recently described solvent systems are summarized in Table 7.1. For the separation of the 3asulfates of lithocholic acid and its glycine and taurine conjugates, Cass ef al. [ 1451 have developed thin-layer chromatograms on Silica Gel H-CaS04 (9: 1) with the solvent

71

TABLE 7.1 hRF VALUES OF BILE ACIDS IN TLC ON SILICA GEL G Solvent systems: 1 = 2,2,4-trimethylpentane-ethyl acetate-acetic acid (5:s: l), RF relative to deoxycholic acid [ 2731 ;2 = ethyl acetate-methanolacetic acid (7:2:1) [ 10841 ;3 = chloroform-ethyl acetate-acetic acid (9:9:2) [ 7431;4 = diethyl ether-acetic acid (249:1) [407];5 = 2,2,4-trimethylpentane-diisopropyl ether-acetic acid-isopropanol(2:l:l:l) [373];6 = chloroform-methanol-7 N NH,OH-water (21:15:1:2),RF relative to cholic acid [ 10211;7 = chloroform-methanol-acetic acid (80:12:3), RF relative to cholic acid [967];8 = 2,2,4-trimethylpentane-acetic aciddiisopropyl ether--isopropanol(10:6:5:1) [375];9 = 2,2,4-trimethylpentane-ethyl acetate-acetic acid-butanol(20: 10:3:3) [ 10251 ; 10 = propanolacetic acid-water (95:4:1) [305];11 = butanol-acetic acid-water (100:7:5)[54];12 = isopropanol-ethyl acetate-water-NH,OH (20:25:6:4) [3661; 13 = 2,2,4-trimethylpentane-diisopropyl ether-acetic acid-butanol-water (10:5:5:3: 1) [476]; 14 = 2,2,4-trimethylpentane-diisopropyl etheracetic acid-butanol-isopropanol-water (10:5:5:3:6:1) [476];15 = chloroform-methanol-water (70:25:3) [477]. Solvent system

Position Free

3 6 7 12 1

2 3 4 5

6 7 8 9 10 11

12 13 14 15

a -

rauro

Glyco a

-

-

-

0

160 98 53 84 90 116

117

73

-

52 98 67 50 50 80 75

-

65

-

-

-

59

a

a -

a a

o -

-

a

-

a -

@

-

0 0

0 0

0

-

100 88 93 93 39 31 44 44 74 67 102 102 164 163 57 51 3 6 30 9 3 9 3 64 64 48 48 37 27 72 66 86 53

78

50

91

52

89

-

-

-

44

-

24 48

37

61 102

-

-

a

a -

-

46

-

-

24 62 59

102 142 40

-

-

- -

48 21 -

-

-

-

a

a

-

-

-

-

-

-

-

-

-

-

-

-

12 52 52

-

-

-

-

a -

a -

&

-

a

-

22 83 9 3 0 6 42 53 100 113 100 26 1 0 2 6 72 84 62 61 42 19 12 52 62 38 18

*Substituents are a-or p-oriented OH groups or 0 (carbonyl) groups at the positions shown.

a

a

a

-

-

-

a

a -

a a

-

-

-

-

-

-

-

-

-

25 0

-

8

-

-

56 1

-

-

30 102

26 102

10 100

-

-

-

3 132

15 7 81 57 46

-

4 0

73 8

-

46 12

7 78 56 48 8 44 12

44 40 3 28 5

0

-

-

0

12 0

-

-

128

0 128

113

-

0 68 34

0 60 27

0

51 0

12 18

8 12

-

0 63 26 53 0 7 12

-

0 0 -

16 49 0 3 8

THIN-LAYER CHROMATOGRAPHY

73

0 2 5

oZ5 0'6

oZ5 o~~ 025

BE-1

1 BE-2

€A-1

I

EA-2

CE-1

0"0 7

I

CAE

CE-2

Fig. 7.1. Separation of bile alcohols in different solvent systems by TLC. Solvent systems: BE-1 = benzene-ethyl acetate (3:2); BE-2 = benzene-ethyl acetate (2:3); EA-1 = ethyl acetate-acetone (4: 1); EA-2 = ethyl acetate-acetone (7:3); CE-1 = chloroform-ethanol (9: 1);CE-2 = chloroform-ethanol (4: 1); CAE = chloroform-acetone-ethanol (14:3:3). Compounds: 1= scymnol; 7 = 24-deoxyscymnol; 16 = SP-cholestane-3a,7a,l2a,26-tetrol; 25 = 5P-cholestane-3ol,7a,l2a-triol.(Reproduced from Steroids, 3 (1964) 62, with permission; [547].)

system chloroform-methanol-acetic acid-water (65:24: 15:9). Comparatively,little work has been reported on TLC of bile alcohols. Some of the results obtained by Kazuno and Hoshita [547] are shown diagrammatically in Fig. 7.1. Masui and Staple [699] have separated the stereoisomers Sj3-cholestane-3~2,7c,l 2~2,2401tetrol and S~-cholestane-3~2,7a,12ar, 24P-tetrol by TLC with benzene-isopropanol-acetic acid (30: 10:1). Solvent 12 in Table 7.1 does not separate the bile acids from each other, but serves t o separate this group from the neutral lipids prior to the quantitative analysis of total bile acids. Similar results are obtained with a 90:S4:11 mixture of chloroform, methanol, and 7.7 N NH40H [35S]. Preliminary fractionation of bile lipids is achieved when a 4:2: 1:1 mixture of 2,2,4-trimethylpentane-isopropanol-ethyl acetate-acetic acid is allowed to rise 13 cm past the origin and, after the plate has dried, a chloroform-methanol-water (65:35:4) mixture ascends only 6 cm [365]. A good preparative separation of individual free bile acids is obtained by continuous ascending development (see p. 000) with 2,2,4trimethylpentane-ethyl acetate-acetic acid (10:3:2 at S0C or 10:5:2 at room temperature) for 4-6 h [ 10111. The zones can be located by allowing water to rise on the inverted finished chromatogram, their position being revealed by an indentation in the advancing wet front.

14

BILE ACIDS AND ALCOHOLS

Another nondestructive method for detecting the bile acids on a thin-layer plate depends on viewing it under a UV lamp (350 nm) after spraying it with 0.05% pyrene in petroleum ether [256]. This indicator is easily removed by developing the plate with a 2:3 mixture of diethyl ether and petroleum ether (b.r. 60-80°), which does not affect the bile acids. When a 0.005% methanolic solution of the sodium salt of 8-hydroxy-l,3,6pyrenetrisulfonic acid is used in place of the pyrene reagent, the unconjugated bile acids can be eluted from the plate with acetone, while the fluorescent dye remains bound to the silica [549]. Characteristic colors are seen in daylight and at 366 nm when the thin-layer chromatograms are sprayed with a reagent composed of 0.5 ml anisaldehyde, 50 ml glacial acetic acid, and 1 ml conc. HzS04, and then heated at 125°C for 10 min [598]. Other detecting reagents for bile acids containing conc. sulfuric acid are: a mixture of 20 g SbC13 in 50 ml butanol, 10 ml HzSO4, and 20 ml acetic acid; a mixture of 2 g FeClj in 8 3 ml butanol and 15 ml HzS04 [28] ;a solution of 150 mg benzoic acid in 0.1 5 ml H2S04 and 20 ml acetic acid [363] ;and a mixture of 50 mg MnC12 *4H20 in 15 ml water and 0.5 ml conc. H2S04 [364]. A 1 :1 mixture of the ceric ammonium sulfate and molybdenum reagents differentiates between deoxycholic acid and chenodeoxycholic acid [ 10261, Bile acids may be determined by direct densitometry, after staining the thin-layer chromatograms by spraying with conc. H2SO4 and heating at 60-80°C for 20 min [409], by spraying with 20% phosphomolybdic acid in ethanol and heating at 110°C for 15 min [956], or by spraying with the anisaldehyde reagent described above and heating at 100°C for 4 min [823]. For direct fluorimetry, the chromatograms are sprayed with ca. 30% Hap04 and heated at 110°C for 20-30 min [99]. The colorimetric determination of bile acids requires nondestructive detection methods, such as spraying the plates with water [1167], or pilot chromatograms [330]. The adsorbent containing the zones is treated directly with 65% H2S04 at 60°C for 1 h [328], with a salicylaldehyde reagent, or with Liebermann-Burchard reagent [3311. Errors due to impurities were noted and reduced by purifying the silica gel [597]. The anisaldehyde reagent permits the sometimes incompletely separated chenodeoxycholic and deoxycholic acids to be determined individually [ 11671. The Pettenkofer reaction may also be used, but impurities in the silica gel, notably iron, interfere with the method [257]. A specific assay is based on the conversion of the bile acids to the 3-keto analogs by a 3a-hydroxysteroid dehydrogenase [ 127,2961.

7.3. GAS CHROMATOGRAPHY The applications of GLC to bile acids have been reviewed by both Sjovall [975,977] and Kuksis [6 1 1-6 131. After the conjugated bile acids have been isolated by LC or TLC, they are first hydrolyzed, and then the carboxyl group of the free bile acids is methylated. A convenient procedure [ 10911 for generating diazomethane involves the introduction of a sohtion of 4.28 g Diazald in 20 ml diethyl ether dropwise into a flask containing a mixture of 7 ml carbitol, 7 ml diethyl ether, and 10 ml60% aq. KOH. The flask is equipped with an inlet and outlet tube, through which diethyl ether-satd. nitrogen, flowing at a rate of 6 ml/ min, flushes the diazomethane into a solution of the bile acids in 10 ml diethyl ether-

GAS CHROMATOGRAPHY

75

methanol (9: 1). When the solution becomes yellow, the reaction is complete and the excess diazomethane can be destroyed with a drop of diethyl ether-acetic acid (9: 1). Subsequently, the hydroxy compounds may be converted to suitable derivatives, such as TMS ethers, TFA esters, etc. (see p. 31). Briggs and Lipsky [lo91 have studied the rate of TMS ether formation for the different hydroxyl groups in bile acids. When they were dissolved in dioxane (instead of pyridine) and allowed to react with HMDS and TMCS at room temperature, it was possible to derivatize the equatorial hydroxyl groups exclusively. Table 7.2 represents a summary of the retention behavior of common bile acid methyl esters observed with some of the currently used GLC methods. The mixed-phase column was prepared by coating 15 g Gas-Chrom P, which had been acid-washed and deactivated with 1% dimethyldichlorosilane in toluene, with a mixture of 0.8 g QF-1 in 60 ml acetone, 0.1 g NGS in 100 ml chloroform, and 0.1 g SE-30 in 50 ml toluene [8 121. On the fluorosilicone, QF-1, the order of elution of monohydroxy methyl cholanoates depends on the position of the hydroxyl group (12 > 7 > 6 > 3) and on its conformation (axial OH eluted before equatorial OH). The silicone OV-1 is less selective, and the phenylmethylpolysiloxane OV-17 approaches the performance of QF-1 but does not separate the conformational isomers as well. The monoketo esters are generally eluted in the order: 12 > 7 > 6 > 3. On OV-17, the 3,7-dione is eluted before the 3,12-dione, whereas the order is reversed on QF-1. With mixed functions and a greater number of functional groups the relations become more complex. Generally, QF-1 gives the best separation. Sandberg et al. [922] have also reported retention times of bile acid methyl esters on CNSi columns, and retention data for QF-1 columns may also be found in Refs. 249,812, and 1082. VandenHeuvel and Braly [ 11051 have added retention data of methanesulfonate and mixed (TMS t CMDMS) ether derivatives of methyl cholanoates on SE-30 columns to the identification methods for bile acids. Incomplete esterification of hyocholic acid by trifluoroacetic anhydride can be avoided by heating the reaction mixture to 80°C for 30 min [748]. The TFA derivatives are very valuable for the quantitative analysis of bile acids. Quantities as small as 0.1 ng of bile acids (or 0.2 ml of serum) can be determined with a 63Ni-ECDin the pulsed mode [531]. Computerized GC-MS (882, p. 431 has been applied to the analysis of bile acids in plasma [40]. The combination of mass fragmentography with a computer system, also called mass chromatography, as been further refined by Miyazaki et al. [724]. The technique, which involves the use of D-labeled conjugated bile acids as internal standards, permits the simultaneous qualitative and quantitative analysis of bile acids (e.g., in bile) and corrects for contaminants in chromatographic peaks. Of the many applications of chromatographlc methods to the assay of bile acids in biological materials only the analysis of feces will be specifically mentioned. This is one of the most difficult problems and one of the greatest achievements in this field [987]. Eneroth et al. [275-2771 have succeeded in the quantitative analysis of fecal bile acids by use of a series of steps, involving solvent extraction, saponification, preliminary purification on silicic acid columns, conversion to the TFA methyl esters, and GLC on QF-1 columns. Grundy et al. [376] have devised a similar procedure, which also includes TLC. In the method of Evrard and Janssen [290] the methyl esters are converted t o their keto analogs by mild Cr03 oxidation and then chromatographed on a 1% JXR column.

76

BILE ACIDS AND ALCOHOLS

TABLE 7.2 RELATIVE RETENTION TIMES OF METHYL CHOLANOATES -

1; I

Conditions* Orientation at C-5 Substituents**

a p

-

-

-

-

-

a

-

-

-

P

-

-

-

-

a P

-

o

-

-

a

a

-

-

-

a

p

-

-

a

-

a

-

P

-

a

-

a P

-

a

-

-

a

P a a 0

-

-

a a

o -

-

-

o o

-

o -

a a

a p

a a

-

a

o

-

-

0

a

-

O

a a

0

a

a

p

-

a

P

P

-

a

-

P a

-

a a

a a

a a 6

-

0

-

a 0 0

-

p a 0 O a 0 0 0

a 0 a a 0 0 0 0

TMS

1.00 1.77 1.42

2 P TMS

3 P TFA

4 P TFA

5 P OH

6 P TFA

7 P OH

8 P TFA

1.oo -

0.44 1.06 0.89 -

0.35 0.81 0.76

0.31 0.37 0.34

0.7 1 1.13 1.08

0.34 -

0.22

3.39 1.93 1.50 2.12 1.00 -

2.22 1.62 1.53

-

1.69 1.28 1.17 1.40

-

5.54 1.64 1.40 2.04 1.30 5.45 5.16 -

1.35

-

1.31 0.93 2.6 1 1.58 0.97 4.01 4.10

4.47 4.44 -

-

0.45 1.42 1.13

-

-

0.22 0.53 1.58 1.28 1.10 1.23 1.00 -

1.77 1.00 3.80 -

1.21 1.00

1.22

1.30 -

-

1.44

1.41

-

1.50' 1.51

-

1.oo

-

2.00

1.09 1.97 -

-

3.86*** 4.13*** 3.34*** 3.36*** 3.31*** 4.27*** 4.17***

-

0.11 0.45 0.43 0.40 0.47 0.30 0.84 0.64 -

0.58+ 0.94 0.37 0.78+1.19 0.67 0.60 1.33 1.oo

3.85*** 2.5 1 4.62***

*Conditions: 1 = 0.5% HI-EFF-8B on GasChrom P, 9 ft. X 3 mm,245"C, relative to methyl cholanoate [693]; 2 = same as 1, except at 280°C; 3 = 0.4% NGS on Anakrom A, 145 cm X 0.4 cm, 210"C, relative to methyl deoxycholanoate [lo811 ; 4 = sameas 3, except 0.6% CNSi, 150 cm x 0.4 cm, 216°C; 5 = same as 3, except 0.4% CNSi, 75 cm X 0.4 cm, 230°C; 6 = same as 3, except 1%SE-30,200 cm X 0.6 cm, 220°C; 7 = QF-1 + NGS + SE-30 (see text), 6 ft. X 5 m m I.D., 210"C, relative to methyl deoxycholanoate [812]; 8 = same as 7; 9 = same as 7 [813]; 10 = 3% QF-1 on GasChrom Q, 6 ft. x 4 m m I.D., 230°C, relative to methyl deoxycholanoate [269]; 11 = same as 10, except OV-1; 360°C; 12 = same as

9 P TMS

-

0.20 -

0.26 0.24+0.56 0.2 2+ 0 .SO 0.28+0.54 0.22+0.45

0.3 1+ 0.4 7 0.5 1 0.23+0.47+ 1 . 3 ~ -

-

-

GAS CHROMATOGRAPHY

10 P OH

11 P OH

12 P OH

0.15 0.49 0.44 0.34 0.36 0.27 0.31 1.00 1.50 1.20 1.18 0.94 1.27 1.oo 0.86 0.70 1.80 1.61 2.40 1.82 1.10 3.05 2.86 3.14 2.42 2.67 2.33 2.33 1.90 2.20 3.85 3.51 4.79 4.55

0.35 0.62 0.64 0.60 0.59 0.55 0.56 0.7 1 1.20 1.11 1.08 1.06 1.06 1.oo 1.00 0.9 1 1.03 1.00 1.23 1.07 1.13 1.04 1.05 1.85 1.84 1.69 1.68 1.66 1.63 1.52 1.73 1.59 1.88 1.so

0.20 0.52 0.5 1 0.44 0.46 0.39 0.4 1 0.61 1.32 1.21 1.14 1.10 1.13 1.oo 1.00 0.84 1.20 1.12 1.43 1.17 0.88 1.21 1.29 2.56 2.65 2.25 2.30 2.26 2.08 2.07 2.57 2.27

-

2.79 2.18

-

6.40

__

1.39

2.25

77

13

14

15

Q

0

Q

OH

OH

OH

0.18 0.50 0.55 0.40 0.43 0.37 1.06

0.40 0.72 0.72 0.64 0.67 0.60 0.76 1.18 1.18 1.12 1.16 0.99 1.23

0.24 0.58 0.60 0.5 1 0.52 0.49

-

1.22 1.29 1.07 1.16 0.90 1.97 2.32 2.23 1.56 3.96 3.52 -

-

2.10 2.68 2.78 -

-

4.62 4.50 6.10 -

-

9.24

-

1.27 1.19 1.09 1.17 1.15 -

1.78 1.77 1.88 -

.-

1.95 1.85 1.96

-

1.84 1.64

-

0.71 1.27 1.34

-

1.19 1.20 0.99 1.42 1.60 1.41 1.13 1.52 1.51 2.34 2.73 2.71 2.89 2.87 3.12 3.04 2.98

16 P TMS

17 P TMS

18 P TMS

0.93 0.86 0.63 0.75 0.62 0.56 1.08 0.98 1.oo 0.87 1.14 1.oo 0.90 0.60 1.98 1.91 1.84 1.67 1.35 -

-

-

0.91 0.83 0.65 0.7 1 0.59 0.55

0.85 0.81 0.61 0.71 0.58 0.54

1.20 1.08 1.09 0.96 1.24 1.00 1.02 0.72 3.65 3.19 3.81 4.10 2.74

1.12 1.10 1.34 0.96 1.21 1.oo 1.oo 0.69 1.34 1.33 1.17 1.09 0.93

-

-

1.38 1.05 1.88 1.44 1.05 0.96

-

4.37 4.95

-

5.84 4.55 12.5

-

-

-

1.15 1.12 1.92 1.45 1.09 0.96 1.72 1.59 1.35 1.88 1.56 -

-

1.11 0.84 1.71 1.18 0.90 0.79 1.97 2.03 1.68 3.38 3.10 -

19

20

21

Q

0

OL

TMS

TMS

TMS

-

0.72 0.82 0.57 0.78 0.60

0.92 1.20 0.57 0.83 0.64 0.92 1.oo 1.22 1.19 0.53 2.30 1.60 1.85 1.54 -

0.89 1.20 0.62 0.81 0.66 -

-

1.00 1.14 0.93 1.35 0.63 4.16

-

0.84 1.08 1.18 1.23 0.63 1.41

-

--

4.12 3.04 3.42 -

1.08 1.17 0.97

-

1.41 1.00 1.18 5.45 7.00 5.07 12.7

-

11, except OV-17; 13 = same as 10; 14 = same as 11; 15 = same as 12; 16 to 21 = relative to bis-TMS ether of methyl deoxycholanoate; 16 and 19 same as 10; 17 and 20 = same as 11; 18 and 21 = same as 12. **In the vertical row the position ofhydroxyl (a! and P ) and carbonyl(0) substituents is given. The horizontal row shows whether the hydroxyl groups are free (OH) or converted to derivatives (TMS, TFA). ***Column temperature 220"~.

-

-

-

-

1.44 1.05 1.01 1.62 1.96 1.09 1.78

-

-

1.17 0.87 0.79 1.95 2.46 1.45 3.76 -

This Page Intentionally Left Blank

Chapter 8

Estrogens The estrogens are characterized by an aromatic A ring, and some use may be made of the phenolic properties of this class in chromatographic analysis. Individual estrogens differ from each other in the number, location, and nature of oxygen functions. In addition to the 3-hydroxyl group, they usually also have an oxygen at C-17. In body fluids, the hydroxyl groups may be conjugated with sulfuric or glucuronic acid.

8.1. LIQUID COLUMN CHROMATOGRAPHY

Hahnel [384] introduced the use of DEAE-Sephadex columns in the analysis of urinary estrogens. By eluting them with a suitable concentration gradient of NaCl, it was possible to separate not only estrogen glucosiduronates from pregnanediol glucosiduronate [385], but to obtain free estrogens, estrogen 3-glucosiduronates, estrogen 16 (or 17)-glucosiduronates, and estrogen sulfates in separate fractions [386]. Using a linear NaCl gradient from 0 t o 0.8 M, Hobkirk et al. [446] and Hobkirk and Nilsen [447] further showed that DEAESephadex is capable of separating (in the order of elution): estradiol, estrone glucosiduronate, estradiol 17-glucosiduronate,estrone sulfate, estradiol 3-sulfate, estradiol 3-sulfate 17-glucosiduronate, and estradiol3,17-disulfate.More recently, Van der Wal and Huber [ 11391 have determined the suitability of a number of dextran and cellulose ion exchangers for HPLC. Fig. 8.1 shows a chromatogram obtained by use of Cellex E, an ECTEOLA-cellulose. Just how much of these separations is due to ion exchange is not clear. Certainly, ordinary gel filtration through Sephadex can also effect some fractionation. As early as 1963, Beling [58] observed two separate estrogen peaks when he washed a Sephadex (2-25 column, to which a sample of pregnancy urine had been applied, with water. The first peak contained estriol3-glucosiduronate and the second a mixture of estriol 16- and 17-glucosiduronates. Gel filtration is a very valuable step prior to the enzymatic hydrolysis of urinary estrogens, because it eliminates enzyme inhibitors [5,720]. It has also been used for the isolation of estriol glucosiduronate on a preparative scale [ 1931. A method

I

1

1

1.5

I

,

I

2 .o T i m e (hours)

,

-

1

25

1

1

1

10

Fig. 8.1. HPLC of estrogen glucosiduronates on a column of Cellex E, particle size 9 pm, 25 cm X 0.3 cm, at 4O"C, eluted with 0.5 MCI- + 0.05 M OAc- at pH 4.5. T = Testosterone, E, = estrone, E , = estradiol, E = estriol, G = glucosiduronate. Estriol 17-glucosiduronate and estriol 16-glucosiduronate are incompletely separated. (Reproduced from J. Chrornatogr.,102 (1974) 373, with permission; [1139].) 79

80

ESTROGENS

for separating the four estriol conjugates known to occur in urine was published by Tikkanen and Adlercreutz [ 10551. A 10-ml aliquot is filtered through a Sephadex G-25 column, 100 cm X 1 cm, according to Beling's method (see above). The first fraction is chromatographed on a 4-g Sephadex LH-20 column with a 0.01 MNaCl solution in chloroform-methanol (2:3), yielding estriol3-glucosiduronate, and then with methanol, yielding estriol3-sulfate 16-glucosiduronate. The second fraction, when chromatographed on a 4-g Sephadex LH-20 column with satd. NaCl solution in chloroform-methanol (2: l), yields, in succession, estriol 16-glucosiduronate and estriol3-sulfate. For the separation of equine estrogen 3-sulfates, 14 g of Sephadex G-15 in a column, 11 mm I.D., was eluted NaCl [491]. The elution volumes were: estrone, 73; equilin, 90; 17P-dihydrowith 5 X equilin, 100; equilenin, 125 ;17/3-dihydroequilenin, 160 ml. Eechaute and Demeester [262] have used Sephadex G-25 t o isolate estrogens in 95% yield after the hydrolysis of urine [471]. Sephadex G-15 is even more suitable [ 10901. In 1971, Horst [4701 demonstrated that estrone, estradiol, and estriol can be completely separated on Sephadex G-10. Up to 200 ml of urine, saturated with Na2S04 and adjusted to pH 4.6 k 0.2, was applied to a 600-mm X 12-mm Sephadex G-10 column. The estrogens, which were adsorbed near the top of the column, were subsequently eluted (in the order: estriol, estrone, estradiol) by a convex gradient, produced by adding 0.1 N NaOH to 16% Na2S04 in a 50-ml mixing vessel. Van Baelen et al. [ 10891 have compared the adsorption of estrogens on Sephadex G-25 and LH-20. Water eluted estriol, then estrone, and finally estradiol from the Sephadex LH-20 column, whereas estriol and estrone remained unresolved by Sephadex G-25. Using 0.02N NaOH as the eluent, Horst et al. [472] were able to separate estradiol and estriol epimers on a Sephadex LH-20 column, 60 cm X 1 cm. At 25"C, they detected first estriol, then estrone, followed by estradiol-l7a, and finally estradiol-170. At 55'C, 16-epiestriol emerged first, followed by estriol, and finally a mixture of 17-epiestriol and 16,17-epiestriol. The equine estrogens were successfully resolved on Sephadex LH-20 with cyclohexanebenzene-methanol (20:6:3) as the eluent [602]. Lisboa and Strassner [665] have investigated the behavior of twenty-three estrogens on Sephadex LH-20 with six nonaqueous solvent systems and used gel chromatography to test their radiochemical purity. Adsorption chromatography of estrogens has a long history. Only the most recent results can be mentioned here. Eechaute et al. [263] have chromatographed an extract from 3 ml of hydrolyzed urine, after gel filtration (see above), on a column, 5 cm X 1 cm I.D., containing 2.5 g silica gel. The column was eluted with 15 ml2.5% ethanol in chloroform, followed by 10 ml 20% ethanol in chloroform, and the eluates were collected in seven 3-ml fractions. After the first 2 ml of effluent had been discarded, estrone was recovered from fractions 1 and 2, estradiol from 3 and 4, and estriol from 6 and 7. With the advent of HPLC, Huber et al. [478] demonstrated the fractionation of urinary estrogens by liquid-liquid partition [479]. Later, Butterfield et al. [134] applied a variety of partition systems to the separation of equine estrogens by HPLC. Dolphin [241] obtained a complete chromatogram within 6 min on a 1-m X 2.2-mm I.D. column of Corasil I, 37-50 pm, with a hexane-ethanol(24: 1) eluent. Similarly, Lotscher and Kern [667] separated estrone, estradiol, and estriol in 5 min on a 250-mm X 2.1-mm column of Micropak SI-60 with a linear gradient from 5 to 12% of chloroform-isopropanol(1:l) in heptane.

THIN-LAY ER CHROMATOGRAPHY

81

8.2. THIN-LAYER CHROMATOGRAPHY Although some work with polyamide layers has been reported [183,474,475,1082] and some advantages have been claimed for alumina layers without binder [ 10051, silica gel is the adsorbent of choice for TLC of estrogens. Continuous development has been advocated [600,677]. For the separation of the equine estrogens, four developmen'ts with cyclohexane-ethyl acetate (7:3) on layers prepared from a slurry of 40 g Silica Gel H in 100 ml of 7.5% aq. AgN03 [211], or three developments with chloroform-cyclohexaneacetone-NH40H (15:27:4:4) on Silica Gel Fzs4 [943] were found necessary. Table 8.1 lists some ~ R values, F determined for various natural estrogens. Generally, the polarity of the substituents is in the order: 6-0 < 6 a < 60 < 110 and 17-0 < 17a < 170. The resolution of the estrone-equilin-equilenin triad on Silica Gel G is usually unsatisfactory. For the separation of the four epimeric estriols by TLC, Hertelendy and Common [430] have converted them to their methyl ethers, and Touchstone et al. [ 10601 prepared the acetonides. However, Lisboa [653] was able to effect the resolution of the free estriols by triple development with cyclohexane-ethyl acetate (1 :1). Data on the chromatographic properties of estrogen acetates may be found in Lisboa's paper [653], and various esters and ethers have been studied by Rajkowski and Broadhead [876]. There are also publications on TLC of estrogen dyes, produced by coupling with Fast Violet Salt B [884], of oximes and D-homo-aza-lactams [701], and of Dns derivatives [253]. Fishman et al. [313] have used two-dimensional TLC to separate the glucosiduronates of estradiol and estriol. The separation of various estrogen sulfates on AgN03-impregnated Silica Gel G [212] and on activated Silica Gel H [316] has also been reported. Lisboa and Diczfalusy [662] have made a comprehensive study of detection methods for TLC of estrogens. Amounts of estrogens as small as 1 pg can be detected in W light, if the plates have been sprayed with fluorescein. Reactions based on the phenolic nature of estrogens are: the Folin-Ciocalteau; K3Fe(CN),-FeC13 ;coupling with Fast Black Salt K, Fast Blue VB Salt, and Fast Red Salt GG; Feigl's Na3Co(NOz)a; and the nitroso reaction. The o-dihydroxyphenols also react with phloroglucinol and with NH4V03. Unsaturated compounds give the Os04, C(NOz)4, and Tortelli-Jaffd reactions, ketones the Gornall-McDonald and the Zimmermann reactions, and a$-diketones the Ischidate and p-aminodiethylaniline-SOz reactions. Some of the nonspecific detection methods for steroids have already been mentioned (p. 18). The phosphomolybdic acid test becomes specific for phenols when the plate is afterwards treated with alkali [450]. Varon et al. [112] have compared fourteen methods of detecting estrogens and their acetates on thinlayer chromatograms. They found the following spray reagent most sensitive, convenient, and reliable for both free and acetylated estrogens: A solution of 2 g sodium p-phenylsulfonate in 100 ml 85% H3P04 is diluted with nine volumes of 95% ethanol. A method for the quantitative estimation of nanogram amounts of estrogens by TLC is based on the electrical conductivity of phenols [768]. They are detected as they pass a pair of Pt electrodes during continuous development with ketonic solvents in a BN chamber. A so-called photogrammetric procedure [509,5 101 is based on photographing in W light the fluorescent zones produced by spraying estrogens with H2S04. The strip of developed film is then evaluated densitometrically. If the layers have been prepared with a slurry of silica gel in 10%phosphomolybdic acid, heating the chromatograms at

TABLE 8.1

hRF VALUES OF ESTROGENS IN TLC ON SILICA GEL G

m

w

Solvent systems: 1 =ethyl acetate-cyclohexane-ethanol:9:9:2) [653]; 2 =ethyl acetate-cyclohexane (1:I) [653]; 3 = chloroform-ethanol (9:l) [653]; 4 = ethyl acetatehexane-ethanol-acetic acid (144:27:9:20) [653]; 5 = benzeneethanol (4:l) [6531;6 = benzene-ethanol (9:l) [653]; 7 = chloroform-ethanol (19:l) 16531; 8 = ethyl acetate-hexane-acetic acid (15:4:1) [653];9 = hexane-ethyl acetate (3:1) [6531; 1 0 = chloroform-diethyl ether (3:l) 16531; 11 = benzene-ethyl acetate (3:l) 1653); 12 = benzene-ethyl acetate (1:l) 16531; 13 = benzene-diethy1 ether (1:l) 16531; 14 =dUsopropyl ether-ethanol (93:7) [1064]; 15 = diisopropyl ether-acetone (4:l) [1064]; 16 = ethylacetate [1064]; 17 = chloroform-acetic acid (1O:l) 184). Substituents'

Double bond (s)"

2

3

6

11

16

17

-

OH OH

-

-

-

o

-

0

7 6

-

-

OH

OH OH

0 -

-

&

-

-

8

-

-

-

OH

8 -

-

OH

-

OMe

OH

OH

-

OH -

OMe

-

OMe

OH OH OMe

OH OH OH OH OH

OH OH OH

OH OH OMe OMe OMe OH OH

OH

-

-

OH OH OH

-

0

o

0

-

-

o o

-

-

-

-

o

-

-

-

-

-

-

-

I

-

8

-

-

j

-

-

0 (I

-

8

-

-

(

-

-

O

-

-

-

-

P

-

-

a

-

-

7

-

p p

-

0

0

-

-

-

P

-

-

8 P

-

-

-

-

3

-

-

a

p

-

-

-

P

O

-

-

a

&

-

-

p

a P

-

-

-

-

-

p

8

-

-

p

-

-

-

-

-

_

-

a a

p p

-

-

a

p

-

-

--_

2

69 53 68 63 47 6 0 3 59 34 66 43 47 13 46 12 56 27 51 32 68 50 61 40 61 43 5 7 3 6

-

0

O

a

-

-

-

-

1

0

0

Solvent system

4

65 66 63 3 61 64 35 34 50 58 73 52

80 59 78 77 58 74 -

2 1 1 2 1 29 6

10

11

12

13

14

15

51

46

69

20

50

44

66

58

-

-

-

-

60 60

56

-

55

-

-

16

17

30

22

44

38

-

-

-

-

-

-

36

39

58

44

65

45

-

-

-

-

-

-

-

-

-

-

-

-

-

-

26 54 - - 33 66 69 74 53 42 30 61 55 75 52 45 34 62 5 3 7 4 5 0 4 1 3 0 5 9

4 6

-

-

-

21

21

37

-

-

-

-

-

-

-

-

58

93 78 70

-

-

-

65 62 72 68

-

_

-

9

5

-

-

8

-

32 55 69 2 1 2 4 7 0 1 2 2 3 6 1 12 23 61 8 2 9 5 4 3 6 21 48 1 8 2 6 20 30 58 8 18 50 36 58 72

.

7

58

-

-

6

-

59 4 7 45 44 34 29 4 3 45 34 58

_

-

5

3

-

-

-

3

5

-

-

-

-

42 45 38 41 45

-

-

51

42

-

_

50

-

-

-

-

-

-

65

-

16

-

-

-

-

-

18 10

11 7

-

-

- 4 7 2 1 2 4 4 10 43 9 2 0 9 3 4 0 38 17 7 29 5 4 - - 1 7 21 -

-

53

-

-

-

-

45

41

57

--

-

-

-

4 - 2 4 - 43 -

__

-

1

-

-

-

8

-

-

-

30 35 43 23 -

-

-

1 8

-

24 27 32 19 -

44 51

38 43

51 53

52 45 41

-

-

-

51 40

-

-

-

-

35

42

39

66

-

-

2 - 5

-

-

-

-

-

-

-

-

7 16

-

32 21 37 3 2 8 12 5 18 12 7 20 -

34 -

-

-

-

4 11 - 15 -

-

l a 1 0 - 1 3 2 6 -

-

-

-

-

-

-

-

-

9

7

-

_

6

'Substituents on estra-l,3,5(10)-triene are shown as OH, OMe, and 0 (carbonyl) at the positions indicated. For hydroxyl groups the orientation ((I or p ) is given. "The position of double bonds in Ring B is shown.

-

43

-

62 -

-

GAS CHROMATOGRAPHY

83

1 10°C for 20 min will produce colored zones that can be read directly by a densitometer [1059]. Alternatively, chromatograms on ordinary silica gel may be dipped in a 5% ethanolic solution of phosphomolybdic acid and then heated [ 11721. A simple, yet highly specific and sensitive method for the determination of individual estrogens in 1 ml of urine is based on coupling them with Fast Black Salt K and chiomatographing the azo dyes on aluminum foil coated with silica gel [ 115 11. Alternatively, the dyes can be formed on the foil, if the layer is treated with Fast Black Salt K [ 1 1521. They are separated by a 15-cm development with diethyl ether, followed by a 7-cm development with chloroform-dimethyl formamide-acetic acid (80:5 : 1) and assayed in a densitometer. Similarly, estrogens can be coupled with Fast Violet Salt B, the azo dyes separated with a butyl acetate-benzene (1 7:3) developer, and then scanned [ 1058,10661. Esters of estrogens, prepared with azobenzene-4-sulfonyl chloride, are also suitable for densitometry after separation by, e.g., chloroform-benzene-ethanol (18:2: 1) [847]. Dns derivatives of estrogens separate in the same solvent system and can be estimated in nanogram quantities by direct fluorometry [808,848,849}. The fluorescence is excited at 362 nm and measured at 5 17 nm. The spontaneous decomposition of estrogens on silica gel plates is particularly undesirable when TLC is used to purify picogram amounts for protein-binding methods. It is generally assumed that oxygen is responsible for this decomposition, because when plates and solvents are freed of contaminants [858], when plates are kept under nitrogen [206], or when reducing agents, such as mercaptoethanol [239] or ascorbic acid [346,744], are incorporated into the chromatographic systems, destruction is prevented. However, recent evidence indicates that spontaneous decomposition of estrogens must be due t o atmospheric pollution [619]. The determination of estrogens after elution from thin-layer plates requires nondestructive methods. Iodine is not suitable for this purpose [722]. Sodium fluoresceinate, added t o the silica during preparation of the layer at a level of 1 pg per g of Silica Gel G, permits the detection of estrogens at 254 nm [886]. If the estrogens are chromatographed in the form of Dns derivatives, they can be located by their fluorescence at 366 nm [252]. Similarly, the PIPSYL derivatives are detected at 254 nm [503].

8.3. GAS CHROMATOGRAPHY The determination of estrogens in biological fluids by GC has been reviewed by Adlercreutz et al. [ 121 and by Morvay [747]. Although difficult, GC is now the preferred method of estrogen analysis in many clinical laboratories. It may involve a number of steps, such as hydrolysis, extraction, LC, and derivatization. In a procedure for the analysis of 25 estrogens in urine, Knuppen e t al. [585] used multiple PC, acetylation, TLC of the acetates, and then GC. For the analysis of estrone and estradiol in children’s urine, Eberlein [259] had to isolate the estrogens first by passing a methanolic solution through an anion exchanger in the carbonate form. They were then eluted with aq. methanol and subjected to, in sequence, KBH4 reduction, TLC, acetylation, and GC. The silica gel for TLC had to be specially purified [745]. For less exacting problems, of course, simpler purification procedures will suffice [689]. Thus,

84

ESTROGENS

for the analysis of estrogens in plasma, the estrogens have been isolated by TLC and purified by rechromatographing them in the form of their acetates [620]. The method was applicable to estradiol and estriol in urine, but estrone had to be purified in the form of its methyl ether by chromatography on an alumina column [ 10511. Methods for GC of free estrogens are known [32,33,604,710,1065], but their derivatives are much more volatile, less adsorbed, better separated, and easier t o detect. TABLE 8.2 RETENTION TIMES RELATIVE TO CHOLESTANEFOR SOME ESTROGEN DERIVATIVES 17351 Substituents* 2

3

4

OH OTMS OAC OH OTMS OTMS OAC OAC OH OAC OMe OMe OMe OMe OH OTMS OAC OH OTMS OH OH OTMS OMe OMe OMe OMe OMe OMe OMe OAc OMe OMe OMe OMe OMe

-

-

-

-

OH OH OH OH

-

OH OAc

16

17

2F-1

5e-30

2.5 1.6 3.9 1.8 0.89 0.63 2.6 1.7 2.8 3.9 1.3 0.89 1.8 1.8 3.5 1.5 3.5 35 4.9 1.9 2.7 2.1 2.3 2.0 1.2 2.6

0.45 0.5 1 0.54 0.46 0.65 0.55 0.68 0.55 0.68 0.42 0.54 0.55 0.38 1.2 1.5 0.94 1.1 0.89 1.4 0.76 0.92 0.50 0.57 0.67 0.60 0.57 0.67 0.72 -

-

2.8 2.7 2.0

*Substituents on estra-l,3,5 (lO)-triene; 0 = carbonyl. .**Columns, 6 ft. X 0.101 in., packed with 3% of the stationary phases shown on Chromosorb W, 60-80 mesh, at 255"C, except in case of TMS ethers at 258°C.

GAS CHROMATOGRAPHY

85

TABLE 8.3 RETENTION TIMES________ RELATIVE TO CHOLESTANE FOR ESTROGENS TMS ETHERS [7] 1 . -

Substituents*

2

3

OMe

OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH

-

OMe

I-I

Column**

____._

6

16

17

0 a-OH -

0 0 0

0

0 C&H

-

0-OH 6-OH o-OH @-OH CY-OH

0

a-OH 0-OH 0-OH 0-OH &OH @-OH @-OH D-OH' a-OH a-OH 0-OH

1

2

3

4

1.58 2.32 1.76 5.30 0.76 0.85 2.26 3.02 1.26 1.30 1.40 1.58 1.36 1.20 2.12

1.37 2.32 1.74 3.64 0.70 0.79 2.12 2.43 1.25 1.26 1.34 1.59 1.36 1.16 2.08

3.44 5.02 3.53 11.80 0.90 1.07 4.02 3.80 1.23 1.92 1.64 1.76 1.54 1.28 2.74

0.60 1.18 0.98 1.14 0.62 0.70 1.08 1.38 1.00 0.99 1.23 1.30 1.14 1.04 1.86

*Substituents on estra-l,3,5(1O)-triene; 0 = carbonyl. **All columns were 6 ft. X 4 mm and stationary phases were on GasChrom P, 100-140 mesh: 1 = 2% QF-1 at 203°C; 2 = 1% XE-60 at 205°C; 3 = 1%Z at 195°C; 4 = 3% F-60 at 230°C.

The acetates [676,1174,1175],methylethers1606,6781,TMS ethers [ 3 , 7 , 6 8 5 ] , and methyl ether acetates or methyl ether TMS ethers [205] have been used earlier. Halogenated derivatives, such as the monochloroacetates [875], TFA [439,440], and HFB [ 4 3 9 4 1 , 8941 derivatives, can be assayed in quantities of less than 1 ng by the ECD. A convenient method for the preparation of HFB derivatives is the vapor-phase acylation described by Rigby et al. [894]. An alcohol solution, containing up to 5 pg of estrogens, is evaporated on a steel gauze, which is then placed in a vial. This vial, together with a vial containing HFBA and a third one containing PzOs, are heated in a glass-stoppered bottle at 45'C for 2 h. At the end of the incubation, the HFB derivatives are washed off the steel gauze with benzene. A derivative which can be detected in picogram amounts with the AFID is the dimethylphosphinic ester [ 11331. These esters are easily prepared in quantitative yield by heating 50 p1 of estrogen solution in anhydrous acetonitrile with 10 p1 dimethylaminodimethylphosphine under nitrogen at 90°C for 2 h and have excellent GC properties. The retention times relative to cholestane for a series of derivatives of natural estrogens, determined by Montalvo and Wheeler [735], are shown in Table 8.2!. The differences in R T values are much smaller on the nonpolar SE-30 than on the polar QF-1 column. For QF-1 the RT values are in the order ether < alcohol
86

ESTROGENS

analysis of estrogens in biological materials by GC-MS. They involve gel filtration, enzymatic hydrolysis, solvent partition, LC, Girard separation, methylation, acetonide formation, and preparation of TMS ethers before individual fractions can be introduced into the gas chromatograph. In that way, nanogram amounts of the various estrogens can be identified and quantitatively determined, even if they are incompletely separated. The determination of estriol in pregnancy urine or plasma, which is clinically important, is relatively simple although estriol shows a longer retention and lower detector response than most estrogens. The urine is hydrolyzed and extracted, and the extract is used for silylation [723,930] or acetylation [ 10421, usually without any preliminary purification. Plasma requires a purification of the extract by TLC before it is silylated [310,879]. Except during late pregnancy, the amounts of estriol in plasma are so low as to require analysis by an electron-capturing derivative, such as the 16,17-diheptafluorobutyrate of the 3methyl ether [ 171. The simultaneous analysis of estriol and estetrol (estra-l,3,5(10)triene-3,l 5a,16a117fl-tetrol)in pregnancy urine is difficult, requiring elaborate purification [424], a combination column prepared from a 2: 1 mixture of 3% SE-30 and 3% QF-1 on Diatomite CQ [634], or a mass spectrometer [552]. Similarly, the analysis of 2-hydroxyestriol involves two LC and two PC steps [347]. The equine estrogens, especially the estrone-equilin pair are difficult to separate by GC. Schroeder et al. [944] tried to solve this problem by epoxidation of the equilin. Some improvement in resolution was achieved by chromatographing the MO-TMS derivatives on a 3% OV-225 column, 12 ft. X 1/4 in. O.D., at 225°C [683]. Roman et al. achieved separation of the TMS derivatives on a 1% DEGS column, 8 ft. X 2 mm O.D., at 200°C [900] and identified the equine estrogens by MS [899]. The correlation between the structure and behavior in GC of mono- and disubstituted 2- and 3-oxygenated estratrienes has been studied on the basis of the steroid number (see p. 48) concept [766]. GC has been used to determine the solubility of estradiol in organic solvents [912]. Finally, two GC techniques should be mentioned which have been proposed for estrogens. One is solid sample injection [137,605] and the other is a method for collecting effluent fractions in dioxane at 7°C [603].

Chapter 9

Androstane derivatives The CI9 steroids may be said to have very few chemical properties useful for chromatographic analysis. All of the biologically important ones carry oxygen atoms at C-3 and usually at C-17. The 17-ketosteroids are quantitatively the predominant group of urinary steroid metabolites, which are usually conjugated with sulfuric or glucuronic acid.

9.1. LIQUID COLUMN CHROMATOGRAPHY One of the oldest methods of fractionating steroids, the chromatography of 17-ketosteroids on alumina columns, is still with us. In its modern form, gradient elution [93] and fully automated multiple capillary columns [ 11211 are used in conjunction with a computerized photometric read-out system [ 11 191 (see p. 9). Column chromatography on silica gel has also been modernized by gradient elution [520,1157] and use of capillaries [ 10171. Florisil has been recommended for the separation of free 17-ketosteroids from their sulfates and glucosiduronates by elution with 4, 20, and 75% methanol in benzene, respectively [ 10571. Other sorbents which have been successfully applied to the fractionation of androstane derivatives are: partially esterified Amberlite IRC-50 [954], DEAE-Sephadex [445], Sephadex LH-20 [674], and Lipidex [83]. Fitzpatrick et al. [3 151 have adapted HPLC to the analysis of 17-ketosteroids by converting them to 2,4-dinitrophenylhydrazonesto make them W-absorbing. The 1-m X 1/8-in. O.D. column was packed with 1.5% P,$-oxydipropionitrile on Zipax, and the mobile phase was isooctane. For the 1 l~-hydroxy-l7-ketosteroids, which were too strongly retained on this column, a reversed-phase system was used. They were chromatographed on a 1.2-m X 1.8-mm I.D. Corasil CI8 column with 50%aq. ethanol as the mobile phase.

9.2. THIN-LAYER CHROMATOGRAPHY The application of TLC to CI9 steroids has been reviewed by Lisboa [657]. Aside from a few experiments with alumina [399,776,825 J and polyamide 18461 layers, most of the reported work has been done with silica gel. In a few cases, silica has been used as a carrier for the stationary phase of partition systems [ 178,529,7411, but the predominant mode of operation is adsorption on Silica Gel G [61,254,302,350,672,829,883]. Table 9.1 lists some ~ R values F determined in Lisboa’s laboratory [647,649,650,663]. Lisboa’s papers as well as that of FehCr [299] also list RM values and give generalizations about the relation between structure and chromatographic behavior. For the difficult separation of testosterone and epitestosterone, Remmers et al. [885] use five repeated developments with dichloromethane-ethyl acetate (9: 1). Two-dimensional TLC was applied to the also difficult [369] resolution of 16-unsaturated CI9 steroids [82]. Some work has been reported on TLC of C19 steroid oximes and lactams [360,702],

88

ANDROSTANE DERIVATIVES

TABLE 9.1

hRF VALUES OF ANDROSTANE DERIVATIVES IN TLC ON SILICA GEL G (Data from Refs. 647,649,650,663) Solvent systems: 1 = cyclohexane-ethyl acetate-ethanol (9:9:2); 2 = cyclohexane-ethyl acetate (1: 1); 3 = chloroform-ethanol (9: 1); 4 = chloroform-ethanol (19:l); 5 = ethyl acetate-hexane-acetic acidethanol (144:27:20:9); 6 = ethyl acetate-hexane-acetic acid (15:4: 1); 7 = benzene-ethanol (4: 1); 8 = benzene-ethanol (9:l); 9 = benzene-ethanol (19: 1). Substituents*

1

3

Solvent system

4

5

-

0 0

-

A A

O 0 O

A A

A

O

A

-

-

0

-

p

-

0

a

p 0

-

0

-

P p @

A -

a a

-

p

-

A

-

-

A A

O 0 0 O

A A A

A

0 O

A A

-

a

-

-

0

-

a

-

-

-

-

-

-

P

@ a 0 &

-

A

69 66 67 60 51 50 56 43 58 56 50 57 58 56 57 32 26 31 63 63 56 53 55 46 62 60 57 64 62 55 48 43 59 31 27 48 33 39

64 63 58 55 47 48 50 48 33 29 25 34 30 29 30 15 12 13 42 42 38 37 39 31 36 30 44 40 39 44 39 49 37 33 44 34 32 41 29 36

-

0 0

-

-

-

0 -

-

-

-

p

-

p

-

p

-

p p

-

p

-

-

o

r

-

-

P

-

-

p

-

p p p

-

-

-

-

-

-

-

-

-

-

-

-

r

-

p

a

-

p 6 0 0 0 0 0 0 0 0

-

-

a

-

-

-

/3

-

-

-

-

-

-

-

a

-

-

A A

p

-

-

-

a a

-

-

-

P p p p

-

-

-

-

0

78 78 75 68 62 70 60 70 68 63 70 70 69 71 43 37 47 73 71 70 64 64 58 65 74 70 71 75

53 49 45 42 32 35 36 30 18 16 13

31 56 55 55 50 51 41 46 56 52 53 57 53 53 53 54 4 38 7 36 32 7 7 38

69 67 67 65 58 61 58 53 32 29 23 32 32 31 32 7 49 46 45 43 43 34 38 50 44 43 50 43 52 40 31 46 30 25 42 26 42

66 67 66 64 58 60 66 56 48 49 48 50 50 50 50

6 35 30 22 23 14 33 60 24 36 34 30 36 26 14 9 27 8 9 17 8 11

74 74 76 73 71 71 69 67 50 47 45 50 47 48 49 21 17 22 61 61 60 88 60 55 55 49 63 58 58 62 57 67 59 52 63 52 49 58 49 59

0 0 0 0 0 0 0 3 p

a

01

-

46 43 38 30 20 22 25 14 26 25 17 30 26 26 26

-

-

o p a

-

66 64 61 53 49 49 52 9 52 51 46 53 50 49 50

-

A A

A

A A A A

-

-

-

0 0 0 0

-

-

-

0

9

0

a

-

8

-

A

-

7

-

a

-

6

A

p

A A

5

-

-

0 0

4

a a p

-

-

3

-

-

P o

-

2

-

-

r

1

-

-

a

1 1 1 6 1 7 1

-

-

A

6

0 0

-

0

4 0 p 0 5 0 0 p 0 4 a 0 3 a 0 -

-

-

73 68 60 61 15 64 49 58

-

33 57 59 -

52 52 49 58 56 55 59 55 64 59 55 64 54 54 55 50 53

-

17 17 18 4 31 28 23 23 16 -

29 23 27 33 18 15 20 29 18 27 16 14 21 13 19 -

89

THIN-LAYER CHROMATOGRAPHY

TABLE 9.1 (continued) Solvent system

Substituents*

4

6

1 1 1 6 1 7 1

A

-

0

OL

-

I

3

5

-

p

-

a

-

(Y

-

p

-

-

P

-

-

O O

A A

-

-

0

A

-

-

O P

A -

A

-

Q!

-

P

-

-

@

-

-

P

-

P o

-

@

0

1

-

-

A a a p A

-

p p -

-

A A

-

r

p

-

-

OL

-

p -

-

0 -

-

-

(Y

-

P -

-

-

a

a

A

A A A

0 0 0 O P

P p 0 0 0 fl -

4 7 49 45 4 4 31 18 19 22 4 7 43 3 7 6 8 6 6 6 6 6 6

2

3

4

1 9 5 9 4 1 17 51 29 13 43 22 1 2 4 5 2 4 5 4 1 1 9 5 34 11 2 2 6 5 2 2 6 9 2 2 7 9 1 7 4 8 2 5 16 46 30 1 0 3 8 1 7 5 9 5 1 5 6 5 3 -

5

6

7

8

9

62 66 68 64 46 27 33 35 62 67 50 -

48 53 51 48 26 12 15 17 49 51 39

51 57 53 52 47 45 33 38 36 50 51 40 -

37 34 28 30 26 22 13 18 14 29 19 20

21 13 13 9 7 2 5 4 4 14 8 36 25 30 27

-

-

-

-

-

-

~~

*Substituents: Hydroxyl groups are indicated by a and p , depending on orientation, at the positions listed. However, at C-5, or and p are used to designate the orientation of hydrogen. Carbonyl groups are indicated by 0 and double bonds by A .

but the most useful derivatives for TLC are the 2,4-dinitrophenylhydrazones.They are conviently prepared, stable compounds that are easily separated by TLC and assayed after elution by spectrophotometry at 390 nm [ 1006,1075] or by direct spectrodensitometry [573,575]. Hakl [390] has devised a two-dimensional TLC procedure which separates the 2,4-dinitrophenylhydrazonesof the seven physiologically most important 17-ketosteroids. The chromatogram is developed twice in the first dimension with diethyl etherpetroleum ether (7:3) and then once in the second dimension with dichloromethaneacetone (47:3). He has also used multiple development in structure correlation studies [39 11. Other derivatives that have been used for quantitative analysis are the isonicotinic acid hydrazones [go], the Dns hydrazones [29], and the TMS ethers [485]. Reagents for the detection of androstane derivatives on thin-layer chromatograms have been described by Lisboa [647,649,650]. In addition to the nonspecific tests, such as the anisaldehyde-HZS04 and vanillin-HZS04 reactions, there are the ketosteroid tests, such as the Zimmermann, Dragendorff, isonicotinic acid hydrazide, Bodansky-Kollonitsch, and Ishidate reaction. A combined Tetrazolium Blue-Zimmermann reagent has also been described [ 10761. A number of preliminary separation schemes, based on TLC of crude biological extracts, are available [ 177,228,7851. A suitable nondestructive detection method is the examination in UV light when sodium fluoresceinate [887] or other fluorescent materials are present in the adsorbent. Pyridinium salts of 17-ketosteroids can be observed in UV light during development [ 5461. A simple analysis of androsterone, etiocholanolone, and dehydroepiandrosterone in

TABLE 9.2

\o

0

RETENTION TIMES OF ANDROSTANE DERIVATIVES RELATIVE TO CHOLESTANE 14151 Steroid 3% SE-30 3% NGS 3% XE-60

5a-Androstane $3-Androstane 5a-Androstan-17p-ol 50-Androstan-170-01 5a-Androstan-1"-one 50-Androstan-17-one 5 a-Androstan-3p-01 50-Androstan-3p-01 5a-Androstan-3-one 5p-Androstan-3-one 5a-Androstane-3a,l7pdiol 5p-Androstane-3a,l7p-diol Sa-Androstane-3&17pdiol 5p-Androstane-3p,l7p-diol 3a-Hydroxy-5a-androstan-1 %one 3a-Hydroxy-5p-androstan-17-one 3p-Hydroxy-Sa-androstan-l7-one 3p-Hydroxydp-androstan-1 %one 17p-Hydroxy-5a-androstan-3-one 17p-Hydroxy-5p-androstan-3-one 17a-Hydroxy-Sa-androstan-3-one 17a-Hydroxy-50-androstan-3-one Sa-Androstane-3,17-dione 50-Androstane-3,I 7-dione 3p-Hydroxy-5-androsten-17-one

OH

OAc TMS TFA OH

0.21 0.20 0.32 0.41 0.32 0.42 0.31 0.29 0.33 0.32 0.54 0.51 0.59 0.49 0.51 0.47 0.55 0.59 0.54 0.56 0.53 0.58 0.50 0.61

0.39 0.33 0.52 0.49 0.39 0.34 0.35 0.31 0.89 0.58 0.87 0.61 0.98 0.69 0.86 0.56 0.58 0.47 0.56 0.46 0.66 0.55 0.76 0.64 0.69 0.59 0.75 0.60 0.61 0.47 0.64 0.55

-

~

0.24 0.22 0.28 0.32 0.36 -

0.27 0.25 0.60 0.89 0.65 0.95

0.65 0.53 0.77 0.68 1.67 1.68 1.89 1.56 2.00 0.28 1.90 0.30 2.10 - 1.98 0.35 2.58 0.33 2.44 2.03 0.33 2.14 - 2.64 - 2.48 0.33 2.01

OAc TMS OH ___ 0.09 0.08 0.71 0.38 0.47 1.04 0.52 0.84 0.39 - 0.71 0.70 0.39 0.47 0.59 0.32 0.38 - 0.49 - 0.42 2.05 0.49 2.27 1.95 0.50 2.08 2.21 0.66 2.44 1.90 0.5 1 1.93 2.14 0.87 1.83 2.15 0.94 1.80 2.44 1.16 2.10 2.98 0.86 1.88 2.81 1.32 2.61 2.53 1.18 2.20 2.32 1.16 2.22 2.14 0.91 2.05 - 2.27 - 1.98 2.38 1.10 2.09

3% HI-EFF-8B

OAc TMS TFA OH

0.44 0.80

-

-

0.20 0.16 - 0.32 I

-

0.47 0.21 0.18 - 0.13 0.37 1.69 0.31 0.29 1.89 0.35 0.34 2.20 0.44 0.41 1.66 0.28 0.28 1.61 0.58 0.67 1.76 0.72 0.73 2.00 0.91 0.94 2.38 1.07 1.18 1.98 0.91 0.97 2.12 0.94 1.76 0.62 0.50 -

-

-

1.99 0.90

-

0.84

0.13 0.12 0.71 1.48 0.61 1.15 0.75 0.61 0.73 0.67 3.94 3.85 4.19 3.50 3.73 3.58 3.97 4.61 4.18 4.36 3.97 4.00 3.64 4.00

OAc TMS TFA

0.58 0.22 0.16 1.27 0.71 0.24 ' 0.20 0.15 0.57 2.48 0.28 0.27 2.82 0.37 0.30 3.18 0.42 0.24 2.42 0.28 0.23 2.62 0.74 0.80 2.86 1.03 0.6 1 3.44 1.20 0.67 3.88 1.27 1.24 3.44 1.13 1.14 3.31 1.09 2.91 0.81 0.78 3.38 1.16 1.02

$ $

a 4

P

z M

U t z P

T

zs

Cn

GAS CHROMATOGRAPHY

91

urine is based on TLC with dichloromethane-diethyl ether (19:l) [1023]. For the determination of the eight most important urinary 17-ketosteroids, two-dimensional TLC on A 1 2 0 3 has been used, with dichloromethane-ethyl acetate (80:l) in the first dimension and ethanol-hexane-benzene (3:27:70) in the second [591]. There are numerous other schemes, most of them based on spectrophotometry after the fractions have been treated with Zimmermann reagent I8281 or on fluorimetry after treatment with ethanolic H2S04 [481]. Chromatograms stained with Zimmermann reagent can also be evaluated by direct spectrophotometry [402,437]. The clinically important specific determination of testosterone in blood and urine has been the subject of numerous reports. Detection on thin-layer plates is facilitated by the W absorption at 254 nm. Eluates from TLC plates are assayed by spectrophotometry [293,618] or fluorimetry [486,805], as above, or by scanning with a spectrophotometer [1016]. Testosterone may also be chromatographed and determined in the form of its 2,4-dinitrophenylhydrazone [553,1034].

9.3. GAS CHROMATOGRAPHY Methods of GC are very readily applicable to the CI9 steroids. Initially, 1-3% SE-30 [918] and 1.5% NGS [lo021 were used as stationary phases for the uncombined steroids, but better separations were obtained by chromatographing the derivatives instead. The TMS ethers may be separated on 0.3% NGS at 180°C [ 152,636,637],0.6% XE-60 at 210°C [317,436,911,921],or 1%QF-1 at 190°C [91l].Table9.2 istakenfromapaper by Hartman and Wotiz [415] on the relation between structure and retention behavior. It shows retention times relative to cholestane for a series of CI9 steroids on four columns, all 5-6 ft. X 1/8 in. I.D. The SE-30 and XE-60 columns were operated at 26OoC, HI-EFF8B at 238"C, and NGS at 232OC. On the last two columns, the retention times are in the order OH > OAc > TMS > TFA, whereas on SE-30 the order is OAc > TMS 2 OH > TFA, and on XE-60 it is OAc > OH > TMS. Thus, the polarities of the columns are in decreasing order: HI-EFF-8B = NGS > XE-60 > SE-30. Further studies on the retention behavior of androstane derivatives have been published by Hiscoe et al. [443]. Other derivatives useful for GC of androstane derivatives are the n-butyrates [916], formates [690], chloroacetates [826], CMDMS ethers [ 1053],O-methoximes [294], and 0-butyl- and 0-pentyloximes [45]. The extent of preliminary purification depends on the biological source and concentration of the CI9 steroids [705]. For the quantitative estimation of the major urinary 17ketosteroids, no purification was used [906] or the urine extract was fractionated by TLC in ethyl acetate-benzene (2:3) [68]. The dehydroepiandrosterone zone which is eluted also contains androsterone and etiocholanolone. Before GC analysis of 5a-androstane-3a,l7pdiol, the urine extract was purified by alumina column chromatography as well as silica gel TLC [72]. Combinations of TLC and GC were used to resolve the four epimers of 5aandrostane-3,17-diol [74] and other urinary androstanediols [73,761]. The determination of androsterone and dehydroepiandrosterone sulfates in serum by GC was preceded by chromatography of an acetone-ethanol extract on methylated Sephadex G-25, solvolysis, and another column chromatogram on silicic acid [980]. Similarly, dehydroepiandrosterone

92

ANDROSTANE DERIVATIVES

sulfate was isolated first by chromatography on the anion-exchange resin Amberlyst XN1006 19841. The analysis of testosterone in urine by GC involved gradient elution from an A1203 column [920] or two A1203 chromatograms, one of the free steroid and one of its TMS ether [1122]. In another approach, the acetate was purified by gradient elution from a disposable SiOz column [ 1 1491. Purification by TLC can be simple if the urine extract has gone through a Girard separation [338]. Otherwise, two developments by horizontal TLC [673], or TLC of testosterone followed by TLC of its acetate [493,999], may be required. Demish and Staib [229] found that the TMS ethers of testosterone and its hydroxylated metabolites are adequately purified by TLC with cyclohexane-ethyl acetate (4: 1). The testosterone concentration in plasma is so low that it can only be determined by the ECD. Labeled testosterone may be added to trace the steroid in the purification process and to correct for manipulative losses. The plasma extract is usually subjected to TLC, the eluted testosterone is converted to an electron-capturing derivative, and then the derivative is purified by TLC [ 1951, but two alumina columns may be similarly employed [ 10521. Suitable derivatives are: the heptafluorobutyrate [617], the monochioroacetate [ 1261, the hexadeca- and eicosafluoroundecanoate [563], the iodomethyldimethylsilyl ether [ 10521, and the 0-methyldichlorotetrafluoroketal[753]. The bromomethyldimethylsilyl ether of testosterone is suitable for the estimation of testosterone by GC-MS, because it is formed quantitatively and shows a relatively intense molecular ion [180]. When 'H-labeled testosterone is added to the plasma as an internal standard, no elaborate purification is necessary, but a simple extraction, followed by derivatization, permits the accurate determination of as little as 0.8 pg testosteroneflO0 ml plasma by multiple peak monitoring. Dehydroepiandrosterone in plasma can be determined by the ECD if it is oxidized t o 4-androstene-3,6,17-trione, which is electron-capturing [525].

Chapter 10

Pregnane derivatives This chapter deals with the chromatography of CZl steroids, with the exception of the corticosteroids, which are treated in Chapter 1 1, but it includes the Czl alkamines. The pregnane derivatives are relatively stable and nonpolar compounds, substituted a C-3 and often at C-20 as well. They are metabolically related t o progesterone, which contains an a$-unsaturated carbonyl group, like most steroid hormones. The chief urinary metabolite, 5/3-pregnane-3a,20cw-diol,is excreted as the glucosiduronate. There have been very few publications on LC in the past ten years. A method for the determination of urinary pregnanediol was based on the centrifugal microparticulate-bed chromatography described on p. 10 [926]. Gel chromatography on Sephadex LH-20 was used in the isolation of Sa-pregnane-3a,ZOa,21-triol [979] and hydroxyalkoxypropyl Sephadex in the protein-binding assay of 17-hydroxyprogesterone [456]. King er al. [554] have developed a convenient assay method for progesterone in oil, based on HPLC. The progesterone is extracted with 85% ethanol, and the extract is injected into a 1-m X 2.1-mm I.D.column, packed with spherical glass microbeads, 30 pm in diameter, which have been coated with 1% octadecylsilane. Reversed-phase partition with an 18% aq. isopropanol solution at 42-44°C separates progesterone from the other ingredients in the preparation.

10.1. THIN-LAYER CHROMATOGRAPHY The definitive work on TLC of 21-deoxypregnane derivatives was done by Lisboa [652,656,658]. Table 10.1 excerpts some of the ~ R values F from that work. In many cases where resolution was not possible by a single development, it was accomplished by the simple expedient of multiple development. For unsaturated steroids, argentation TLC frequently proved to be effective. The order of mobilities was generally for oxygen functions at C-20: 0 > /3 > a , and for oxygen functions at C-3: 3/3(50) > 3a(5a) > 3/3(5a) > 3a(5P), i.e., as in other series,'the steroids with axial hydroxyl groups were more mobile than those with equatorial ones. In addition to the nonspecific detection methods, Lisboa used the 2,4-dinitrophenylhydrazine reagent for ketonic steroids, the UV absorption on Silica Gel GFZ54 plates for 4-en-3-ones, and the nitroprusside reaction for the methylketones. A highly specific brickred color is produced when 1 1/3-hydroxyprogesterone on Silica Gel G is sprayed with conc. HZSO4and heated at 70-72°C for 0.5-1.0 min [1073]. TLC is used extensively for the quantitative estimation of 5P-pregnane-3a,20a-dioland its congeners in the urine. This usually involves enzymatic or acid hydrolysis and extraction of the urine, followed by TLC with, e.g., chloroform-acetone (9: 1). The steroid may be located with water or iodine vapor, eluted with chloroform-methanol (1 : l), and determined colorimetrically [309,806,1022]. Alternatively, the H,S04 reaction can be performed directly on the scraped-off silica gel [49,453,810,844,933,1048].The direct densitometric procedures are more convenient [79,536]. TLC was also used in the

93

94

PREGNANE DERIVATIVES

TABLE 10.1 ~ R F V A L U E SOF PREGNANE DERIVATIVES IN TLC ON SILICA GEL (Data from Refs. 652,656,658)

Substituents*

Solvent system*

3

4

5

6

a a

-

a

-

-

-

a

-

-

-

P P

-

a a

-

-

-

a a

-

p p

-

P P P

-

P P

-

a

-

a P

P P

-

a

-

a

a

-

a

-

a

-

a a

-

p p

a -

P

-

P

-

-

-

-

a

P P P P P 0

-

-

p

p

P

-

-

a a

a P

a

-

-

-

-

0

-

-

a a

-

a

-

a

-

P

-

P

-

-

a

-

p

-

-

a

-

a

-

a P a

a

-

p

a

-

-

6 P 0

-

O

A

-

-

0 O 0 a

-

a P 0

P

-

a

a

-

P 0

-

o

-

p

-

o

0

-

0

0

-

p a

-

0 o

a

-

o

O

A

-

O

A

-

O

A

-

-

0

P

-

-

P

a a

P a

0 P

P P P &

a a

-

a

P

a

P

-

a

&

a -

P

-

-

-

-

-

-

0

a a

-

0

-

-

-

0 -

a

a

p P

-

-

-

-

-

P

-

-

-

-

-

-

-

a a

p

-

-

a

a

-

-

a

a

1 1 1 6 1 7 2 0 1

-

-

-

-

a a

(Y

a a P 0 0 0 0 0 0 0

0

a a a a -

a 0 0 0 0 0 0 0 0 0 0 0

-

P

-

a

P

59 52 51

49 54 56 59 30 27 24 21 18 15 26 34 32 39 34 36 9 I 57 64 55 59

56 52 26 23 46 50 24 62 66 64 66 42 48 49 45 56 53 53 56 50 44

2

3

4

5

6

7

8

9

32 34 27 31 22 25 34 37 -

55 55 50

31 34 33 35 28 31 37 40 11 13 9 7

68 70 65 70 65 68 70 I0 47 47 39 38 32 30

59 52 56 50 56 59 61 34 34 26 25 18 16 30 39 37 45 41 45 3 4 65 63 25 22 52 58 63 68 70 66

58

35 34 30 33 33 36 40 41 14 13 14 11 10 8 20 26 23 29 25 27

-

-

5 11 8 16 10 13 38 42 33 36 39 29

-

4 15

25

-

38 53 50 38 10 14 8 12 26 22 27 27 24 16

-

52 50 52 57 30 30 26 24 20 16 23 35 32 41 33 38 4 5 -

71 62 61 33 29 42 52 25 75 75 73 71 54 57 44 37 76 13 14 63 60 51

5

4 7 15 12 19 14 18 50 60 45 52 49 45 12 9 20 33 67 70 69 57 38 40 19 26 69 66 66 55

52 27

45 55

51 61 58 13 10 76 71 74 -

38 37 64 68 72 78 76 75 54 59 62 57 68 65 65 70 67 61

40

47 49 41 56 54

56 53 56 52

54 54 57 40 39 41 37 36 32 39 47 44 49 45 17 19 62 65 58 59 61 61 46 43 57 61 68 70 70 54 -

58 -

49 47 67 68 67 56 54

50

55

55

-

-

-

-

-

-

-

47 51 42 47 41 44 18 15 30 36

-

-

-

56 63 63 47 31 33 19 24 53 49 51 38 36 32

42 48 -

-

-

-

-

20 18 -

THIN-LAYER CHROMATOGRAPHY

95

TABLE 10.1 (continued)

_-

-.

Substituents* 3 4 5

6 a

1 -

1

1 -

-

0

-

O O O

A A A

-

O

A

-

-

O

A

-

-

O O O O O

A A A A A

-

0

-

0

O

A

-

O

A

-

O @ 0 P

A -

A A A

-

@

-

A

6 p 6

-

@ P

-

& a

-

@ P P @

-

6

-

-

-

6 -

-

a

A

a

-

-

-

OL

-

-

a a a

-

P 0 -

-

-

A A A

-

A A

-

A A A A

-

A A

-

A

-

-

a

0 p

a

-

A a

-

-

-

a

a a a

-

-

a

-

a a

-

-

a a a

-

-

-

-

0

a

-

-

-

-

-

-

-

-

Solvent system* 1 1 2 0 1 2 3 4 P 39 10 48 19 65 P 34 5 38 11 0 41 12 51 27 a 36 9 48 23 0 59 38 12 68 14 0 49 15 55 31 61 0 46 14 51 40 60 0 31 6 43 24 43 0 29 6 47 35 44 0 56 21 64 52 12 0 43 11 40 20 62 0 26 6 5 6 3 5 44 16 I0 60 62 0 0 54 31 58 48 12 53 31 57 49 71 0 0 35 9 41 21 52 0 51 28 50 33 70 a 44 13 37 13 66 0 34 I 31 11 51 0 35 12 47 26 52 a 50 21 48 33 66 p 53 31 50 36 69 a 30 2 32 11 42 21 2 2 2 8 4 1 a 30 14 38 15 46 25 8 26 10 44 a 38 11 36 14 57 p 41 16 40 22 61 a 29 5 21 6 51

5 44

-*

42 31 46 34

-

-

-

38 58 49 33 44 26 21 27 26 40 43 31

6

7 51 -

-

65 55 54 45 52 58 50 60 56 55 41 53 46 40 49 50 42 48 43 36 44 41 36

8 27 22 32 28 55 30 32 20 25 40 22 29 44 42 42 26 35 31 23 25 33 34 19 16 22 14 23 30 19

*See the footnote and the legend to Table 9.1 (pp. 88 and 89).

specific analysis of urinary pregnanediol by the double isotope derivative dilution technique [265]. In the determination of progesterone and its congeners in plasma similar approaches have been used. For the elution from silica gel, methanol should be used [ 10041. Hobzovi and Novak [448] have chromatographed the 2,4-dinitrophenylhydrazoneof progesterone, which is easily assayed by spectrophotometry. For direct scanning, Keller et al. [548] and Weiss [ 11531 determined the absorption at 254 nm of progesterone on fluorescent silica gel, whereas Egg [264] measured the fluorescence produced when progesterone on an A1203FZs4 plate Type T is heated at 150°C for 20 min. TLC was likewise used for the purification of progesterone in double isotope derivative methods [895,1158]. The separation of CZl steroidal bases by TLC has been investigated by Libler and t e r n 9 [623,624]. Using benzene or diethyl ether, previously saturated with NH40H, in chambers with a NH3-saturated dtmosphere, they systematically studied the effect of substituents on chromatographic mobility. Because the axial substituents were less polar

9 -

-

40 14 14 I 8 22 7

-

21 -

-

-

96

PREGNANE DERIVATIVES

than the equatorial ones, they observed that the order of mobility for alkaloids substituted with a -N(CH3)2 group at C-3 is: 3a,5a > 3a,As > 30,50 > 3a,5p > 3/3,A5 > 30,5a, and for those with a -N(CH3)2 group at C-20 the 0-substituted steroids move faster than the a-substituted ones. Bennett and Heftmann [64,65] used 0.1 N NaOH instead of water in the preparation of the silica gel plates and chromatographed Holarrhena alkaloids with various water-saturated solvent systems.

10.2. GAS CHROMATOGRAPHY

Earlier experiments having shown that progesterone [337] and other pregnane derivatives [773] can be chromatographed on SE-30 and other packed columns, investigators began to apply GC to the analysis of pregnanediol in urine [204,515,840] and bile [6]. The free steroids were used, as well as their acetates [ 11731, TMS [ 15 I ] , and MO [ 101 derivatives. For the analysis of pregnanediol in urine, preliminary purification by preparative TLC, e.g., with chloroform-acetone (9: 1) [63 1] was recommended, although other methods, e.g., precipitation by 70% (NH4)?S04 [ 1811, have been proposed. Nowadays, however, urine extracts are usually not laboriously purified. Pregnanediol diacetate is analyzed on a 6-ft. column of 3% SE-30 at 250°C [ 182,3621, or 1% SE-30 at 270°C [856], on a 5-ft. column of 2% OV-101 at 230-250°C [736], or 3%OV-1 at 240°C [913]. Free pregnanediol can be determined on a column of 0.5% NPGA at 235°C [717] or 3.8% SE-30 with programmed temperature [27]. For routine analyses of pregnanediol, one of the available automatic sample injection systems may be used to increase the processing rate [27,856,913]. Occasionally, other progesterone metabolites may be sought in the urine. For instance, in the presence Metcalf [7 181 has analyzed pregnanolone (3a-hydroxy-50-pregnan-20-one) of pregnanediol on a 2% XE-60 column, but found that pregnanetriol (50-pregnane3a,l7a,20a-triol) interferes. A method for the simultaneous determination of pregnanediol and pregnanetriol by GC was perfected by Rahman [874]. Pregnanediol, pregnanetriol, pregnanetriolone (3a, 17a,20a-trihydroxy-S/3-pregnan-l1 -one), and pregnanetetrol(50pregnane-3ql lp,l7a,2Oa-tetrol), when chromatographed in the form of TMS ethers on 1.5% SE-30 at 219'C gave retention times relative to cholestane of 0.96, 1.45, 1.93, and 2.38, respectively [555]. For an additional estimation of urinary S-pregnene3/3,17a,2ktriol, which gives an R T of 1.65 under these circumstances, that steroid is isolated by preliminary chromatography through an alumina column 15561. Pregnanediol, pregnanetriol, and pregnanetriolone have also been analyzed as the TMS ethers without preliminary chromatography [213] or after fractionation on alumina thin-layer plates [877]. Another approach to the simultaneous analysis of pregnanetriol and pregnanetriolone is to convert them to the acetates after alumina column chromatography and to separate the acetates by GC on a 1.2-m X 3-mm I.D. column of 3.8% SE-30 at 230-238'C [632]. The low concentration of progesterone in plasma makes it necessary to account for losses in the isolation procedure. This may be accomplished by adding radioactive progesterone as an internal standard at the beginning of the isolation [ 196, 11821. Preliminary purification by TLC [675], which is almost invariably needed, utilizes the W ab-

GAS CHROMATOGRAPHY

91

sorption of progesterone 15481 or the radioactivity of the tracer [ 11821 for detection. Use of solid injection into a 1% XE-60 column with a FID was recommended [ 118.51. The ECD may be used if progesterone has been reduced to 20P-hydroxy-4-pregnen-3-one by 200-hydroxysteroid dehydrogenase and the chloroacetate has been prepared [73 11. A more convenient electron-capturing derivative is the HFB 3-en01 ester of progesterone, which is prepared by heating the sample with 10 1.11 abs. tetrahydrofuran, 25 1.11 benzene, and 25 p1 HFBA at 65°C for 30 min [304]. Pregnenolone (3fl-hydroxy-5-pregnen-20-one) in biological samples can be determined in the nanogram range if the sample has been purified by TLC and the steroid enzymatically reduced (see above) [868]. The 3,20-dichloroacetate is then assayed by an ECD. For the analysis of 16a-hydroxyprogesterone in amniotic fluid, the purified steroid was converted to the TMS ether, which was chromatographed on a 6-ft. column of 3% QF-1 or OV-1 [325]. The determinations of progesterone in milk by GC and by radioimmunoassay give essentially the same results [798].

This Page Intentionally Left Blank

Chapter I I

Corticosteroids The adrenocortical hormones are characterized by a A4-3-keto group and an a-ketol side chain. Both features facilitate their detection by TLC and HPLC, but they are rather unstable in GC. The individual hormones differ from each other by oxygen substituents, usually at C-11, (2-17, or C-18. The urinary metabolites are usually saturated compounds, many of which retain the hydroxyl group at C-2 1. They are largely conjugated with glucuronic or sulfuric acid at (2-3. There has been much interest in the analysis of both natural and synthetic corticosteroids, but no comprehensive reviews have been written on this subject.

11.1. LIQUID COLUMN CHROMATOGRAPHY

Silica gel columns are useful, not only for the preliminary fractionation of corticosteroids [786], but also for the automatic determination of individual 17-ketosteroids and adrenocortical hormones [520,626,627]. For that analysis, a column of silicic acid with 60% water content is eluted with a linear gradient of increasing dichloromethane concentration in petroleum ether (cf. also Fig. 2.2). Apparatus has been designed for the simultaneous development of twelve capillary columns filled with silica gel, having ca. 20% water content, by a gradient of acetone in chloroform [1124] (see p. 9). An elegant machine for this type of work was devised by Cavina et al. [ 140,1551. It provides a suitable gradient of methanol in chloroform with solvent delivery at 500-600 p.s.i. to a column, 560 mm X 6 mm, containing silicic acid, 325 mesh, of known water content. The corticosteroids are measured by a UV detector as well as by a FID. Celite has been used for partition columns in corticosteroid chromatography. For instance, the double isotope analysis of aldosterone involves three Celite columns [545]. It should be noted that under these conditions there is some isotope fractionation, the tritiated aldosterone separating from unlabeled aldosterone [ 1631. From the partition coefficients, Weber et al. [ 1 1501 predicted the elution curves of various synthetic corticosteroids on a Celite column. Partially esterified Amberlite IRC-50 was used in the separation of 17-hydroxycorticosteroids and 17-ketosteroids by column chromatography [954]. Subsequently, Seki [953] found that Sephadex LH-20 columns also produce partition chromatograms of 17-hydroxycorticosteroids. Such columns have been used to separate labeled corticosteroids in biosynthetic research [960]. Sephadex LH-20 has also found use in the purification of aldosterone [219,1140] and deoxycorticosterone [ 1491 for radioimmunoassays. A marker dye facilitates the collection of the aldosterone fraction [494]. An automated procedure has been devised for the isolation of corticosterone, 1 1-deoxycortisol, aldosterone, cortisone, and cortisol by chromatography through 15-g Sephadex LH-20 columns of 11 mm I.D. [974]. A battery of six columns is simultaneously eluted with metered quantities of dichloromethane-methanol(49: l), and the eluate fractions are individually collected.

99

CORTICOSTEROIDS

100

$ 0 (u

*0 C

P

P 0, R 0 3 > c

8 2

I

I

’pRetention time (min)

Fig. 11.1. HPLC of a steroid mixture on a column of Spherosil XOA-400 with the organic phase of a 948:35: 17 mixture of dichloromethane, ethanol, and water. (Reproduced from Z . Klin. Chern. Klin. Biochem., 12 (1974) 194, with permission; [433].)

Because corticosteroids absorb UV light and have suitable solubility properties, they are ideal subjects for HPLC. Interest in this application is especially great, because GC is not easily adapted to corticosteroids. The first experiments with silica and bonded layers of octadecyltrichlorosilane or cyanoethylsilicone as column packing materials gave moderately good resolution of adrenocortical hormones [822,1071,1170]. Using Zorbax SIL (porous siiica microspheres) as adsorbent and dichloromethane-methanol-water (48: 1:1) as eluent, adequate separation of cortisone, prednisone, cortisol, and prednisolone (in order of elution) could be accomplished [835], and the procedure was found applicable to the quantitative analysis of plasma 110741. Hesse et al. 14331 have devised a convenient routine method for the determination of nanogram quantities of corticosterone and was packed with cortisol in plasma, based on HPLC. The column, 300 mm X 2 mm I.D., Spherosil XOA-400,4-8 pm, and eluted with the organic phase of a 948:35: 17 mixture of dichloromethane, ethanol, and water at a rate of 1 ml/min. A model chromatogram is shown in Fig. 1 1 . l . A “preparative” application of HPLC was recently announced by Pei el aE. 18421. A Vydac column, 410 mm X 15.8 mm I.D., separated 0.87 mg of a five-component corticosteroid mixture in 30 min by elution with heptane-chloroform-methanol(79: 19:2) at 100 p.s.i. and 15 ml/min. It is fair to say that these accomplishments will be considerably improved upon by the time this book has been published.

THIN-LAYER CHROMATOGRAPHY

101

11.2. THIN-LAYER CHROMATOGRAPHY There have been some attempts to use polyamide layers for TLC of corticosteroids [321,480], and an evaluation of the applicability of silica layers on polyester sheets has been published [869]. However, the bulk of the TLC methods presently used is based on conventional gypsum-bound silica on glass plates. Table 1 1.1 summarizes the results observed on Silica Gel G plates by Lisboa [655,658]. Table 1 1.2 gives additional ~ R F values, obtained with Polygram Sil G sheets by Smith and Hall [993]. The separation of Compound S (1 7a,2 l-dihydroxy-4-pregnene-3,2O-dione) from Comis difficult, but succeeds with a 4: 1 pound B (1 1/3,2l-dihydroxy-4-pregnene-3,20-dione) mixture of diisopropyl ether-acetone [640]. Compound B separates from aldosterone and 18-hydroxycorticosterone with chloroform-acetone (3: 2) ( ~ R values: F 5 6 , 3 3 , and 18, respectively) [336]. Two-dimensional systems for difficult separations have been described [70,870]. Thus, aldosterone can be very neatly separated from other corticosteroids by two-dimensional TLC with ethyl acetate-95% ethanol (7:3) or with chloroform-95% ethanol (49: 1) 19701. Spraying of the chromatoplates with a concentrated solution of ascorbic acid in ethanol will inhibit air oxidation of corticosteroids during radioautography [324]. Because TLC is inadequate and ion exchange is too cumbersome for the separation of conjugated corticosteroids, Kornel et al. [ 5921 have recently devised PC procedures based on continuous development of Whatman No. 3MM filter paper strips, which are in some instances treated with boric acid. The solvent systems are mixtures of petroleum ether, isopropanol, and water, and the method permits closely related conjugates of corticosteroids and of androstane derivatives to be separated. Lisboa [648] has described no less than 34 reactions suitable for the detection of corticosteroids and related compounds on thin-layer chromatograms. In addition to general tests, such as treatment with 50% H2S04, which may nevertheless produce very sensitive and specific responses [235], there are quite a few which are due to characteristic features of corticosteroids, such as the a$-unsaturated carbonyl group or the a-ketol side chain. The former is responsible for the W absorption and for the reactions with tetranitromethane and with Os04 and the latter for the reduction of phosphomolybdic or arsenomolybdic acid and of various tetrazolium salts. The sensitivity of the tetrazolium test is greatly increased when Tetrazolium Blue is incorporated in the silica layer during preparation [ 11 131. Most of the numerous methods for the determination of urinary corticosteroids by TLC are based on enzymatic hydrolysis, extraction, isolation from a thin-layer chromatogram on the basis of W absorption or Tetrazolium Blue reduction, and elution with ethanol or chloroform-acetone (1: 1). The eluate may be rechromatographed and the individual zones separately eluted and analyzed with Tetrazolium Blue [ 162,7841, H2S04 [ 10281, or Porter -Silber reagent [97,482]. An exacting technique and apparatus permits the accurate determination of corticosteroids in nanogram quantities [308]. The steroids are detected in W light on layers of Macherey-Nagel HRzS4 silica gel and are then eluted for fluorimetric analysis with minimal interference. An interesting approach to the estimation of corticosteroids is the reaction of 14C-labeledTetrazolium Blue with the zones on a paper chromatogram, which are then

TABLE 11.1 hRpVALUES FOR CORTICOSTEROIDS IN TLC ON SILICA GEL G (Data from Refs. 655 and 658) Substituents*

Solvent system*

3

4

6

O

A

-

O

A

-

O O O O O

A A A A A

-

O O O O O

A A A

-

A

D

A

O

A

O

A

O O O

A A A

-

O O O O O

A A A A A

11 0

16

17

18

-

-

-

-

-

-

-

a

a

0 O O D -

-

a

-

6

-

-

21

1

2

3

4

5

6

7

8

9

0 0 0 0 0 0 0 0 P

OH OH OH

40 22 13 14 38 31 16 16 9 9 12 15 3 28 16 18 21 21 17 -

13 9 -

62 31 20 22 41 38 30 26 16 17 14 21 4 18 11 16 29 14 49 34 16 24

51 15 7 9 22

55 37 22 26 57 42 30 26 20 21 26 28 9 44 28 23 44 39 37 47 31 35

46

55 38 26 31 49 42 31 32 25 25 24 30 12 37 25 26 42 43 28

38 16 9 11 23 22 16 16 13 12 6 10 -

20 5 8 -

a

-

OH 0

a

-

a

-

a

-

-

a

-

0 P

a a

-

P

a

-

0 0 0 0 0 0 0

a

-

0

a

-

0

a

-

a

-

-

a

-

-

P -

p O O O O

a a

a a

-

-

-

a

20

-

-

19

OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH

*See the footnote and the legend to Table 9.1 (pp. 88 and 89).

4 5 -

-

9 39 24 15 13 -

8

26 14

28 21 23 29 20

-

-

23 17 -

-

8 5 -

cl 0 ;a 4

ii

3m F

s

GAS CHROMATOGRAPHY

103

eluted and counted [ 1 1 141. The double isotope analysis of aldosterone involves both TLC and PC [ 1101. A rather complex procedure is evidently necessary for the specific determination of the very low quantities of aldosterone in blood. The direct densitometric evaluation of corticosteroids on thin-layer chromatograms requires staining with Tetrazolium Blue if the instrument is a spectrophotometer [400]. With a fluorometer, the quenching of the background fluorescence can be read directly [ 10831. The fluorometer can also be used for corticoid metabolites lacking the a,p-unsaturated carbonyl group by scanning a layer containing a fluorescence indicator before and after it has been treated with Tetrazolium Blue [ I ] . The analysis of aldosterone in urine requires rather efficient TLC procedures. Ripa [896] purified the urine extract by chromatography on a silica gel column and applied the eluate to a thin layer of kieselguhr. Two-dimensional chromatography with chloroform-ethanol (9: 1) and diethyl ether-benzene-acetone (5:3:2) was effective in isolating aldosterme. In the method of Gerdes and Staib [351], the aldosterone zone from TLC on silica gel with chloroform-ethanol (9: 1) is eluted, the eluate acetylated, the product chromatographed with cyclohexane-chloroform-acetic acid (4:5: l), again eluted, and chromatographed a third time with chloroform-ethanol (9: 1). Cavina et al. [ 1531 use a silica column, followed by TLC with chloroform-methanol-water (225 :25:2), but then resort to two-dimensional TLC with ethyl acetate-methanol-water (85: 15: 1) in the first dimension and continuous development for 4%h with benzene-acetone-acetic acid (140:60: 1) in the second. Other examples of the application of TLC to the analysis of specific adrenocortical steroids are the determination of 6P-hydroxycortisol [7 11 and of corticosterone [915], TLC is now being replaced by radioimmunoassay methods for hormone analysis, but continues to be useful in the preliminary purification of biological extracts. Material interfering with the radioimmunoassay of urinary aldosterone, which was not eliminated by two silica gel chromatograms, with dichloromethane-96% acetic acid-methanol (1 8:2: 1) and with chloroform-95% methanol (23:2), was finally removed by TLC on a cellulose layer with 95% ethanol [937]. PC is now eclipsed by TLC, but continues to find use in the analysis of conjugated hormones. Aldosterone 18-glucosiduronate, tetrahydroaldosterone glucosiduronate, and aldosterone sulfate have been isolated from urine by PC [374].

11.3. GAS CHROMATOGRAPHY The analysis of corticosteroids by GC has recently been reviewed by Menini [712]. Early attempts to exploit the thermal decomposition of corticosteroids during GC for analytical purposes having failed [368], Tsuda et al. [ 10801 experimented with H I 0 4 and NaBi03 oxidation prior to injection. The 17-ketosteroids produced upon injection of the tetrahydro derivatives can be utilized for quantitative analysis [670], but in this case it is also preferable to carry out a NaBi03 oxidation before GC [ 10001. Complete schemes for the determination of corticoids in biological fluids, based on either NaBiOJ 1411 or HI04 [564] oxidation or both [53], have been presented. The procedure of Craig and Chamberlain [207] involves NaBH4 reduction, followed by HI04 oxidation, and finally acetylation.

c

P

TABLE 11.2

hRF VALUES FOR NATURAL CORTICOSTEROIDS IN TLC ON POLYGRAM SIL G [993] Solvent systems: 1 = 1,2-dichloroethane-ethanol(7: 1); 2 = 1,2dichloroethane-rert.-butylalcohol (17:3);3 = 1,2-dichloroethane-acetone (67:33);4 = 1,2-dichloroethanedioxane (3:l);5 = 1,2dichloroethane-acetic acid (31:9);6 = 1,2dichloroethane-pyridine;I = propyl acetate; 8 = isobutyl methyl ketone; 9 = nitromethane-ethanol (37:3);10 = anisole-ethanol (9:l);1 1 = diisopropyl ether-ethanol (9:l);12 = diisopropyl ether-tert.-butyl alcohol (4:l); 13 = diisopropyl ether-acetone (7:3);14 = diisopropyl ether-dioxane (4:1); 15 = diisopropyl ether-acetic acid (3:1); 16 = diisopropyl ether-pyridine (4:l). Substituents*

3

4

5

O

A

-

a

-

a O a a O a a

A A A

p a p

O

a f

0 a O a

a

O 0 O

f

Solvent system

f

p

6 f

17

18

-

a a a

-

-

-

-

-

0

-

0

a

-

o

-

-

P

0

-

p

A

a -

-

-

p

A -

-

A

-

p

A A

-

-

11

p (Y

p

0 o p p p p

-

-

D

20

0 0 0 O O O O O O 0 0 0 0 0 0 (Y 0 a 0 a 0 a! 0 0 0 0 O O O

*Substituents, see Table 9.1 (pp. 88 and 89).

21

1

2

3

4

5

6

7

8

9

1 0 1 1 1 2 1 3 1 4 1 5 1 6

OH OH

66 44 50 42 26 30 55 28 33 38 26 27 36 21 26 18 19 23 14 27

61 45 55 43 25 33 50 24 30 34 22 25 34 17 22 12 15 20 10 23

66 50 59 52 28 37 51 25 31 40 24 29 41 19 27 14 17 21 13 21

82 70 75 64 41 51 72 45 53 51 41 46 50 25 34 18 23 28 13 36

66 61 65 50 41 44 54 41 44 45 37 40 33 22 24 16 19 15 6 30

69 51 59 55 32 40 59 30 38 43 26 31 48 24 33 18 20 30 19 34

53 45 57 54 43 55 32 18 24 34 26 32 41 21 39 30 36 26 26 18

53 42 59 54 40 56 35 16 24 34 25 32 41 22 37 26 34 25 24 12

58 45 51 48 29 41 54 35 39 43 28 30 45 27 33 20 22 27 21 25

60 47 54 46 30 37 49 31 36 41 31 34 36 21 31 20 24 27 19 29

OH H H H OH OH

OH OH OH OH OH

OH OH OH OH

OH OH H

46 49 54 47 46 53 17 16 20 29 27 32 33 30 34 30 34 21 22 14

53 61 70 62 64 72 23 19 29 39 40 48 44 43 49 50 56 33 35 21

55 51 61 48 42 52 33 20 28 36 29 38 34 24 33 26 31 22 18 20

42 37 49 35 27 38 20 12 18 22 18 27 20 12 17 14 19 14 10 16

37 48 57 35 42 50 15 17 20 20 26 29 18 21 20 24 27 10 9 9

58 55 62 47 43 47 36 30 34 39 33 39 31 25 29 24 26 21 14 31

8

2

; 4

E! U

CA

GAS CHROMATOGRAPHY

105

Many other derivatives of corticosteroids have been proposed for use in GC. Steroids with a dihydroxyacetone side chain react with formaldehyde to produce thermostable bismethylenedioxy derivatives [560]. Acetylation of the side chain also increases the thermal stability of corticosteroids [ 11 11. The acetates and chloroacetates may be analyzed with the ECD [878]. Rosenfeld [903] prepared the TMS ethers of cortisol metabolites and found them suitable for GC on 3% QF-1 at 220°C and on 3% SE-30 at 235°C. The TMS ethers of corticosteroid methyloximes are quite stable on 1% SE-30 at 220°C 14.591, although the cortisone and corticosterone derivatives are not separated [411]. To prevent sample losses due to acidic materials in the column, the first 3 cm should be packed with 10%SE-30 and the sample should contain an excess of TMSI [ 10501. Instead of using a carbonyl reagent for corticosteroids with ketol or dihydroxyacetone side chains, Chambaz et al. [ 166,1691 have made use of the enolization of the keto group in the presence of nucleophilic agents to produce the TMS-enol-TMS derivatives. The reaction was carried out overnight in a closed vessel at room temperature. First, 0.2 ml of a solution of 50 mg potassium acetate per milliliter methanol was evaporated in the vessel, then 50 yl of a solution of 1 mg steroid per milliliter ethanol was added and likewise evaporated. Finally, 200 yl BSTFA was added. Upon completion of the reaction, 2-5 pl of this solution were directly injected into the gas chromatograph. The derivatives, which formed in quantitative yield, were shown to be the TMS ethers of the 20-enols. The 3-en01 ether group was hydrolyzed when the reaction mixture was evaporated t o dryness. Single peaks for each of the derivatives were obtained on a 12-ft. 1% OV-101 column at programmed temperatures. Bailey [42] has prepared the 20,2 1-acetonides of 20,2 1-dihydroxycorticoids and found them useful for the separation of the 20-dihydrometabolites of cortisone and cortisol. Kelly [550,551] described the preparation and properties of siliconides formed by the action of dimethyldiacetoxysilane and triethylamine on steroids with a dihydroxyacetone side chain. Brooks and Harvey [ 1 151 have compared the GC properties of various cyclic boronates. The methylboronate of 4 y g of Compound S was formed in 85% yield in 15 min when it was kept at room temperature in a closed vessel with 4 molar equivalents of methylboronic acid in 6 yl of ethyl acetate. Comparison of various corticosteroid derivatives by GC-MS showed that boronates could be estimated in the subnanogram range [46]. The sensitivity, specificity, and versatility of GC is nowhere better illustrated than in the determination of aldosterone. Relatively simple but less sensitive methods are based on the use of a FID. For instance, by isolating aldosterone from urine with an Amberlite XAD-2 column and purifying the eluate by TLC, a concentrate is obtained which may be oxidized with HI04.The aldosterone ylactone is isolated by TLC and is determined by GC with a sensitivity of about 1 yg/l, 10%precision, and 95-99% accuracy [608,638]. Similar results were reported with a method in which the preliminary purification involved in succession, PC, acetylation, PC, and TLC [ 1 1651. Greater sensitivity is achieved with the ECD. Fabre et al. [292] have estimated the concentration of tetrahydroaldosterone in a 10-ml sample of blood by preliminary purification with TLC, followed by HI04 oxidation to the ylactone, and conversion of the product to the 30-chlorodifluoroacetyltetrahydroaldosterone ylactone, which was accurately determined with the ECD. Aldosterone was assayed by the same procedure after enzymatic reduction to the tetrahydro derivative.

106

CORTICOSTEROIDS

Other electron-capturing derivatives of aldosterone that have been tested include the HFB [698] and MO-HFB [465] esters, The most specific assays are based on GC-MS. Amounts of aldosterone down to 100 pg can be measured in 10 ml of plasma by a method involving chromatography on Amberlite XAD-2, preparation of the acetal and its purification by TLC, preparation of the HFA derivative of the acetal, and GC-MS [969]. The amount is estimated by addition of 4-['4C]-aldosterone to the plasma and measuring the 12C/'4C ratio by MS. Using mass fragmentography, highly specific and sensitive analyses can be performed with a minimum of purification. The 18-hydroxycorticosteroids [865,866] and the hydrogenated metabolites of corticosterone [ 1011 in animal tissues as well as the cortisol level in the plasma [85] have been determined in that manner. Obviously, only the cost of the instruments stands in the way of widespread adoption of this technique.

Chapter 12

Miscellaneous steroid hormones 12.1. INTRODUCTION Individual groups of natural steroid hormones are discussed in Chapters 8- 11 and 14, but Chapter 12 deals with the chromatographic analysis of biological samples for steroid hormones belonging to different classes and with the chromatography of synthetic steroid hormones. Surveys of these methods are scattered in various textbooks and symposium volumes on clinical and pharmaceutical analysis, chromatography, and MS. Among recent review articles in readily available journals, special mention should be made of those dealing with the applications of GC [ 108,168,730,850,968,1027]. General schemes for the work-up of complex biological mixtures in preparation for GC have been published [562,67 1,8391. They consist of various combinations of solvent partition, LC, PC, TLC, and derivatization. Global hormone analysis has been proposed by two developments of a thin-layer plate with benzene-ethanol (19: 1) [961] or by elution of 7 g Sephadex LH-20 in a chromatographic tube, 2 cm I.D., with 50 ml of a 100:100: 1 :1 mixture of heptane-chloroform-methanol-water (androgens + progesterone), followed by 60 ml of a 19: 1 mixture of dichloromethane-methanol (estrogens) [416]. TLC has been useful in the purification of various steroid derivatives, synthesized for conjugation to protein in radioimmunoassays [50]. R F values have been reported for the hemisuccinates, hydrazones with p-hydrazinobenzoic acid, and other derivatives of steroids.

12.2. HORMONES IN URINE The determination of steroid hormones in urine by GC has been reviewed by Gleispach [358]. As is evident from Chapters 8-1 1, chromatography is extensively used in the extraction of steroid hormones from urine. TLC [561,853], LC on A1203 [216,938], and combinations of both [526] have been standard procedures for the isolation of 17-ketosteroids,pregnanediol, and other relatively stable urinary metabolites prior to GC. The method of isolating steroid conjugates with the aid of a neutral resin, Amberlite XAD-2, is due to Bradlow [103]. A chromatographic tube, 35 cm X 7 cm, is filled with a slurry of 1 kg of the resin in water and back-flushed to remove fines. When 2 1 of urine are percolated through this column, all the steroid conjugates are retained, and impurities may be washed out with 4 1 of water. The conjugates are then quantitatively recovered by elution with 5 1 of methanol. Acidic impurities remaining after hydrolysis of the conjugates may be removed by passage through Amberlyst A-26 [959] . Steroid N-acetylglucosaminides can be isolated on Amberlite XAD-2 prior to GC analysis [762]. Group separations prior to GC by means of Sephadex LH-20 have been described by Setchell and Shackleton [9571 and by B6gue et al. [56]. Janne [511,512] has used Sephadex LH-20 to separate mono-, di-, and trisulfates of C19 and CZl steroids, which were subsequently hydrolyzed. In the work of Anderson et al. [2 1,221 the steroid conjugates were subjected to differential

107

MISCELLANEOUS STEROID HORMONES

108

% 0

10 Q L

b"

c

aJ

c 13 a,

10

15

20

25

30

35

Retention time (min)

Fig. 12.1. High-resolution gas chromatogram of MO-TMS derivatives of the urinary steroids of a healthy man. 1 = Androsterone; 2 = etiocholanolone; 5 = 11-ketoetiocholanolone; 7 = 11-hydroxyandrosterone; 9 = allopregnanediol; 10 = pregnanediol; 14 = pregnanetriol; 17 = cholesterol; 18 = tetrahydrodehydrocorticosterone; 19 = tetrahydrocorticosterone; 20 = allotetrahydrocorticosterone; 21 = tetrahydrocortisone; 22 = tetrahydrocortisol; 23 = allotetrahydrocortisol; 24 = cortolone; 25 = p-cortolone. (Reproduced from Z. Klin. Chem. Klin. Biochem., 9 (1971) 45, with permission; [ 11311.)

hydrolysis and then fractionated on lipophilic Sephadex. There are many reports on GC of underivatized urinary steroids [680,923], but most investigators prefer to use the TMS ethers or MO-TMS [342] derivatives. The BO and BO-TMS derivatives are especially advantageous, because they are better resolved in GC and give a characteristic MS [232]. The MO-HFB derivatives, which exhibit very good thermal stability, are especially suitable for the analysis of nanogram quantities by the ECD [708]. Ros [YO21 preferred the enol-TMS derivatives to the double derivatives. With the advent of high-resolution capillary GC and greater availability of the GC-MS combination, there has been much interest in urinary steroid patterns or profiles. Vollmin [1129-11311 has presented some impressive examples of the power of this new tool, one of which is shown in Fig. 12.1. The technique is described in Chapter 4. Similar metabolic profiles have been obtained by Luyten and Rutten [679], by German and Horning [353] and by Horning et al. [463], who used the MO-TMS as well as the BO-TMS derivatives. For the separation of the dihydroxysteroids in urine, the TMS, TFA, and HFB derivatives were used [75]. In view of the speed and the accuracy of the analysis, Bailey et al. [43] have recommended an automatic version with solid sampler for routine application. GC has also been applied to the analysis of synthetic steroids and their metabolites in urine. Following the administration of spironolactone, the aldadiene levels in urine and plasma were determined by GC [ 1711. The urinary metabolites of Nilevar [123], 16achloroestrone methyl ether [763], dimethisterone and norethisterone [ 10121, and methandienone [242,306] have been identified by GC.

HORMONES IN OTHER BIOLOGICAL SPECIMENS

109

12.3. HORMONES IN OTHER BIOLOGICAL SPECIMENS For the determination of steroid hormones in blood the purification methods are similar to the ones described for urine, but electron-capturing derivatives are usually preferred because the concentrations are quite low [732]. Sjovall and Vihko [982,983] separated the steroid monosulfates from disulfates in blood on Sephadex LH-20 and determined the steroids separately by GC-MS. In preparation for radioimmunoassays, a column of 2.5 g of Lipidex separated the following steroids in an extract of 1-2 ml of (19: 1): progesterone plasma by elution with petroleum ether (b.r. 66-68")-chloroform in 10-20 ml, testosterone in 48-72 ml, and 17-hydroxyprogesterone in 72-105 ml [513]. Similarly, a combination of chromatography on Amberlite XAD-2, Lipidex, and Sephadex LH-20 was used for the GC-MS analysis of neutral steroids in plasma by Axelson and Sjovall [38]. A micro column of 2 ml Celite-ethylene glycol ( 2 :1, w/v) also proved effective in separating estrogens, androgens, and progesterone in connection with competitive protein-binding assays and radioimmunoassays [928]. With single-ion monitoring the GC-MS analysis has become so specific that no elaborate purification of the blood extract is necessary. Testosterone and progesterone were determined in 1 ml of plasma by adding 4-methyl-19-nortestosterone as an internal standard for testosterone and 4-ethylandrostenedione as an internal standard for progesterone [223]. After simple extraction, the dienol-HFB derivatives were prepared and peak heights were determined by single-ion detection. The results agreed well with those of radioimmunoassays [224]. Selective ion detection has been used to determine norethisterone [ 10131 and mass fragmentography t o determine megestrol acetate [ 1 11 in plasma by a GC-MScomputer system. For the analysis of adrenal and sex hormones in cow's milk and tissues by TLC, Duthie et al. [250] have devised new solvent systems. A scheme for the analysis of human ovarian tissue, based on TLC before and after acetylation and on GC, was published by Poteczin el ai. [864]. Axelson et al. [37] have determined neutral tissue steroids by a procedure involving chromatography on Lipidex, preparation of MO derivatives, chromatography on Arnberlyst A-26, trimethylsilylation, and computerized GC-MS. Lipidex chromatography, TLC, and GC-MS were also involved in-the identification of metabolites in placental rnicrosomes [ 1071. High-resolution capillary GC was successfully applied to the trace analysis of steroids in tissues [226,721]. GC has also found use in the determination of some synthetic glucocorticoids in rat muscle [973]. The CI9O2 and CZ1O2steroid mono- and disulfates [622] and glucosiduronates [621] were analyzed by separating them first on Sephadex LH-20 and then hydrolyzing them prior to GC. Other applications of GC include the analysis of amniotic fluid [929] and feces [286,287].

12.4. HORMONES IN PHARMACEUTICALS The structure of the 17a-ethynyl steroids suggests, of course, the use of argentation chromatography. They have, in fact, been chromatographed on a silver-impregnated

110

MISCELLANEOUS STEROID HORMONES

Florisil column [6 161 and on a silver-sulfoethyl cellulose column [845]. A reversed-phase partition column, with heptane held stationary on silanized Celite and with ethanol as the mobile phase, was used for the analysis of testosterone propionate in oil injectables [989]. For the automatic analysis of steroid pharmaceuticals, Cavina etal. [ 156,1571 have used elution of a silicic acid column with a gradient of diethyl ether in petroleum ether (b.r. 65-75") and continuous monitoring of the eluate (see p. 9). Many analysts are beginning to use HPLC for pharmaceuticals. Corticosteroid creams and ointments have been assayed by eluting a silicic acid column with dichloromethane95% ethanol (19: 1) [339], a column of fl,fl'-oxydipropionitrile on Zipax with a hexaneethyl acetate-acetonitrile (474:25: 1) mixture [733], or a column of silanized Porasil with a 2,2,4-trimethylpentane-isopropanol-acetonitrile (1 38:37:25) mixture [628]. Permaphase ODS columns have been used for a variety of synthetic steroid hormones [44], including the synthetic estrogens [901]. Other column materials included Permaphase ETH [ 135,9011 and a spherical porous copolymer of styrene-divinylbenzene [333]. For TLC of pharmaceutical preparations, some use of polyamide [320] and polyvinylpyrrolidone [217,218] plates has been reported, and continuous [154] as well as twodimensional [ 159,10331 development was occasionally used. Silver nitrate-impregnated silica gel has proved selective for unsaturated steroids [ 10091. In the case of the 1 7 ~ ethynyl estrogens, argentation TLC has been advocated I2841 but found to result in some TABLE 12.1

~ R VALUES F FOR ESTROGEN AND PROGESTERONE ANALOGS Solvent systems: 1 = heptane-acetone (4:l) [60]; 2 = cyclohexane-ethyl acetate-acetone (15: 15:2) [60]; 3 = benzene-methanol (19:l) (972];4 = benzene-acetone (4:l) [972]; 5 = chloroformmethanol (9: 1) [972] ; 6 = dichloromethane-methanol-water (300: 18:l) [972]. Compound

Progesterone 17-Hydroxyprogesterone acetate Medroxyprogesterone acetate Norethindrone Norgestrel Allylestrenol Lynestrenol Ethylestrenol Norethynodrel Vinylestrenolone Ethynodiol diacetate Estrone Estradiol Estradiol benzoate Ethynylestradiol Mestranol Chlormadinone acetate Dimet histerone Megestrol acetate

Solvent system -1 2 3 28 19 22 15 16 57 42 53 24 29 40 18 12 22 13 32 -

32 26 30 22 26 66 60 59 38 39 50 33 22 32 26 48 -

-

29 37 -

61 42 -

77 23 57 55 40 52

4 37 45 63 -

51 68 -

5

6

-

-

-

-

57 59

48 53

65

69 57 84 39 68 74 57 70

-

63

-

76

-

-

-

-

43 56 47 46 43

46 65 72 61 71

-

HORMONES IN PHARMACEUTICALS

111

TABLE 12.2 h R F VALUES FOR SYNTHETIC CORTICOSTEROIDS Solvent systems: 1 = 1,2-dichloroethane-methanol-water (475:25: l ) , relative to hydrocortisone acetate = 100 [ 1921 ; 2 = 1,2-dichloroethane-2-methoxyethyl acetate-water (80:20: l), relative to hydrocortisone acetate = 100 [ 192); 3 = cyclohexane-ethyl acetate-water (25:75:1), relative to hydrocortisone acetate = 100 [192]; 4 = heptane-chloroform-acetic acid (1:1:2) [1127]; 5 = chloroform-acetic acid (9:l) [ 11271 ; 6 = cyclohexane-ethyl acetate (3:17) [ 11271 ; 7 = 1,2-dichloroethane-methyl acetate-water (2:1:1), relative to cortisone acetate = 100 [ 3921; 8 = 1,2-dichloroethane-dioxane-water (2: l : l ) , relative to hydrocortisone = 100 [ 3921 ; 9 = dichloromethane-diethyl ether-methanol-water (385:75:40:6) [59] ;dichloromethane-dioxane-water (2:l: 1) [59]. Compound

Solvent system 1

2

3

4

5

6

7

8

9

10 -

Deoxycorticosterone acetate Deoxycor ticosterone Cortisone acetate Prednisone acetate Dexamethasone acetate Fludrocortisone acetate 6a-Methylhydrocortisne 21-acetate Hydrocortisone acetate Methylprednisolone acetate Prednisolone acetate Triamcinolone acetonide Cortisone F’rednisone 6a-Methylhydrocortisone Dexamethasone Hydrocortisone Fludrocortisone Betamethasone Methylprednisolone Prednisolone 16a-Hydroxyfludrocortisone Triamcinolone

244 176 125 116 111 103 100 100 92 78 59 51 43 27 27 24 24 22 22 19 14

8

251 196 145 124 114 114 110 100 90 82 73 63 51 31 35 29 33 35 24 22 18 14

142 98 102 89 118 116 108 100 100 92 74 53 44 50 68 45 63 69 45 39 31 27

-

82 -

-

58 76 -

68 88 64 50 32

192 100 79 79 87 66 53 50 19 19 -

26

-

59

188 -

87 82 77 79

-

-

-

75 73 70 47 62 35 50 38 44 42 37

-

139 137 121 105 100 100 100 89 83 -

77 70 65 66 64 60 55 42 50 43 40 44 36 35 25 -

decomposition [39]. A spray of 0.1% AgN03 in 0.5 M NH,OH, followed by a spray of 0.02% eosin or sodium fluoresceinate in ethanol is a useful detection reagent for these compounds [897]. The RF values of a number of substituted estrone and estradiol analogs have been reported [ 10351. Table 12.1 gives hRF values for some estrogen and progesterone analogs. TLC has been used to identify 21-aminocorticosteroids [361], prednisone and prednisolone in oily solutions [774], impurities in prednisolone preparations [787], and the etiocholenic acids produced by the oxidation of corticosteroids [251]. Table 12.2 lists the hRF values of a few synthetic corticosteroids in TLC on silica gel. Some natural corticosteroids are included for reference. Rosetti [905] has studied the effect of multiple development with water-saturated diethyl ether of silica plates with different water content on the resolution of synthetic corticosteroids. Two or three developments of plates kept at 20% humidity gave the best results.

112

MISCELLANEOUS STEROID HORMONES

Structure-mobility correlations have been attempted for many synthetic steroids [243, 404,4051, including androgen [834] and progesterone [867] analogs. For quantitative analysis, not only elution and colorimetry [148] or IR spectrometry [76] have been used, but also direct densitometry [966]. GC has not been applied extensively to pharmaceutical analysis. Schulz [946] has compared GC with TLC in the assay of 17a-ethynylestradiol3-methylether. Both methods were equally accurate, but GC was significantly faster. A method for the analysis of ethynylestradiol by GC was published by Talmage et al. [ 10391. Adhikary and Harkness [4] have explored the carbon skeleton chromatography of synthetic steroid hormones. In this method, the steroids are subjected to high-temperature catalytic reduction prior to GC. Synthetic steroids can be isolated from oil solutions in an adequate state of purity for GC by a simple partition between hexane and 85% ethanol [ 1611. TLC was used to isolate estradiol monoesters from oil solutions prior to analysis by GC [ 160,7371. The GC-MS combination has been applied to fluorinated corticosteroids [ 1201.

Chapter 13

Vitamins D The chromatography of the vitamins D p r e s a t s major problems in addition t 6 the difficulties of separating a group of sterols differing in only minor structural details of the side chain. They are unstable to light, air, heat, and strong adsorbents. On the other hand, they are easily detected because they absorb UV light. Many aspects of the chromatography of vitamins D have been reviewed [227,540,758]. Norman and DeLuca [791] have devised a preparative method for separating the products of W irradiation of ergosterol and 7-dehydrocholesterol. By eluting a column, 58 cm X 1.5 cm, of 24 g silicic acid with 10% diethyl ether in petroleum ether (b.r. 6570'), they were able to separate at least 25 mg of an irradiation mixture into vitamin D2, tachysterol, , and ergosterol. Subsequently, Dollwet and Norman [240] accomplished the resolution of vitamins D2 and D3 by reversed-phase partition chromatography. When a column, 214 cm X 0.8 cm I.D., of Factice was developed with acetone-water (19: l), separate bands of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol emerged, in that order. By using 29 g of silica gel containing 12% of alumina in a 60-cm X 1-cm column, Mermet-Bouvier [7 161 was able to fractionate the photochemical isomers of ergosterol with diethyl ether to yield, in the order of elution: toxisterol, A, previtamin D2, lumistero12, tachystero12, and ergosterol. To isolate metabolites of vitamin D3, Holick and DeLuca [455] have used a 600-mm X 1 1-mm column of 20 g Sephadex LH-20, which was eluted with chloroform-petroleum ether (b.r. 65-70') mixtures, a 13:7 mixture being particularly useful. As one would expect, HPLC is eminently suitable for the fractionation of fat-soluble vitamins [ 11601. Applications to the vitamin D field are just beginning to appear. Krol et al. [601] have shown that the separation of calciferol from precalciferol by HPLC on a Vydac column takes 1.2 min, whereas an equivalent analysis on a Lipidex column takes more than 1 h. Use of a 500-mm X 2.1-mm I.D. column of Permaphase ODS with a linear elution gradient of methanol in water allowed Matthews e t al. [704] to separate a number of hydroxylated cholecalciferol metabolites in a matter of minutes (see Fig. 13.1). Solvent systems for TLC of vitamins D and related sterols include: petroleum ether (b.r. 65-7O0)-acetone (9:l) for the separation of vitamin D2 from ergosterol [791], hexane-acetone-water (45 :5:1) for the separation of vitamin D3 from 7-dehydrocholesterol [833], hexane-ethyl methyl ketone (5: 1) for the isomers of vitamin D2 [586], carbon tetrachloride-acetone (10: l ) , or benzene-acetone (1 0: 1) for the separation of the photochemical isomers of ergosterol on either silica or alumina plates [714], and petroleum ether-benzene (1 :1) or hexane-chloroform (1 : 1) for ergocalciferol and related sterols on alumina plates [860]. The incorporation of BHT and squalane in the solvents inhibits air oxidation [403]. Chen [ 1841 has studied the use of various fluorescent dyes for the detection of vitamin D on thin-layer plates. An SbC13 spray produces a variety of colors with vitamin D analogs [860], A recurrent problem in the analysis of biological extracts for vitamin D is the removal of cholesterol. This may be accomplished by TLC with 1,2-dichloroethane-isobutylmethyl

113

VITAMINS D

114

L

I

5

10

I

I

I

15

20

25

Time in minutes

Fig. 13.1. HPLC of hydroxylated cholecalciferol (HCC) derivatives on Permaphase ODs. A linear solvent gradient was used from methanol-water 3:7 to 8: 2 at the rate of 2%/min. (Reproduced from FEBS Lett., 4 8 (1974) 123, with permission; [704].)

ketone (9:1) [855] or dichloromethane [312], or by TLC on 5% (w/w) AgN03-Silica Gel G with chloroform-acetone (9:1) [985,986]. The quantitative estimation of vitamin D may be based on visual comparison of zones on thin-layer plates after they have been sprayed with 50% H2S04 [418] or SbC13 in chloroform [ 1015,11431 or on elution of the zones and either W spectrophotometry [ 10851 or spectrophotometry after the Nield reaction [403]. A method for the specific determination of nanogram quantities of vitamin D3 is based on isotope dilution [139]. By adding the sample to a mixture of I4Clabeled vitamin D3 and tetracyanoethylene, the amount of radioactive product, which is estimated by scanning a thin-layer chromatogram, is decreased, depending on the amount of vitamin D3 in the sample. The vitamins D undergo thermal rearrangement to the pyro and isopyro compounds during GC, but the peak areas of these products can nevertheless be used for quantitative estimation [36,757,1115].The vitamins have been chromatographed as such or in the form of their TMS or TFA derivatives on 3.4%butanediol succinate, SE-30, SE-52, or mixed SE-52 t cyanoethyl methylpolysiloxane columns. Recent experiments by Fisher et aZ. [311] indicate that it is best to silylate the sample by incubation at 4°C in the dark with 1 volume of pyridine and 0.1 volume of MSTFA and t o separate the products by GC on 4% OV-225.Even better resolution was obtained by GC on 4%Dexsil300 [260]. Another approach has been the conversion of the vitamins to isovitamins with SbC13 [755] and purification of the products by silicic acid column chromatography [285]. Isovitamins D, and D3 were separated by GC on a 3% OV-1 column [756]. To make this method more sensitive, Wilson et al. [ 1 1611 esterified the isotachysterols with HFBA at O°C, thus enabling as little as 10 ng of the vitamins to be determined with a 63Ni-ECD.Sheppard el d . [964] preferred acetyl chloride to SbC13 as a reagent for complete isomerization of the vitamins D. The isotachysterols were then chromatographed on 3% JXR [963]. The separation of eight photochemical isomers, produced by prolonged irradiation of ergosterol, has been accomplished without derivatization 17 151. From a Pyrex column, 10 ft. X 0.08 in. I.D., packed with 3%XE-60 on Gas-Chrom Q, at 225"C, three toxisterols, pyrocalciferol, lumisterol, isopyrocalciferol, ergosterol, and tachysterol were eluted, in that order, and the eluate fractions were analyzed by W spectrometry.

Chapter 14

Molting hormones This is a short chapter, because molting hormones have been rather recently discovered. The reason for this is that they are sterols of unusually hydrophilic character, which were apparently discarded in earlier work on sterol isolation. The molting hormones owe their hydrophilic character to the numerous hydroxyl groups, both in the nucleus and in the side chain. Like most steroid hormones, they contain an a,@nsaturated carbonyl group, a useful feature for W analysis. Several dozen representatives of this subgroup of sterols are known, differing in both the number of carbon atoms and the number and distribution of hydroxyl groups. Hori [458] used a 150-cm Amberlite XAD-2 column for the automatic analysis of molting hormones by linear gradient elution with from 20 to 70% ethanol in water. The elution, monitored by a UV detector, took 12 h and yielded the following sequence: ecdysterone, inokosterone, cyasterone, pterosterone, ponasterone A, i.e., the hormones emerged in decreasing order of polarity. An Amberlite XAD-2 column was also used by Schooley et al. [941] for the isolation of insect-molting hormones from a plant extract. A 500-g column retained the hormones in 4 1 of plant extract in 20% aq. methanol solution. After it had been washed with 3 1 of 33% methanol, the column was eluted with 3 1 of 70% methanol to recover 99% of the ponasterones A and B. HPLC with Poragel PN and a linear gradient of methanol in water was used for hormone analysis [940]. Nigg et al. [782] used a 3-m X 2-mm I.D. column, packed with Corasil II,37-50 pm, and various mixtures of chloroform and 95% ethanol to separate up to nine of the hormones. The molting hormones are separable by TLC [924], but, being hydrophilic, they can be very well analyzed by PC [357]. The solvent systems for PC were prepared by equilibrating toluene or benzene with 40-50% aq. 2-butanol or isopropanol. A number of color reactions, suitable for TLC of molting hormones, have been described [45 11. They may be assayed on the thin-layer plate by a densitometric method [924]. For GC, the insect-molting hormones are silylated by heating 1 mg of the steroid with 0.5 ml BSA and 0.1 ml pyridine at 80°C for 1 min [541]. Ikekawa et al. [501] and Miyazaki et al. [725] have prepared the HFB derivatives by an exchange reaction, heating 0.5 mg of steroid, 20 pl TMSI, 20 pl HFBI, and 2 p1 HFBA at 50°C for 2 h. The TMS ethers were separated on 1.5% OV-101 at 275°C and the HFB esters on 1% OV-101 at 260°C. Picogram amounts of the HFB-TMS derivatives could be determined by an ECD, and the TMS derivatives were suitable for mass fragmentography. Morgan and Woodbridge [740] have chromatographed the MO-TMS derivatives of molting hormones, but subsequently Poole et al. [862] found that their TMS ethers can be detected at the picogram level by the ECD, probably owing to the 7-en-6-onegroup. The results of GC analysis agree well with those obtained by radioimmunoassay 1981.

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Chapter 15

Steroid sapogenins and alkaloids The C27 sapogenins and alkaloids occur in plants in the form of their glycosides, the saponins and glycoalkaloids. Because of their hydrophilic nature, these glycosides are sometimes still separated by PC. The sapogenins are relatively stable and inert compounds, having a hydroxyl group at C-3 and differing from their analogs only in the number and location of additional oxygen functions and in the junction of Rings A and B. The alkaloids are too weakly ionized to be effectively separated by ion-exchange and electrophoretic methods and are treated together in this chapter because they are isolated and chromatographed along with the sapogenins. The isolation of sapogenins and alkaloids from plant material almost invariably involves hydrolysis, extraction, and column chromatography. Suitable column materials are A1203 [830], Si02 [413], and ion exchangers [24-261. HPLC has also been applied to the steroidal alkaloids 14891. The chromatographic column, up t o 16 ft. long and 3/8 in. O.D., was packed with Porasil A, 37-75 pm, and eluted with a concave gradient of 97% aq. acetone in a 2: 1 mixture of acetone-hexane. Analysis of the effluent by TLC showed the elution sequence: tomatidine, solanidine, solasodine, rubijervine, veratramine, jervine. TLC of saponins has recently been reviewed by Hiller and Woitke [438]. Table 15.1 shows the hRF values observed with various solvent systems. Additional data were published by Blunden and Hardman [90] and by Elmunajjed e t al. [270]. The separation of Sol- and 5-unsaturated sapogenins is generally poor, unless AgN03-impregnated layers are used [297], Ronsch and Schreiber [898] and Schreiber et al. [942] had observed earlier that silica and alumina layers, impregnated with 7-1 5% AgN03, can accomplish the separation of steroidal sapogenins and alkaloids belonging to the As- and Sol-series. Subsequently, Rozumek [909] has determined that the optimum resolution of such pairs is obtained with a AgN03 content of 4.2-6.3%. Continuous development improves the separation. Another possible solution for this problem is the addition of bromine to the solvent system [298]. Table 15.2 lists the hRF values observed by Hunter et al. [490] for a series of steroidal alkaloids. Additional data for TLC of Veratrum alkaloids may be found in Zeitler’s article [ 1 1901. The quantitative analysis of individual sapogenins and alkaloids by PC or TLC requires detection, usually with iodine [2], and elution [ 13,380,668,83 11. Densitometric estimation involves a staining process, a SbC13 spray being the preferred method [91,92, 105, 6663. Boll [95] has separated glycoalkaloids on Silica Gel G plates with the organic phase of either of two solvent systems: ethyl acetate-water-pyridine (3:3: 1) or chloroform-water1% NH40H (2:2: 1). Another suitable solvent system, 95% ethanol-acetic acid (3: I), was described by Paquin and Lepage [832]. For the fractionation of steroid saponins, a mixture of 2.5 g cellulose powder, pretreated with 2% HCI, and 0.3 g gypsum was slurried in 1 1 ml water. The slurry was used to make thin-layer plates, which were developed with butanol, saturated with 5% acetic acid [688]. Kawasaki and Miyahara [544] used silica gel plates for the saponins and their acetates, whereas Paseshnichenko and Borikhina [837] have

117

STEROID SAPOGENINS AND ALKALOIDS

118 TABLE 15.1

hRF VALUES OF SAPOGENINS IN TLC Solvent systems 1-7 were used on silica gel layers, 8-12 on alumina-sintered glass plates, 13-18 on silica-sintered glass plates, 19-22 on AgN0,-impregnated alumina, and 23-26 on AgN0,-impregnated silica gel. 1 = Chloroform-ethanol (19: 1) [ 10371 ; 2 = chloroform-acetone (9: 1) [ 10371 ; 3 = benzeneacetone(l7:3) [1037];4 = benzene-methanol(23:2) [1037];5 = hexane-ethyl acetate (1:l) [1037]; 6 = hexane-acetone (4:l) [1037]; 7 = benzene-ethanol (17:3) [703]; 8 = benzene-ethanol (17:l) [818]; 9 = benzene-ethyl acetate (4:l) [818]; 10 = chloroform-acetone (9: 1) [818]; 11 = chloroform-ethanol (9:l) [818]; 12 = chloroform-methanol (9:l) (8181; 13 = benzene-ethanol (17:l) [814]; 14 =benzeneethyl acetate (4:l) [814] ; 15 = benzene-ethyl acetate (2:l) [814]; 16 = chloroform-ethyl acetate (4: 1) [814]; 17 = chloroform-acetone (9:l) [814]; 18 = chloroform-acetone (4:l) [814]; 19 = hexaneethyl acetate (20: 1) [ 2971 ;20 = cyclohexane-ethyl acetate (10: 1) [ 2971 ; 21 = hexane-ethyl acetate (5:l) [ 2971; 22 = cyclohexane-chloroform-ethyl acetate (20:4:3) [297] ; 23 = hexane-ethyl acetate (5: 1) [ 2971; 24 = cyclohexane-chloroform-ethyl acetate (20:4:3) [ 2971; 25 = toluene-chloroform (10:3) [297]; 26 = toluene-chloroform-ethyl acetate (20:4:3) [297]. Compound*

Sarsasapogenin Smilagenin Diosgenin Tigogenin Pennogenin Gentrogenin Hecogenin Convallamarogenin Isorhodeasapogenin Rhodeasapogenin Y onogenin Gitogenin To korogenin Metagenin Kitigenin Kogagenin Chiapagenin Isochiapagenin Chlorogenin p-Chlorogenin Digitogenin Neogitogenin Neotigogenin Neoyonogenin

Solvent system 1

2

65

39

-

-

59 59 53 49 46 39 39 37 21 19 9 6 4 3

35 35 26 24 22 21 18 18 11 7 2 1 1 1

-

-

-

-

-

-

-

-

-

-

3

4

46 51 41 42 39 39 30 42 25 32 22 37 24 30 25 38 25 32 11 17 11 16 2 1 2 1 9 1 1 0 1 8 -

5

-

-

51 46 46 35 26 21 28 28 28 13 9 3 2 0 0 -

-

-

-

-

-

-

-

-

-

-

-

6

7

8

42 67 - 83 34 57 73 29 53 77 22 50 75 16 42 80 16 50 76 22 42 77 22 47 22 47 9 30 34 11 35 28 4 2 7 6 1 2 3 1 2 0 0 23 2 - 69 69 46 - 60 - 18 26 - 76 - 38

9

1 0 1 1 1 2

-

80 61 67 56 70 59 50

75 60 67 55 62 58 42 -

9 6 0 0 41 41 7 15 0 6 66 8

-

19 40 41 18 20 21 20 1 1

0

-

0 11 10 1 3 0 1 41 1

-

6 5 0

0 39 40 7 13 0 5 65 7

85 85 83 81 82 81 77 54 56 30 16 69 68 48 56 48 58 81 55

*Compounds chromatographed as genins, except in Solvent systems 19-26, where the acetates were chromatographed.

119

STEROlD SAPOGENINS AND ALKALOIDS

13

14

15

16

17

18

71 71 63 63 53 46 46 44

58 61

66 67 64 59 52 45 40 39 15 10 3

65 69 65 60 52 47 44 37 12 9 1

70 73 68 65 58 55 51 47 20 16 4 0 38 38 10 15 32 16 66 19

80 87 75 77 75 71 69 67 -

-

25 18 16 2 31 31 12 16 3 18 63 26

55 50 36 29 24 24 6 3 2

-

-

-

0 15 15 2 5 0 5 48 7

0 32 32 6 10 0 10 62 15

-

0 30 29

6 10 9 60 12

-

40 41 17 '8 60 60 35 43 42 41 72 41

19

20

21

22

23

24

25

26

120

STEROID SAPOGENINS AND ALKALOIDS

TABLE 15.2

hRF VALUES OF STEROIDAL ALKALOIDS IN TLC ON SILICA GEL [490] Solvent systems: 1 = hexane-ethyl acetate (1:l); 2 = hexane-ethanol (1:l); 3 = dichloromethanemethanol (23:2); 4 = dichloromethane-methanol(9: 1); 5 = dichloromethane-acetone (4: 1); 6 = hexane-acetone (1: 1); 7 = dichloromethane-methanol-acetic acid (85: 13:2); 8 = dichloromethanemethanol-NH,OH (100: 100:1). Compound

Tomatidine Tomatidenol Soladulcidine Solasodine Veramine 9a-Hydroxytomatidine 7wHydroxytomatidine 7a, 1la-Dihydroxytomatidine 9 q 1 1a-Dihydroxytomatidine Demissidine Solanidine S p-Solanidan-3-one

4-Solaniden-3-one Rubijervine Isorubijervine Veralobine Verarine Cyclopamine Jervine Veratramine Veramarine Solanocapsine Verazine Tomatillidine Etioline Veralkamine

Solvent system 1

2

3

4

5

6

7

47 47 26 27 10 8 4 0 0 58 59 75 63 15 19 11 3 3 0 5 3 0 69 69 1 3

90 92 87 85 75 86 88 69 69 85 83 85 83 73 81 75 60 46 35 16 29 2 92 91 47 52

49 46 40 41 21 21 20 5 5 21 42 61 48 17 21 43 22 25 26 28 12 0 59 61 12 19

66 66 53 59 35 31 35 6 6 33 61 50 50 13 33 43 17 21 19 17 8 0 64 69 14 14

37 40 23 23 9 7 5 0 0 19 15 43 39

87 89 85 82 60 60 53 10 10 87 95 0 0 56 60 64 19 17 14 36 11 0 84 84 9 22

73 94 73 96 71 91 70 96 68 93 45 91 37 93 10 93 10 93 57 97 81 89 91 100 71 89 55 82 43 91 55 100 64 13 51 81 53 83 31 91 27 60 5 1 2 92 97 92 97 47 77 60 73

I 11 17 3 5 3 11 5 0 83 82 3 5

8

preferred alumina plates. There has been very little research activity in the application of GC to steroid sapogenins and alkaloids. Cooke [ 1981 and Rozanski [908] have shown that a crude Dioscoreu extract can be assayed for diosgenin very simply by injecting it in a column of 2.5% SE-30. Cowley et ul. [203] have prepared the TMS ethers of sapogenins by heating them in 1 ml tetrahydrofuran with 0.4 ml HMDS, 0.1 ml TMCS, and 0.05 ml pyridine. Good separation and quantitative data were obtained by chromatographing the TMS ethers on a 9-ft. X 1/8-in. column of 1.2% SE-30 at 26OoC. A method for the analysis of permethylated derivatives of glycoalkaloids by GC on OV-1 or Dexsil300 columns has recently been published [428].

Chapter 16

Cardenolides and bufadienolides The cardenolides and the homologous bufadienolides do not occur together, but they are discussed together because the same chromatographic methods are applicable to both groups of steroids. In plants, they occur in the form of glycosides, which are quite hydrophilic and therefore still frequently analyzed by PC. The genins, which contain at least two hydroxyl groups, at C-3 and at C-14, and an unsaturated lactone in the side chain, are also rather polar. The lactone ring may be exploited for detection, but it makes the molecule relatively unstable. The older preparative methods for isolating cardiac glycosides from plants with silicic acid columns [96,202,528] have been modified by automation [589] and by use of socalled dry columns [590], containing finer silica particles. A column of Sephadex (3-200, eluted with 30% methanol, was used for the isolation of heart glycosides from animal tissues [370]. El-Olemy and Stohs [272] found that a Sephadex LH-20 column, eluted with 95% ethanol, separated cardiac glycosides from aglycones. The first application of HPLC to heart glycosides was reported by Evans [288]. An SCX ion-exchange column, 3 ft. X 1/4 in. O.D., eluted with 4%amyl alcohol at 45"C, produced in consecutive fractions: digitoxigenin, its monodigitoxoside, its bisdigitoxoside, digitoxin, and lanatoside A. Only a few examples of the use of PC for the quantitative analysis of cardiac glycosides can be given. Lanatoside C was isolated by continuous PC with benzene-ethyl acetateformamide (30:69: 1) on formamide-impregnated paper, which was subsequently treated with xanthydrol reagent [267]. Comparing the use of this solvent system on filter paper and on talc layers, it was found that the former is more suitable for quantitative analysis [ 11961. Instead of xanthydrol, dixanthylurea [85 11 or 2,2',4,4'-tetranitrobiphenyl [872] may be used as color reagents. A convenient method of direct densitometry for paper chromatograms has been described [645]. TLC of cardiac glycosides has recently been reviewed [792]. Reviews on cardenolides [811] and bufadienolides [824] are available in Japanese. Earlier methods of TLC were adaptations of PC, and in many cases employed partition systems. Cellulose powder [871], talc [1194,1195], kieselguhr [857,995,1142], and silica gel [lo41 were impregnated with formamide, and thin-layer chromatograms were developed with partially miscible solvents. Impregnation of silica gel with borax was found to retard specifically glycosides with cis vicinal hydroxyl groups [880]. Current TLC methods are based on the adsorption on silica or alumina [349,607,818, 10241. Table 16.1 summarizes a few of the observed mobilities on silica gel. Additional solvent systems have been described for the separation of cardenolides from plant sterols [94], for cardenolide acetates [873], and for the bufadienolides [504,587]. Special solvent mixtures have been recommended for the analysis of cardiac glycosides in Digitalis [136,295,323,629,684],Strophanthus[201,449],Convallaria I6091 ,Apocynum [352], Eiysimum [888], and Acokanthera [62] species. Repeated and successive developments [ 1441 as well as continuous development [434] improve the resolution. The relationship between structure and mobility in TLC has been studied by Nover et al. [793] and by

121

+

TABLE 16.1

N N

~ R VALUES F OF CARDENOLIDES AND THEIR GLYCOSIDES IN TLC ON SILICA GEL Solvent systems: 1 = chloroform-acetone (13:7) [ 11931; 2 = diethyl ether-methanol (9:l) [1193]; 3 = chloroform-isopropanol(9:l) [ 11931;4 = chloroform-ethanol (9:l) [ 11931; 5 = toluene-methanol (4:l) [ 1193);6 = cyclohexane-acetone-acetic acid (49:49:2) [ 11931; 7 = ethyl acetatehexane-acetic acid (8:l:l) [ 11931; 8 = cyclohexane-acetone-acetic acid (65:33:2) [ 1281; 9 = hexane-ethyl acetate-ethanol (3:15:2) + 2.2% water [ 7931 ; 10 = ethyl acetate-pyridine (9: 1) + 2.7% water [ 7931; 11 = ethyl methyl ketone + 5.4% water [ 7931; 12 = toluene-ethyl acetate-propanol (10:33:7) + 2.2% water [793]; 13 = ethyl acefate-chloroform-acetic acid (18:l:l) [144]; 14 = two developments with System 13 [144]; 15 = cyclohexane-acetone-acetic acid (65:33:2) followed by a second development with (49:49:2) [ 1441; 16 = cyclohexane-acetone-acetic acid (65:33:2) followed by a second development with ethyl acetate-chloroform (9: 1) [ 1441; 17 = two developments with chloroform-isopropanol-acetone (16:1:3) [144]. Solvent system Compound ~~

~

1

2

~

-

~

3

4

_

5

_

6

_

7

_

8

9

10

11

12

13

14

15

16

17

43 26 22 19 16 12 10 9 20 12 9 41 18

48 35 34 26 24 23 23 31 25 24 41 29

38

79 73 67 60 51 46 41 36 -

79 60 42 30 38 30 19 12

78 56 45 36 40 29 22 18 -

-

-

-

-

-

-

34

29

-

-

25 23 39

68 64 51 47 50 47 38 36 55 50 38 66 48 32 41 46 33 -

93 85 73 57 71 63 42 28

12 -

62 56 47 39 42 41 32 26 45 46 29 62 41 27 38 38 27 -

14 70 60 57 68 59 51

21

53 44 35 26 24 9 30 12 33 15 22 23 12 35

71 62

33 27 28 24 19 17 30 25 18 40 32 18 26 26

45 33 21 15 23 19 11 8 23 19 8 44 23 16 12 23

68 70 59 53 55 55 44 42 55 57 44 72

-

38 30 28 22 20 19 20 29 26 24 36 33 26 29 24 33

-

~

.

~

~

~

Digitoxigenin Digitoxigenin monodigitoxoside Digitoxigenin bisdigitoxoside Digitoxin Digoxigenin Digoxigenin monodigitoxoside Digoxigenin bisdigitoxoside Digoxin Git oxigenin Gitoxigenin monodigitoxoside Gitoxin Gitaloxigenin Periplogenin Strophanthidol Strophanthidin Sarmentogenin Diginatigenin Acovenosigenin

35 19 15 9 16 11 7 4 18 9 4 32 16 12 24

43 34 30 24 26 23 17 14 28 22 14 42 29 23 31 ~

-

30

-

-

-

55

43 51 53 36 -

51

66 65 49 76 62 48 67 62 55

-

44 31 43 36 22 15

-

-

-

-

-

-

-

-

-

-

-

CARDENOLIDES AND BUFADIENOLIDES

123

Ziillich et al. [ 1 1931. For the detection of cardiac glycosides, sulfuric acid or aldehyde reagents, such as anisaldehyde, in sulfuric acid are generally used. Fluorescence may be produced by a perchloric acid spray [523] or modified Jensen reagent [519]. It is prepared by adding 2.1 ~ 1 3 0 % HzOz to a mixture of 20 mg ascorbic acid in 19 ml methanol and 30 ml conc. HCI and permits the detection of as little as 0.01 pg of the steroids. For quantitative analysis, visual comparison of the fluorescence can be used, which is produced by a spray reagent consisting of 60 ml acetic acid, 5 ml HzSO4, and 1 ml9% FeC13 [246]. Zones detected by their fluorescence or by other nondestructive methods can be eluted and determined colorimetrically [81,200,348,435] or fluorimetrically I5191 The UV absorption of bufadienolides at 355 nm 2 h after the addition of 1 N methanolic KOH can also be used for estimation [1007]. Kartnig and Danhofer [537] assayed Strophanthus glycosides by TLC on MgO by scraping off the adsorbent and heating it with 0.2 ml conc. HCl + 3 ml HzSO4 at 60°C for 15 min. The absorption at the maximum around 400 nm was then determined. The dersitometric estimation of cardenolides [289, 7 131 and bufadienolides [588] has also been described. The fluorescence, produced by the Jensen reagent [238] or by HCl vapors [326,327] can be measured by a scanning fluorimeter. Jelliffe and Blankenhorn [5 181 introduced GC into the analysis of cardenolides. which were separated in the form of their TMS ethers. The application of the GC-MS combination to these derivatives was later studied by Maume etal. [709] and several modifications were published [ 1162,1163j. Tan [ 10401 has proposed the TMS ethers of the 14-anhydrocardenolides as derivatives, and Watson and K,ilman [ 11471 and Watson et al. [ 1 1481 have been able to measure nanogram quantities of Ihe HFB esters with the ECD. The HFB derivatives are also suitable for the identification of cardenolides by GC-MS [ 1 1461.

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List of abbreviations Proportions of solvents are v/v, unless otherwise stated.

A a

abs . AFID aq. atm BDSA BHT BN BO b.r. BSA BST F A BTPPC ca . cm CMDMS conc. Dns DEAE DMH DMS e ECD ECTEOLA FID ft. g GC GC-MS HFB HFBA HFBl HM DS HPLC h hRF I.D. in.

IR

Angstrom, 10-l0m axial absolute alkali flame-ionization detector aqueous atmosphere bis(dimethylsily1) acetamide butylated hydroxytoluene Brenner-Niederwieser benzyl oxime boiling range N, 0-bis(trimethylsily1) acetamide N, 0-bis (t r imethylsilyl) t rifluoroacet amide benzyltriphenylphosphonium chloride about centimeter, 10-'m chloromethyldimethylsilyl concentrated 1-dimet hylaminonapht halene-5 -sulfonyl die thylaminoethyl N, N-dimethylhydrazone dime thylsilyl equatorial electron-capture detector epichlorohydrin of triethanolamine flame-ionization detector feet gram gas chromatography (ic) gas chromatography-mass spectrometry heptafluorobutyrate heptafluorobutyric anhydride hexafluorobutyrylimidazole hexamethyldisilazane high-pressure liquid chromatography hour R F X 100 internal diameter inches infrared

125

LIST OF ABBREVIATIONS

126

1 LC

M m mCi ME mg Pg min ml P1 mm Pm mmole MO MS MSTFA N ng O.D. PC PFBO Pg PIPSYL p.s.i. PTFE PTV

RI rPm satd. sec TFA TLC TMCS TMS TMSDEA TMSI

uv vlv wlv

WIW

liter liquid column chromatography molar meter millicurie, 1O - 3 ~ i methyl ether milligram, 1O-3g microgram, 10-6g minutes milliliter, 10-~1 microliter, millimeter, I r 3 m micrometer, 1W6m millimole methyl oxime, methoxime mass spectrometry

N-methyl-N-trimethylsilyltrifluoroacetamide normal nanogram, w 9 g outside diameter paper chromatography pentafluorobenzyl oxime picogram, lO-''g p-iodobenzenesulfonyl pounds per square inch poly(tetrafluoroethy1ene) per cent total bed volume refractive index rotations per minute saturated seconds trifluoroacetate thin-layer chromatography trimethylchlorosilane trime thylsilyl trime thylsilyldiethylamine trime thylsilylimidazole ultraviolet volume/volume weight/volume weightlweight

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Subject index

A Acetates 29, 67 Acovenosigenin 122 Adrenocortical hormones, see Corticosteroids Adrenosterone 21,49, 88 Aldadiene 108 Aldosterone 8,99-106 Alkaloids, C,, 23, 95,96 _ _ _ , C,, 23,117,120 Alkamines, see Alkaloids Allopregnanediol 38,52,94,108 Allopregnanolone 13, 21, 35,48,49,52,94 Allotetrahydrocorticosterone 104, 108 Allotetrahydrocortisol 104, 108 Allylestrenol 110 Alumina 3, 15 Androgens, see Androstane derivatives 1,4-Androstadiene-3,17-dione 20, 34, 88 5J6-Androstadien-3p-01 89 1,4-Androstadien-3-one,17phydroxy- 20, 88 _ _ _ , 17p-hydroxy-l7-methyl-, see Methandienolne 4,6-Androstaddien-3-one, 17p-hydroxy- 88 Androstane derivatives 87-92, 107, 109 _ _ _ blood 91, 92 _ _ _ GC 53,90-92 -_- HPLC 10,87 _ _ _ LC 87 _ _ - TLC 20,21,87-91 __- , urinary 89,91, 92 So-Androstane 4, 50, 90 5p-Androstane 90 Sa-Androstane-3p,l6pdiol 20, 49 5a-Androstane-3a,l 7pdi01, see Dihydroan droster o ne 5a-Androstane-3p,17p-diol 20, 49,52, 88, 90 5p-Androstane-3a,l7pdiol 88,90 5p-Androstane-3p,l7pdiol 88,90 Sa-Androstane-3,16-dione 20, 49 Sa-Androstane-3,17-dione 20,48,49, 52, 88, 90 _ _ _ , llp-hydroxy- 88 5p-Androstane-3,17-dione 20,48,88,90 5a-Androstane-ll,17-dione,3a-hydroxy; see 11-Ketoandrosterone 5PAndrostane-ll,l’I-dione, 3a-hydroxy-, see 11-Ketoetiocholanolone

193

Sa-Androstane-3a,l lp,l7p-triol 88 Sp-Androstane-for,llp,l7p-triol 88 Sa-Androstane-3,11,17-trione21 5P-Androstane-3,11,17-trione 88 5a-Androstan-301-01 20 5a-Androstan-30-01 20, 90 Sp-Androstandp-01 90 Sa-Androstan-1701-01 6 5a-Androstan-17p-01 4, 6, 20, 90 5pAndrostan-170-01 90 Androstanolone 35,88,90 5a-Androstan-3-one 90 _ _ _ , 17a-hydroxy- 90 _ _ _ , 170-hydroxy-,see Androstanolone Sp-Androstan-3-one 90 - _ _ , 17a-hydroxy- 90 _ _ - , 17p-hydroxy- 20, 88, 90 Sa-Androstan-16-one, 3p-hydroxy- 20 5a-Androstan-17-one 4, 20,49,52, 90 _ _ _ , 3tuJ lpdihydroxy-, see 11-Hydroxyandrosterone --- , 3p,llpdihydroxy- 35,89 _ _ - , 3a-hydroxy-, see Androsterone _-- , 3P-hydroxy; see Epiandrosterone $3-Androstan-1Tone 90 _ _ _ , 3a,l lp-dihydroxy-, see 1lp-Hydroxyetiocholanolone _ _ _ , 3a-hydroxy-, see Etiocholanolone -_- , 3Phydroxy- 6, 13, 88,90 1,4,6-Androstatriene-3,17-dione88 1,4,6-Androstatrien-3-one, 17p-hydroxy- 88 4-Androstene-3p,l7p-diol20,88 5-Androstene-3p,16pdiol 20 5-Androstene-3p,l7adiol 20, 35, 88 5-Androstene-3p,17p-diol 20, 35,49, 52, 88 _ _ _ , 17a-methyl-, see Methandriol l-Androstene-3J7-dione 20, 88 4-Androstene-3,16-dione 20, 49 4-Androstene-3,17-dione 20, 34,48,49, 52, 88 __- , 6tu-hydroxy- 88 _-- , 6p-hydroxy- 88 -_- , lla-hydroxy- 49,88 -__ , 1lp-hydroxy- 21,49,52,88 _ _ _ , 16a-hydroxy- 88 5-Androstene-l1,17-dione,30-hydroxy- 89 5-Androstene-3p,1lp,l7p-triol 88 5-Androstene-3p,l6a,l7a-triol35

194

4-Androslene-3,11,17-trione, see Adrenosterone 5a-Androst-l6en-3a-01 89 5a-Androst-16en-30-01 89 5p-Androst-16en-3a-ol 89 Androstenolone, see Dehydroepiandrosterone 4-Androsten-3-one, 6pJ7pdihydroxy- 89 _-- , lla,l7a-dihydroxy- 21 _ _ _ , lla,l7pdihydroxy- 89 ---, llp,l7p-dihydroxy- 89 _ _ _ , 16a,l7pdihydroxy- 89 -_- , 17aethynyl-l7-hydroxy-6a,2 1-dimethyl-, see Dimethisterone _ _ _ , 17a-hydroxy-, see Epitestosterone , 170-hydroxy-, see Testosterone 4-Androsten-17-one, 3a-hydroxy- 88 _ _ _ , 3P-hydroxy- 88 5-Androsten-ll-one, 3p,l7pdihydroxy- 89 5-Androsten-16-one, 3p,l7p-dihydroxy- 2 1 5-Androsten-17-one, 3p,l ladihydroxy- 89 _ _ _ , 3p,l lpdihydroxy- 89 _ _._ , 3p,16cudihydroxy-, see 16-Hydroxydehydroepiandrosterone _-- , 3a-hydroxy- 8 8 _-- , 3P-hydroxy-, see Dehydroepiandrosterone Sa-Androst-len-3-one, 170-hydroxy- 20, 88 Androsterone 6, 7, 13, 20, 23, 35, 38,49,S2, 88-91,108 --- glucosiduronate 25, 26 _ _ _ sulfate 25, 26 Argentation chromatography 15, 55,56, 5 9 , 6 0 Avenasterol, see 28-lsofucosterol

B Benzoates 11 Benzyloximes 33 Betamethasone 111 BiIe acids I 1 - 77 _ _ _ , fecal 75 _ _ _ GC 74-71 _ _ _ LC 5 , 6 , 7 1 _ _ _ methyl ethers 74, 75 _ _ - TLC 22,71-74 Bile alcohols 7 1, 73 Botogenin, see Gentrogenin Brassicasterol 59,63, 6 4 , 6 6 Bufadienolides 121, 123 Bufogenins, see Bufadienolides

C Campestadienol 63

SUBJECT INDEX Campestanol 6 3 , 6 4 Campesterol 5 6 , 5 8 , 5 9 , 6 3 , 6 4 , 6 6 , 67 5cu,l4pCard-20(22)~nolide,3@,14-dihydroxy-, see Uzarigenin 5P,14pCard-20(22)enolide, 3p,14-dihydroxy-, see Digitoxigenin _ _ _ , 16p-formoyloxy-3p,l4-dihydroxy-,see Gitaloxigenin

_ _ _ , 3p,5,14,194etrahydroxy-,see Strophanthidol

__- , lp,3p, 14-trihydroxy-, see Acovenosigenin _ _ _ , 3p,5,14-trihydroxy-, see Periplogenin

_ _ _ , 3p,l la,Irl-trihydroxy-, see Sarmentogenin _ _ _ , 3@,12p,l4-trihydroxy-,see Digoxigenin

_-- , 3!3,14,16f3-trihydroxy-,see Gitoxigenin _ _ _ , 3p,5,14-trihydroxy-19-oxo-, see Strophanthidin Cardenolides 22, 121-123 Cardiac genins, see Cardenolides, Bufadienolides _ _ _ glycosides 121-123 CentriChrom 10 Chalinasterol, see 24-Methylenecholesterol Chenodeoxycholic acid 22,12, 76, 77 Chiapagenin 118 Chlormadinone acetate 110 16aChloroestrone methyl ether 108 Chlorogenin 118 Chloromethyldimethylsilyl ethers 32 SwCholanic acid 77 5PCholanic acid 22, 76, 77 SaCholan-24-0ic acid, see 5aCholanic acid __- , 3a,ladihydroxy- 77 _-- , 3p,7adihydroxy- 77 __- , 3a,12adihydroxy- 77 _ _ _ , 3P,12~~-dihydro~y71 _ _ _ , 7a,l2adihydroxy- 77 _ _ _ , 3a,12adihydroxy-7-0~0- 7 1 _ _ _ , 3p,12adihydroxy-7-0~0- 77 _ _ _ , 7e,I 2a-dihydroxy-3-0x0- 77 _ _ _ , 3,7-dioxo- 77 _-- , 3,12-dioxo- 77 _-- , 3a-hydroxy- 77 _ _ _ , 3p-hydroxy- 77 _ _ _ , 7a-hydroxy- 77 _ _ _ , 7p-hydroxy- 77 _-- , 12a-hydroxy- 77 _ _ _ , 12a-hydroxy-3,7-dioxo- 77 _-- , 3a-hydroxy-7-0x0- 77 _-- 7 ~ ~ - h y d r O ~ y - 3 - 077 ~0_-- , 12a-hydroxy-3-0x0- 77 _-- , 12a-hydroxy-7~x0- 77 _-- , 50x0- 77 _ _ _ , 3a,6@,7p-trihydroxy- 77 _ _ _ , 3a,7a,12or-trihydroxy- 77 9

SUBJECT INDEX

195

-_--_

, 3p,7a,l2a-trihydroxy- 77 , 3,7,12-trioxo- 17 SpCholan-24-oic acid, see 5pCholanic acid

--_ _-_

-__

, 3a,6a-dihydroxy-, see Hyodeoxycholic acid --_ , 3a,6p-dihydroxy- 76, 77 --- , 3a,7a-dihydroxy-, see Chenodeoxycholic acid -__ , 3a,7pdihydroxy- 72,76, 77 --_ , 3P,7wdihydroxy- 7 1 --_ , 301,12a-dihydroxy-, see Deoxycholic acid - _ _ , 3p,l2a-dihydroxy- 1 7 --_ , 7a,l2a-dihydroxy- 77 --_ , 3a,7a-dihydroxy-12-oxo- 76, 77 --_ , 3a,l2adihydroxy-7-oxo- 72, 76, 77 _ _ _ , 7a,l2a-dihydroxy-3-0~0- 77 --_ , 3,7-dioxo- 76,17 - - _ , 3,12-dioxo- 76, 77 --_ , 3a-hydroxy-, see Lithocholic acid --_ , 3p-hydroxy-, see lsolithocholic acid --_ , 7a-hydroxy- 77 _ _ _ , 7p-hydroxy- 77 _ _ _ , 12a-hydroxy- 76, 7 1 --_ , 120-hydroxy- 71 --_ , 3a-hydroxy-7,12-dioxo- 72,76, 77 --_ , 12a-hydroxy-3,7-dioxo- 77 --_ , 3a-hydroxy-7-0x0- 16, 77 --_ , 3a-hydroxy-12-0x0- 72, 76, 77 _-- , 7whydroxy-3-oxo- 17 - _ _ , 12~-hydroxy-3-0~0-76, 77 _-_ , 12a-hydroxy-7-0x0- 77 __- , 3-0x0- 76, 77 _-_ , 3a,6a,7a-trihydroxy-, see Hyocholic acid -__ , 3a,6&,7@-trihydroxy- 76, 77 _-_ , 3a,6(3,7a-trihydroxy- 76, 77 _ _ _ , 3a,6p,7p-trihydroxy- 76, 77 --_ , 3a,7a,12a-trihydroxy-, see Cholic acid --_ , 3a,7&12a-trihydroxy- 77 --_ , 3p,70r,l2~~-trihydroxy-77 --_ , 3,7,12-trioxo-, see Dehydrocholic acid 5p-Cholan-7a-ol 6 5pCholan-70-01 6 Cholecalciferol 113, 114 Cholecalciferols, hydroxy- 114 3,SCholestadiene 22, 57, 6 2 Sa-ChoIesta-7,22-dien-3p-ol 6 3 _ _ _ , (24R)-24-ethyl-, see d p i n a s te r o l --_ , (24S)-24-ethyl-, see Chondrillasterol 5a€holesta-7,24-dien-3p-o1 56 Sor€hoIesta-8,14-dien-3p-ol 64 5aCholesta-8,24-dien-3p-ol,see Zymosterol 5,7Cholestadien-3p-o1, see 7-Dehyd~ocholesterol ---, (24R)-24-methyl-, see Campestadienol 5,22-Cholestadien-3p-ol, see 22-D~hydrocholesterol

5,24Cholestadien-3p-ol, see Desmosterol 5,25Cholestadien-3p-ol, see 25-Dehydrocholesterol 1,4Cholestadien-3-one 22, 5 7 ,6 2 3,5Cholestadien-7-one 5 7 ,6 2 4,6Cholestadien-3-one 22,57 5aCholestane 4 , 5 0 , 6 8 5pCholestane-3a,l2a-diol 4 Sp€holestane-3,12-dione 4 5~Cholestane-3or,7a,l2a,24a-tetro:73 Sp€holestane-3a,7a,l2a,24p-tetrol 73 5pCholestane-3a,7a,l2a,26-tetrol 73 5a-Cholestane-3p,5a,6p-triol 57, 62 5p-Cholestane-3a,7a,l2a-triol4 , 7 3 Sp-Cholestane-3,7,12-trione 4 Cholestanol 4 ,6 , 7, 16, 22, 34,40, 49,52, 55, 57,59-64,66 5aCholestan-3a-ol, see Epicholestanol 5a-Cholestan-30-01, see Cholestanol _-_ , 5a,6a-epoxy- 22 _ _ _ , (24R)-24-mcthyl-, see Campestanol --- , (24S)-24-methyl-, see Ergostanol SpCholestan-3a-ol, see Epicoprostanol 5pCholestan-3p-ol, see Coprostanol 50-Cholestan-70-01 59 Cholestanone 4 ,6 , 22,49, 52 Sa-Cholestan-3-one, see Cholestanone 5pCholestan-3-one, see Coprostanone 5aCholestan-6-one, 3p,Sadihydroxy- 5 7 , 6 2 _-- , 30-hydroxy- 22, 5 7 ,6 2 5,7,22Cholestatrien-3p-ol 64 _-- , (24S)-24-methyl-, see Ergosterol 5,7,22(9p,lOa)€holestatrien-3p-ol, (24R)-24methyl-, see Lumisterol, 5,22,24Cholestatrien-3p-ol 64 SCholestene, 3p-methoxy-, see Cholesterol methyl ether 5Cholestene-3p,4pdiol 22, 57,62 5Cholestene-3P,7adiol 6, 5 7 ,6 2 5-Cholestene-3p,7p-diol 6 , 5 7 ,6 2 5Cholestene-3p,24a-diol, see 24a-Hydroxycholesterol 5-Cholestene-3p,24pdiol,see 240-Hydroxycholesterol 5aCholest-8ene-3p,6adiol, see Peniocerol _-_ , 14a-methyl-, see Macdougallin 4Cholestene-3,6-dione 2 2 ,5 7 ,6 2 SChoIestene-16,22-dione, 3p,26-dihydroxy-, see Kryptogenin 4Cholesten-3p-ol 59 5Cholesten-3a-ol, see Epicholesterol

, (24R)-24-ethyl-, see Stigmasterol , (24S)-24-ethyl-, see Poriferasterol _ - _ , (24S)-24-methyl-, see Brassicasterol

196 SCholesten-3p-ol, see Cholesterol -__ , (24R)-24ethyl-, see Sitosterol _-- , (24S)-24ethyl-, see Clionasterol _-- , (E)24(28)ethylidene-, see Fucosterol _-- , (Z)24(28)-ethylidene-, see 28-lsofucosterol ---, (24R)-24-methyl-, see Campesterol __- , 24(28)-methylene-, see Methylenecholesterol (22R ,23R,24R)-5Cholesten-3&01, 22,23methylene-23,24-dimethyl-, see Gorgosterol ---, 22,23-methylene-24-methyl-, see 23-Demethylgorgosterol 5-Cholesten-3p-ol, 7-0x0-, see 7-Ketocholesterol 5aCholest-7en-3p-01, see Lathosterol _-- , (Z)24(28)-ethylidene-, see Citrostadienol --_ , 4a-methyl-, see Lophenol _-- , 14amethyl- 64 SaCholest-7-en-3p-ol, (24S)-24ethyl-, see 7-Chondrllasten-30-01 50Cholest-7en-3po1, see 7Coprostenol Sa-Cholest-8en-30-01 56,64 __- , l4a-methyl- 64 5aCholest-8(14)-en-3pol 63 S~Cholest-d(l4)-en-3p-ol,see 8(14)-Coprostenol 5or-Cholest-14en-3p-ol 63 4-Cholesten-3-one 6, 22,49, 52, 68 5-Cholesten-3-one 6, 22, 57,62 5Cholesten-7-one, 30-hydroxy- 22, 57, 62 hexahydroxy-, see Pterosterone

-_- , 2p,3&14,20,22,25-hexahydroxy-,see Ecdysterone

_-- , 2&30,14,20,22,26-hexahydroxy-,see Inokosterone

_-- , 2a,3a,l4,20,22-pentahydroxy-,see Ponasterone B

-__ , 2p,3P,14,20,22-pentahydroxy-,see Ponasterone A Cholesterol 3,5-7, 13,22, 23,34, 37,49, 52, 55-64,66,68,108,113 -_- blood 61,68,69 -__ methyl ether 4 _-_ oxidase 29 Cholesteryl esters 3-5, 56-58,61 Cholic acid 22, 72, 76, 77 7-ChondrilIasten-3po1 65 Chondrillasterol 63,65 Citrostadienol 65 Clionasterol 65 Coated capillaries 37-41, 108 Compound A, see Dehydrocorticosterone Compound B, see Corticosterone Compound E, see Cortisone Compound I:, see Cortisol

SUBJECT INDEX Compound S, see Deoxycortisol Conjugates 101, 107 Continuous development 17 Convallamarogenin 118 Coprostanol 4,6, 22,40,49,57,59,62-64, 68 Coprostanone 4, 22,68 7Coprostenol 64 8(14)-Coprostenol 64 Cortexolone, see Deoxycortisol Cortexone, see Deoxycorticosterone Corticosteroids 8, 10, 11, 99-106 --- blood 100, 103, 105 _-- GC 103,105,106 _ _ _ HPLC 100,110 --- LC 99,100 _-- pc 101 --- TLC 21,22,101-104, 111 _-- , urinary 103, 105 Corticosterone 8, 21, 99-102, 104 Cortisol 8, 21, 22, 99, 100, 102, 104, 111 Cortisone 8,21, 99, 100, 102, 104, 111 (a)Cortol 22 (or)Cortolone 21, 108 PCortolone 21, 108 Crustecdysone, see Ecdysterone Cyasterone 115 Cycloartanol 65 Cycloartenol 65 3,5Cyclocholestan-6-one 22 9p,19-Cyclo-5a-cholestan-3p-ol, 14a-methyl-, see Pollinastanol Cycloeucalenol 65 9p, 19Cycl0-5crlanostan-3p-ol, see Cycloartanol _-- , 24-methyl-, see 24-Methylcycloartanol _-- , 24-methylene-, see 24-Methylenecycloartanol see Cyclo9p, 19-Cyclo-5orlanost-24-en-3~-ol, artenol 9p,19-Cyclo-5c~-lanost-25en-30-01,(24s)-24methyl-, see Cyclolaudenol Cyclolaudenol 65 90, 19Cyclo-31-nor-5a-lanostan-30-1, 24-methylene, see Cycloeucalenol Cyclopamine, see 1 1-Deoxojervine

D 7-Dehydrocholesterol 22, 34,56,57,59-62, 64,113 22-Dehydrocholestero1 59,63,64 24-Dehydrocholesterol, see Desmosterol 25-Dehydrocholesterol 59,64,66

SUBJECT INDEX Dehydrocholic acid 72,76,77 11-Dehydrocorticosterone 8,21, 102, 104 Dehydroepiandrosterone 6, 13,20, 35, 38, 49,52,88-92 _ _ _ glucosiduronate 25, 26 _ _ _ sulfate 25, 26, 91 23-Demethylgorgosterol 65 Demissidine 49,120 Densitometry 24 11-Deoxojervine 120 Deoxycholic acid 22, 72,76, 77 Deoxycorticosterone 8,21,99, 102, 105, 111 Deoxycortisol 8,21,99, 101, 102, 105 24-Deoxyscymnol 73 Derivative ratio analysis 44 Derivatives 25-27, 29-33,43 Desmosterol 22,49,56,59-64,66 Desoxy-, see DeoxyDexamethasone 111 Digilanides, see Lanatosides Diginatigenin 122 Digitogenin 118 Digitoxigenin 22,121, 122 Digitoxin 121, 122 Digoxigenin 22, 122 Digoxin 122 Dihydroandrosterone 20, 35,49,52, 88, 90, 91 17a-Dihydroequilenin 20 17p-Dihydroequilenin 80 17p-Dihydroequilin 80,82 Dihydrotestosterone, see Androstanolone 24-Dihydrolanosterol 22,64 24-Dihydroobtusifoliol 64 7a,l la-Dihydroxytomatidine 120 9a,l la-Dihydroxytomatidine 120 Dimethisterone 108, 110 Dimethylhydrazones 33 Dimethylsilyl ethers 32 2,4-Dinitrophenylhydrazones 11, 26,89 Diosgenin 23, 118

E Ecdysones, see Molting hormones Ecdysterone 115 Elatography 26 Electron-capture detector 4 1 Epiandiosterone 6,7, 13,35,49,52, 88, 90 Epicholestanol 6 , 7 , 4 0 , 4 9 , 5 2 , 5 7 , 6 1 , 6 2 , 6 4 Epicholesterol 6 , 7 Epicoprostanol 6,22,40,59 16-Epiestriol 20, 80,82,85

197 17-Epiestriol 82, 85 16,17-Epiestriol 80,82, 85 Epipregnanolone 6,13,35,94 Epitestosterone 35, 38,87,88 Equilenin 20,49,80-82 Equilin 20,80-82,86 Ergocalciferol 113, 114 5,7-Ergostadien-3p-o1 64 Sa-Ergosta-7,22-dien-3p-o16 3,64 _-_ , 14a-methyl- 64 5a-Ergosta-7,24(28)dien-3p-o1 64 _ _ _ , 4a-methyl-, see 24-Methylenelophenol 5a-Ergosta-8,14dien-3p-o1 64 _ _ _ , 4a-methyl- 65 5or-Ergosta-8,22-dien-3p-ol,l4a-methyl- 64 5a-Ergosta-8,24(28)dien-3&01 64 _ _ _ , 4a,14adlimethyl-, see Obtusifoliol _ _ _ , l4a-methyl- 64 5,22-Ergostadien-3&01,see Brassicasterol 5,24-Ergostadien-3p-o1 63 5,24(28)-Ergostadien-3P-o1,see 24-Methylenecholesterol Ergostanol 66 5,7,(E)22-Ergostatrien-3p~l, see Ergosterol 5a-Ergosta-7,9( 11),22-trien-3p-o1 64 5a-Ergosta-7,14,22-trien-3p-o1 64 9p,l Oa-Ergostad ,7,22-trien-3p-ol, see Lumisterol, 5-Ergosten-30-01 64 5a-Ergost-7en-3p-ol 64 -__ , 14a-methyl- 65 5a-Ergost-8en-3p-ol 64 _ _ _ , 4a,l4adlimethyl-, see 24-Dihydroobtusifoliol --_ , 4a-methyl- 65 _ _ _ , 14a-methyl- 64 5a-Ergost-8(14)en-3p-ol 63,64 Ergosterol 22,34,58,63,64,66, 113, 114 Ergosteryl esters 58 Estetrol 86 Estradiol, see Estradiol-170 _ _ _ , benzoate 110 ___ , glucosiduronates 79 ___ , sulfates 79 Estradiol-l7a 20, 79,80,82,85 Estradiol-17P 4, 20, 35,49,52, 79, 80,82-85, 110 1,3,5( 10),6,8-Estrapentaene-3,1Ila-diol, see 17a-Dihydroequilenin 1,3,5( 10),6,8-Estrapentaene-3,17p-diol, see 17p-Dihydroequilenin 1,3,5(10),6,8-Estrapentaen-l7-0ne,3-hydroxy-, see Equilenin 1,3,5(10) ,6-Estratetraene-3,17pdiol 20

198

SUBJECT INDEX

1,3,5(10),7-Estratetraene-3,17pdiol,see 17pDihydroequilenin 1,3,5(1 0),7-Estratetraen-l7-one, 3-hydroxy-, see Equilin 3-methoxy1,3,5(1 O)-Estratriene-2,17pdiol, 84 1,3,5( lO)-Estratriene-3,16adiol 20 1,3,5(10)-Estratriene-3,17adiol,see Estradiol-l7a 1,3,5(1 O)-Estratriene-3,17pdiol, see Estradiol-170 _ _ _ , 17crethyny1, see Ethynylestradiol -_- , 2-methoxy-, see 2-Methoxyestradiol 1,3,5 (lO)-Estratriene-4.17p-diol,3-methoxy- 84 1,3,5( 1O)-Estratriene-l6a,l7adiol, 3-methoxy82 1,3,5(10)-Estratriene-l6a,17~diol, 3-methoxy82 1,3,5(1 O)-Estratriene-l6p,17pdiol, 3-methoxy82 1,3,5(1 O)-Estratriene-6,17-dione,3-hydroxy82,85 1,3,5(10)-Estratriene-ll,17-dione,3-hydroxy82 1,3,5(1O)-Estratriene-16,17-dione,3-hydroxy82,85 1,3,5 (1O)-Estratriene-2,3,16a,l7p-tetrol,see 2-Hydroxyestriol 1,3,5(10)-Estratriene-3,6cr,16ar,l7p-tetrol 82

1,3,5(10)-Estratriene-3,15a,16a,17cu-tetroI,see Estetrol 1,3,5( lO)-Estratriene-2,3,17p-triol 82, 84 1,3,5(10)-Estratriene-3,4,17p-triol 84 1,3,5(1O)-Estratriene-3,6a,17p-triol 82, 85 1,3,5(1 O)-Estratriene-3,6p,17p-triol 82 1,3,5 (1O)-Estratriene-3,11&17p-trio1 82 1,3,5(10)-Estratriene-3,16a,l7~-triol, see 17-Epiestriol _ _ _ , 2-methoxy- 85 1,3,5 (1O)-Estratriene-3,160~,170-triol, see Estriol _ _ _ , 2-methoxy-, see 2-Methoxyestriol 1,3,5( lO)-Estratriene-3,16p, 17a-triol, see 16J7-Epiestriol

1,3,5(10)-Estratriene-3,16p,l7ptriol, see 16-Epiestriol

1,3,5(1O)-Estratrien-3-01 20 1,3,5(10)-Estratrien-17p-01, 2,3-dimethoxy- 84 --_ , 17aethynyl-3-methoxy-, see Mestranol

_ _ _ , 3-methoxy-

82 1,3,5( 10)-Estratriend-one, 3,17pdihydroxy85 _ _ _ , 3,16~,17p-trihydroxy- 82 1,3,5(10)-Estratrien-16-one,3,17p-&hydroxy. 20,35,82,85 1,3,5(10)-Estratrien-17-one,3,4-dihydroxy- 84

__- , 3,6adihydroxy-

82

_-- , 3,6p-dihydroxy- 82 --- , 3,110-dihydroxy- 82

_-_ _--

, 3,16adihydroxy- 82,85 , 2,3-dimethoxy- 84 --- , 3-hydroxy-, see Estrone ---, 2-methoxy-3-hydroxy-, see 2-Methoxyest r one --- , 3-methoxy-2-hydroxy- 84 4-Estrenedp,l7pdiol, 17aethynyl-3p,17pdiacetoxy-, see Ethynodiol diacetate 4-Estrene-3,17-dione 20,49 4-Estren-170-01, 17-allyl, see Allylestrenol _-- , 17-ethyl-, see Ethylestrenol --- , 17-ethynyl-, see Lynestrenol 4-Estren-3-one, 17a-ethy1-17-hydroxy, see Nilevar --- , 17aethynyl-l7-hydroxy-, see Norethisterone --- , D-1 7aethynyl-17-hydroxy-18-methyl-, see D-Norgestrel _-- , 17p-hydroxy-, see Nandrolone _-- , 17a-vinyl-l7-hydroxy-, see Vinylestrenolone 5( lO)-Estren-3-one, 17~ethynyl-l7-hydroxy-, see Norethynodrel _-- , 17p-hydroxy- 20 Estriol 15,20, 23,35,38, 79,80, 82, 84, 85 _-- glucosiduronates 79,80 _-plasma 86 --- 3-sulfate 80 _-- 3-sulfate 16-glucosiduronate 80 _-- , urinary 86 Estrogen glucosiduronates 79,81 _-- sulfates 79, 81 Estrogens 10, 11, 79-8ti, 107, 109 _-- GC 83-86 _-- gel chromatography 79, 80 _-- HPLC 79,80,110 _-- LC 79,80 _-- plasma 84 _-- TLC 20,81-83 _-- , urinary 83 Estrone 4,20,35,49,52,79-86,110 --- glucosiduronate 79 _-- sulfate 25,26,79 Ethylestrenol 110 Ethynodiol diacetate 110 Ethynylestradiol 110, 112 Etiocholanolone 6, 13,23,35,38,48, 88-91, I08 --- glucosiduronate 25, 26 Etioline 120

SUBJECT INDEX

F Factice 56 Flame-ionization detector 9, 41 Flophemesyl ethers, see Pentafluorophenyldimethylsilyl ethers Fludrocortisone 111 Fragment ion chromatogram 43 Fucosterol 59,63,65,66

G Gel chromatography 4-7 Gentrogenin 23, 118 Gitaloxigenin 122 Gitogenin 118 Gitoxigenin 22, 122 Gitoxin 122 Glass-fiber paper 14 Glucosides 13, 25 Glucosiduronates 13, 16, 19, 25, 32, 109 Glycoalkaloids 117, 120 Gorgosterol 65 Gradient elution 8, 9, 99 Group retention factors 47,48

199

18-Hydroxysolanidine, see Isorubijervine 7a-Hydroxytomatidine 120 9or-Hydroxytomatidine 120 Hyocholic acid 75-77 Hyodeoxycholic acid 22, 72, 76,77

I Inokosterone 115 Isochiapagenin 118 Isocholesterol 22 28-Isofucosterol 65,66 Isolithocholic acid 76, 77 Isopyrocalciferols 114 Isorhodeasapogenin 118 Isorubijervine 120 Isotachysterols 114 Isotope effects 55, 99

J Jervine 117, 120

K

H Hecogenin 23, 118 Heptafluorobutyrates 29 High-pressure liquid chromatography 10, 11 Holamine 23 Holaphyllamine 23 Holaphylline 23 Hydrocortisone, see Cortisol 1I-Hydroxyandrosterone 38, 89, 108 24a-Hydroxycholesterol 59 240-Hydroxycholesterol 59 18-Hydroxycorticosterone 101 6~-Hydroxycortisol 103, 104 16-Hydroxydehydroepiandrosterone 38,89 2-Hydroxyestriol 86 1 lp-Hydroxyetiocholanolone 35, 38, 89 160-Hydroxyfludrocortisone 111 16a-Hydroxypregnenolone 38,95 17-Hydroxypregnenolone 25,26,95 1 lp-Hydroxyprogesterone 6,93, 95 16a-Hydroxyprogesterone 95,97 17-Hydroxyprogesterone 21, 35, 93, 95, 109 _ _ _ acetate 110 21-Hydroxyprogesterone, see Deoxycorticosterone 12ar-Hydroxysolanidine, see Rubijervine

Ketals 32 11-Ketoandrosterone 23, 35, 38, 88 7-Ketocholesterol 60 11-Ketoetiocholanolone 35, 38, 88, 108 17-Ketosteroids 87,89, 91,99, 107 Kitigenin 118 Kogagenin 118 Kryptogenin 23

L Lanatosides 121 8,24-Lanostadien-30-01, see Lanosterol 8-Lanosten-30-01,see 24-Dihydrolanosterol Lanosterol 22, 58,65 Lathosterol 22,49,56,57,62-64 Lithocholic acid 22, 72, 76, 77 Lophenol 64 Lumisterol, 34,58, 113, 114 Lynestrenol 110

M Macdougallin 65 Mass chromatography 75

200 Mass fragmentography 42,69 Mass spectrometry 11,42,43 Medrol, see 6a-Methylprednisolone Medroxyprogesterone acetate 110 Megestrol 109, 110 Mestranol 110 Me tagenin 118 Methandienone 108 Methandriol 49 Methostenol, see Lophenol Methoximes, see Methyloximes 2-Methoxyestradiol 82, 85 2-Methoxyestriol 82 2-Methoxyestrone 82 6a-Methylcortisone 111 24-Methylcycloartanol 65 Methylene units 53 24-Methylenecholesterol 59,63,64 24-Methylenecycloartanol 65 24-Methylenelophenol 65 24-Methylenepollinastanol 64 6a-Methylhydrocortisone 111 Methyloximes 33 24-Methylpollinastanol 65 6a-Methylprednisolone 111 Methylsterols 63,67 Molting hormones 115

N Nandrolone 20 Neogitogenin 118 Neotigogenin 13,23, 118 Neoyonogenin 118 Nilevar metabolites 108 p-Nitrobenzoates 11 19-Nor-4-androstene-3,17dione, see 4-Estrene-3, 1'I-dione 19-Nor-4-androsten-3-one, 17aethyl-17-hydroxy-, see Nilevar 24-Nor-5,(E)22-cholestadien-3p-ol 5 9 31-Norcycloartanol 64 Norethindrone, see Norethisterone Norethisterone 108-1 10 Norethynodrel 110 D-Norgestrel 110 19-Nortestosterone, 17aethyl-, see Nilevar

0 Obtusifoliol 64 Ostreasterol, see 24-Methylenecholesterol

SUBJECT INDEX

P Paper chromatography 13, 14 Peak-shift technique 29 Peniocerol 65 Pennogenin 118 Pentduorobenzyloximes 33 Pentafluorophenyldimethylsilyl ethers 32 Periplogenin 122 Phosphinic esters 31 Pollinastanol 64 Polyamide 16 Ponasterone A 115 Ponasterone B 115 Poriferasterol 64 Prednisolone 100, 111 Prednisone 22, 100, 111 1,4-Pregnadiene-3,20dione, gar-fluoro-1lp, 16a,17,21tetrahydroxy-, see Triamcinolone _-_ , ~a-fluoro-~lp,l7,21-trihydroxy-16amethyl-, see Dexamethasone _ _ _ , 9a-fluoro-llp, 17,2 1-trihydr oxy- 166methyl-, see Betamethasone ___ , 6a-methyl-1 lp,l7,21-trihydroxy-, see 6a-Meth ylprednisolone ---, 1lp,l7,21-trihydroxy-, see Prednisolone 4,6-Pregnadiene-3,20dione,17-acetoxyd-chloro-, see Chlormadinone acetate ___ , 6-methyl-1 7-hydroxy-, see Megestrol 4,16-Pregnadiene-3,2O-dione 21,95 1,4-Pregnadiene-3,11,20-trione, 17,21-dihydroxy-, see Prednisone 5,16-Pregnadien-20-one, 3p-hydroxy- 21, 95 5a-Pregnane 4,48 Pregnane derivatives 6,93-112 --- blood 95,96 ___ GC 96,97 ___ HPLC 93 __- TLC 21,22,93-96 _-_ , urinary 93,95,96 Pregnanediol 21,23,35,38,93-96,107, 108 _ _ _ glucosiduronate 79, 93 5or-Pregnane-3or,20adiol,see Allopregnanediol 5a-Pregnane-3a,20p-diol 94 5 a-Pregnane-3@,2 Oa-diol 4 9, 94 Sa-Pregnane-3p,2Op-diol 6, 21, 35,49, 52, 94 Sp-Pregnane-3a,2Oa-diol, see Pregnanediol Sp-Pregnane-3a,20pdiol 94 Sp-Pregnane-3p,2Oadiol 21,94 Sp-Pregnane-3p,ZOpdiol 21, 94 Pregnanedione 21,48,49, 52,94 5a-Pregnane-3,20dione 21,48,49, 52, 94 Sp-Pregnane-3,20-dione,see Pregnanedione _ _ _ , 17,21-dihydroxy- 21

SUBJECT INDEX

--_ , 120-hydroxy-

201

21

_ _ _ , 17-hydroxy- 94 _ _ _ , 21-hydroxy- 49

Sa-Pregnane-l1,20-dione, 3a,2 l-dihydroxy104 _ _ _ , 3p,l7-'-dihydroxy- 94 _-- , 3p-hydroxy- 49,94 Sp-Pregnane-l1,20-dione,3ql7-dihydroxy- 94 _ _ _ , 3a,2 1-dihydroxy-, see Tetrahydrodehydrocorticosterone __- , 3 ~ h y d r o x y - 94 _ _ _ , 3a,l7,2l-trihydroxy-, see Tetrahydrocortisone Sp-Pregnane-3~,1lp,l7,20a,21-pentol, see aCortol Pregnanetetrol 96 Sa-Pregnane-3p,6a,l6p,20a-tetrol94 Sa-Pregnane-3p,6p,16p,2Oa-tetrol94 Sp-Pregnane-3a,l lp,l7,20a-tetrol, see Pregnanetetrol Pregnanetriol 23,35,38,94,96 Sa-Pregnane-3a,6a,2Oa-triol94 Sa-Pregnane-3or,6a,2Op-triol 94 Sa-Pregnane-3p,6p,2O@-triol 94 Sp-Pregnane-3a,6a,2Ocr-triol 94 Sp-Pregnane-3a,6a,2Op-triol 94 Sp-Pregnane-3~~,6p,2Op-triol94 Sa-Pregnane-3p,17,2Oa-triol94 Sa-Pregnane-3p,l7,2Op-triol 94 Sp-Pregnane-3a,17,20a-triol, see Pregnanetriol Sp-Pregnane-3a,l7,20p-triol 94 Sp-Pregnane-3p,17,2Ocu-triol 94 Sp-Pregnane-3p,l7,20p-triol 94 Sa-Pregnane-3a,2Oa,21-triol93 Pregnanetriolone 38,94,96 Sp-Pregnane-3,6,20-trione94 Sa-Pregnane-3,11,20-trione49, 52, 94 Sp-Pregnane-3,11,20-trione 21, 94 Sa-Pregnan-3p-01 21 50-Pregnan-3a-01 21 Pregnanolone 6, 13, 21, 38,94, 96 Sa-Pregnan-3-one, 20a-hydroxy- 94 _ _ _ , 20p-hydroxy- 21,49,52,94 50-Pregnan-11-one, 3or,l7,20a-tetrahydroxy-, see aCortolone __- , 3a,l7,20p,21-tetrahydroxy-,see pCortolone _-- , 301,17,2Oa-trihydroxy-,see Pregnanetriolone Sa-Pregnan-20-one, 3a,6a-dihydroxy- 94 _-_ , 3p,l6wdihydroxy- 21 _-_ , 3or,21-dihydroxy- 104 _-_ , 3a-hydroxy- 13,94 _-_ , 3p-hydroxy-, see Allopregnanolone

_-- , 3a,l lp,l7,21-tetrahydroxy-,

see Allotetrahydrocortisol _ _ _ , 3a,l lp,21-trihydroxy-, see Allotetrahydrocorticosterone _ _ _ , 3a,17,21-trihydroxy- 104 Sp-Pregnan-20-one, 3or,6a-dihydroxy- 94 - _ _ , 3@,16adihydroxy- 21 _ _ _ , 3a,l7-dihydroxy- 94 _ _ _ , 3p,l7-dihydroxy- 21, 94 _-- , 3a-hydroxy-, see Epipregnanolone _ _ _ , 3p-hydroxy-, see Pregnanolone _ _ _ , 3a,1 lp,l7,21-tetrahydroxy-, see Tetrahydrocortisol _-_ , 3a,Sp,21-trihydroxy- 104 _-_ , 3a,l lp,21-trihydroxy- 104 _-- , 3a,l7,21-trihydroxy- 104 4-Pregnen-l8-al, 1lp,2 l-dihydroxy-3,20-dioxo-, see Aldosterone Pregnenediol 6,35, 38,95 4-Pregnene-3p,20pdiol 21 S-Pregnene-3p,2Oor-diol,see Pregnenediol S-Pregnene-3p,20p-diol 6, 21, 95 l-Pregnene-3,20-dione, 60-hydroxy- 21 4-Pregnene-3,1l-dione, 17,20or,2l-trihydroxy102 _ _ _ , 17,20p,2l-trihydroxy- 102 4-Pregnene-3,20-dione, see Progesterone _ _ _ , 17-acetoxy-6a-methyl-, see Medroxyprogesterone acetate _-- , 11p,17-dihydroxy- 95 _ _ _ , lla,21-dihydroxy- 102 _ _ _ , 1 lp,21-dihydroxy-, see Corticosterone _ _ _ , 16aJ7-dihydroxy- 95 _-- , 16a,21-dihydroxy- 102 _ _ _ , 17,21-dihydroxy-, see Deoxycortisol _ _ - , 18,21-dihydroxy- 102 _-_ , 19,21-dihydroxy- 102 _-- , 9a-fluoro- 1lp,l60r, 17,2 1-tetr ahydroxy-, see 16a-Hydroxyfludrocortisone _-_ , 9a-fluoro-1 lp,17,2l-trihydroxy-, see Fludrocortisone _ _ _ , 6p-hydroxy- 95 __- , lla-hydroxy- 6, 21,95 _ _ _ , 1lp-hydroxy-, see 1lp-Hydroxyprogesterone _ _ _ , lSa-hydrOXy- 21 _ _ _ , 16a-hydroxy-, see 16a-Hydroxyprogesterone _-_ , 17-hydroxy-, see 17-Hydroxyprogesterone _ _ _ , 21-hydroxy-, see Deoxycorticosterone _ _ _ , 6a-methyl-1 lp,l7,21-trihydroxy-, see 6or-Methylhydrocortisone _ _ _ , 6p,l lp,l7,21-tetrahydroxy-, see 6pHydroxycortisol

202

SUBJECT INDEX

_ _ _ , 1lp,16a,l7,21-tetrahydroxy- 102 _ _ _ , 6p,llp,21-trihydroxy- 102, 104 _ _ _ , lla,l7,21-trihydroxy- 102

_ _ _ , 1lp,17,2l-trihydroxy-, see Cortisol _ _ _ , 1lp,l8,21-trihydroxy-, see 18-Hydroxycorticosterone _ _ _ , 16a,l7,21-trihydroxy- 102 _ _ _ , 17,19,21-trihydroxy- 102 5-Pregnene-3pJ lp,l7a,20a-tetrol 95 Pregnenetriol 38, 95,96 5-Pregnene-3a,l6a,2Oa-triol95 5-Pregnene-3a,l6a,2Op-triol95 5-Pregnene-3p,16a,20a-triol95 S-Pregnene-3p,16a,2Op-triol95 5-Pregnene-3pJ 7,20or-triol, see Pregnenetriol 5-Pregnene-30,17,ZOp-triol 95 4-Pregnene-3,11,20-trione21,95 __- , 17,21-dihydroxy-, see Cortisone _-- , 21-hydroxy-, see 11-Dehydrocorticosterone _-- , 6a-methyl-17,21-dihydroxy-,see 6 w Methylcortisone _-- , 6p,17,21-trihydroxy- 102 _-- , 16a,17,21-trihydroxy- 102 Pregnenolone 6, 13, 21, 35,95,97 _ _ _ sulfate 25, 26 4-Pregnen-3-one, 6a,20pdihydroxy- 95 _ _ _ , 6&20p-dihydroxy- 94 _ _ - , 1 lp,20pdihydroxy- 95 _ _ _ , 17,20a-dihydroxy- 95 --_ , 17,20p-dihydroxy- 95 _ _ _ 20a-hydroxy- 21, 35,94 _-- , 20p-hydroxy- 21,35,94,96 _ _ _ , lla,17,20p,21-tetrahydroxy-102 _ _ _ , 17,20p,21-trihydroxy- 102 5-Pregnen-ll-one, 3p,l7,20a-trihydroxy- 95 5-Pregnen-20-one, 3a-amino, see Holamine _-- , 3p-amin0, see Holaphyllamine _ _ _ , 30,16adihydroxy-, see 16a-Hydroxypregnenolone _-- , 3p,17-dihydroxy-, see 17-Hydroxypregnenolone _ - _ , 3p-hydroxy-, see Pregnenolone _-_ , 3p-methylamino-, see Holaphylline _-- , 3p,l lp,l7-trihydroxy- 95 _ _ _ , 3p,16a,17-trihydroxy- 95 5a-Pregn-2-en-2 0-0ne 4 8 _-- , 16a-methyl- 48 5p-Pregn-16-en-20-one, 30-hydroxy- 21 Preparative GC 41 _ _ _ HPLC 11 --_ TLC 16 . Previtamins D 113 Progesterone 4 , 6 , 10, 21, 23, 34,49, 93-97, 100,107,109, 110

.

Pterosterone 115 Pyrocalciferols 114

R Radiochromatography 25 Regisil 32 Retention constants 53 Rhodeasapogenin 118 RM Values, GC 46,47 --_ , LC 45 Rubijervine 117, 120

S Sapogenins 23, 117-120 Saponins 117 Saringosterol 65 Sarmentogenin 122 Sarsasapogenin 23,118 Scymnol 73 9,lO-Secod ,7,10(19)-cholestatrien-3p-o1, see Cholecalciferol 9,1O-Seco-5,7,lo( 19),22-ergostatetraene-3p-01, see Ergocalciferol Self-labeling 3 , 4 Silica 3, 14 Sintered-glass plates 15 Sitosterol 22, 34,56,58,59,61,63,65-67 Sitosteryl esters 58,59 Smilagenin 23, 118 Soladulcidine 120 5a-Solanidan-3p-ol, see Demissidine 5p-Solanidan-3-one 120 4-Solaniden-3-one 120 Solanidine 23, 117, 120 Solanocapsine 120 5a-Solasodan-3p-ol, see Soladulcidine Solasodine 23, 117, 120 Sorbents 3-7, 14-16 Spectroradiochromatograph 9 a-Spinasterol 63,65 (25R)-5p-Spirostane-lp,3p-dioI, see Isorhodeasapogenin (25S)-5p-Spirostane-lp,3pdiol, see Rhodeasapogenin (25R)-5a-Spirostane-2a,3p-diol, see Gitogenin (25S)-5a-Spirostane-2a,3p-diol, see Neogitogenin (25R)-5a-Spirostane-3p,6a-diol, see Chlorogenin (25R)-5p-Spirostane-lp,2p,3rr,5-tetrol, see Kogagenin (25R )-Sp-Spirostane-lp,2p,3a-triol, see Tokorogenin

203

SUBJECT INDEX

(25R)-5p-Spirostane-2p,3p,lla-triol, see Metagenin (25R)-5a-Spirostane-2a,3p,l5p-triol, see Digitogenin (25R)-5a-Spirostan-3p-ol, see Tigogenin (25R)-5p-Spirostan-3p-oI,see Smilagenin (25S)-5a-Spirostan-3p-ol, see Neotigogenin (25S)-Sp-Spirostan-3p-o1, see Sarsasapogenin (25R)-Sa-Spirostan-l2-one, 3p-hydroxy-, see Hecogenin (25R)-5-Spirostene-2p,3a-diol, see Yonogenin (25S)-5-Spirostene-3p,12p-diol, see Chiapagenin (25R)-5-Spirosten-3p-ol, see Diosgenin (25S)-5-Spirosten-3p-o1,see Yamogenin (25R)-5-Spirosten-l2-one, 3p-hydroxy-, see Gentrogenin Spore plate 26 Stanolone, see Androstanolone Stationary phases 33-37 Steroid Analyzer 8 Steroid numbers 48-53 Sterols 55-69 _-- , fecal 68 GC 61-69 _ _ _ LC 3-5,55-57 _ _ _ , plant 68 _ - _ TLC 22,57-61 Steryl esters 3,5, 56,58, 67 _-- glycosides 56,67 Sa-Stigmasta-7,22-dien-3p-o1, see a-spinasterol 5a-Stigmasta-7,24(28)-dien-3p-o1, 28-iso- 65 _ - ,~4a-methyl-, see Citrostadienol 5a-Stigmasta-7,25-dien-3p-o1 63, 65 5a-Stigmasta-8,14-dien-3p-ol 65 _ _ _ , 4a-methyl- 65 5,7-Stigmastadien-3pol 63, 65 5,(E)24(28)-Stigmastadien-3p-ol, see Fucosterol 5,(Z)24(28)-Stigmastadien-3@-01,See 28-lsofucosterol 5,25-Stigmastadien-3p-ol 63, 65 Stigmastanol 63, 65,66 5,7,22-Stigmastatrien-3p-ol 65 Sa-Stigmasta-7,22,25-trien-3p-o1 63, 65 5a-Stigrnasta-8,14,24(28)-trien-3p-o1 65 5a-Stigmast-7-en-3p-01 63, 65 5a-Stigmast-8-en-30-01 65 -_- , 4a-methyl- 65 ~ _ , 14a-methyl_ 65 5a-Stigmast-8(14)en-3@-01 65 Sa-Stigrnast-22-en-3p-ol 22 Stigmasterol 22,40, 58, 59, 61, 63, 64, 66,67 Stigmasteryl esters 58 Strophanthidin 22, 122 Strophanthidol 122 Sulfates 7, 25, 109 Sulfuric acid test 18, 20-23, 93

T Tachysterols 113 Testosterone 20, 35, 38,49,52,19, 87, 88, 91,92,100,109 - _ _ propionate 110 Tetrahydrocortisol 104, 108 Tetrahydrocortisone 104, 108 Tetrahydrocorticosterone 104, 108 Tigogenin 23,49, 118 Tokorogenin 118 5a-Tomatidan-30-01, see Tomatidine 5-Tomatiden-3p-1 23, 120 Tomatidine 23, 117, 120 Tomatillidine 120 Toxisterols 114 Triamcinolone 11 1 Trimethylsilyl ethers 26, 27, 30-32 Tubular TLC 16, 24, 25 Twin ion technique 44

U Urinary steroid profiles 108 Uzarigenin 22

V Vapor-phase acylation 85 Vapor-programmed TLC 17 Veralkamine 120 Veralobine 120 Veramarine 120 Veramine 120 Verarine 120 Veratramine 120 Verazine 120 Vinylestrenolone 110 Vitamins D 113, 114

Y Yamogenin 13,23 Yonogenin 118

1

Zone extraction 24 Zymosterol 56,64

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