Analytical approaches to food supplement analysis
Developing new LC-MS methods Comparing characteristics of RP columns
Volume 1 / Issue 1 www.sepscience.com
January 2009
reduce nitrogen costs for LC/MS. Pure and simple Parker Balston is the leading provider of Analytical Gas Systems for the LC/MS instrument market. Generators are specifically designed to meet the stringent gas requirements for all the leading LC/MS instrument manufacturers including Agilent, Thermo Scientific, Waters, Applied Biosystems, Bruker, Varian and Shimadzu. Utilising Parker’s proprietary hollow fibre membrane technology, there are more than 10,000 systems installed worldwide. Membrane technology offers some unique performance benefits for LC/MS including phthalate free nitrogen, silent operation, no moving parts and no electrical requirements. It is technology you can trust.
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separation science
driving analytical chemistry forwards
Analytical approaches to food supplement analysis
contents
Developing new LC-MS methods Comparing characteristics of RP columns
Volume 1 / Issue 1 www.sepscience.com
January 2009
Volume 1 / Issue 1 January 2009
Rr
feature
06
research round-up 06 Comparing separation characteristics of reversed-phase columns
07 The effect of pressure on small molecule retention using RP-UPLC
22 Two analytical approaches to the evaluation of chondroitin sulfate in european food supplements Nicola Volpi and Francessa Maccari
08 Rapid and direct determination of pesticides in water using anionexchange chromatography with coulometric detection
08 Computer-assisted solution for
multicomponent sample identification
09 SPE and GC–microEDC multiresidue analysis of royal jelly
10 Affinity partitioning of plasmid DNA with a zinc finger protein
11 FD-LC-MS/MS in breast cancer studies
Regulars
Tl Cd
11 Improved liquid chromatography
30
chrom doctor In this months column, the chrom doctor examines best practice when developing new methods for LC-MS analysis.
— Online radioactivity detection for metabolite profiling
13 Fast GC–MS pesticide multiresidue analysis of apples
14 Sequential injection methodologies for environmental analyses
15 Simultaneous HPLC and GC analysis of
Tu
lignans in Forsythia leaves
34
technology update An overview of recent technology advances in separation science and instrumentation.
16 Extraction of amphetamines from urine using a monolithic silica disc-packed spin column and HPLC–diode array detection
18 Development of immobilized enzyme reactors for phase I drug metabolism studies
19 Bioactive compound fishing with DNASeparation Science is published by Eclipse Business Media Ltd, TMC House Two, Alvaston Business Park, Middlewich Road, Nantwich, CW5 6PF, UK. Copyright 2009 Eclipse Business Media Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopying, recording or information storage and retrieval without permission from the publisher, Eclipse Business Media Ltd. Applications for the copyright owner’s permission to reproduce any part of this publication should be forwarded in writing to Permissions Dept, Separation Science, Eclipse Business Media Ltd, TMC House Two, Alvaston Business Park, Middlewich Road, Nantwich, CW5 6PF, UK. Separation Science does not verify any claims or other information appearing in any of the advertisements contained in the publication, and cannot take any responsibility for any losses or other damages incurred by readers in reliance on such content.
based bioseparations and chemical analysis
20 MSPD extraction of carbadox and
olaquindox in feed followed by hydrophilic interaction ultra-highpressure liquid chromatographic analysis
for research news, technical articles, product updates, jobs and applications visit. . . separation science — volume 1 issue 1
contents
3
David Hills – Scientific Director
[email protected]
Peter Myers – Chief Scientific Officer
[email protected]
New Year, new ideas…
S
o it’s now 2009 and Separation Science is finally off and running on a regular basis. Before going any further I’d just like to thank all of you who took the time to contact me with your thoughts on the launch issue,
whether that was in person at the ISC meeting in Münster or subsequently by telephone or email. Thankfully, the vast majority of these comments were positive and I’ve done my best to take them all on board prior to our full launch with
David Barrow University of Cardiff, UK
Melissa Hanna-Brown Pfizer, UK
Zongwei Cai Hong Kong Baptist University
Tuulia Hyötyläinen University of Helsinki, Finland
Yi Chen Chinese Academy of Sciences, Beijing, China
Gongke Li Sun Yat-Sen University, Guangzhou, China
Gert Desmet Vrije Universiteit Brussel, Belgium
Yong-Chien Ling National Tsing Hua University, Taiwan
C. Bor Fuh National Chi Nan University, Taiwan Klara Valko, GSK, UK Y.S. Fung Hong Kong University Jean-Luc Veuthey University of Geneva, Switzerland Xindu Geng Northwest University, Xi’an, China Claudio Villani Universita’ degli Studi di Roma “La Luigi Mondello Sapienza”, Italy University of Messina, Italy Cheing- Tong Yan Paul Haddad Center of Environmental Safety and University of Tasmania, Australia Hygene, Taiwan
this issue. Hopefully, you’ll notice some of these changes as you peruse the page of this digital publication. If you’d like to continue receiving Separation Science simply visit www.sepscience.com, click on the subscribe button and fill out a short form and you’ll receive the publication each month – and don’t forget, it’s absolutely free. You’ll also notice that we now offer two different formats for the magazine: the standard digital turning-page version for those of you with broadband; and a static PDF version for those of you without. The choice is up to you. By the way, when you visit www.sepscience.com, you’ll also notice a couple of changes: r5IFA"QQMJDBUJPO/PUF%BUBCBTFIBTJNQSPWFETFBSDI
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functionality – as well as including over 4,000 application notes in PDF format, it is now searchable by multiple keywords
Hian Kee Lee National University of Singapore, Singapore
Edward Browne GSK, Singapore
in both AND and OR logic. r"TFBSDIBCMFKPCTEBUBCBTFmJGZPVSFMPPLJOHGPSBOFXTUBSUJO the New Year why don’t you try it out. Finally, I’d just like to pique your interest by mentioning our upcoming scientific conference – Separation Science Singapore. It will take place between 26-28 August this year at the stateof-the art Biopolis Park in Singapore, and features an array of high-quality speakers from around the world, as well as from the immediate region, covering analytical challenges in food, pharma, enviro, bioscience and energy markets. I’ll bring you more information on this exciting event in later issues but for more of a teaser go to page 29. Enjoy the issue, and as always if you have any questions or comments just send me an email. David Hills
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from the editor
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separation science — volume 1 issue 1
5
Rr Research round-up
Key
Comparing separation characteristics of reversed-phase columns
Email the author
USA Silica-based chemically bonded stationary
are used for alkyl and aromatic ligands,” he
phases continue to dominate the practice of
said.
Article link
Product information
Comment
reversed-phase liquid chromatography, in spite
The key findings of the study are that
of efforts to replace them with polymeric or
for the XBridge C8, C18, C18 Shield, and
other inorganic oxide-based materials.
phenyl chemically bonded stationary
Dr Colin Poole from the Department of
phases, changes in selectivity in going
Chemistry at Wayne State University, USA,
from one phase to the other are small
looked at the differences in the system
and largely independent of the mobile
constants of the solvation parameter model
phase composition used. “Larger changes
and retention factor correlation plots for varied
are associated with weak electrostatic
solutes to study the retention mechanism
interactions and steric repulsion for a few
on XBridge C8, XBridge Phenyl and XTerra
specific compounds,” he added.
Phenyl stationary phases (all Waters Corp.,
“There is a road block in method
Milford, Massachusetts, USA) with acetonitrile–
development for those mixtures that are
water and methanol–water mobile phases
difficult to separate, in that changing the
containing from 10 to 70% (v/v) organic
column type or supplier, will rarely result
solvent. Published in Chromatographia [68
in significant progress as columns become
(7-8) 491-500 (2008)], these stationary phases
more alike. This may largely be a waste
are compared with XBridge C18 and XBridge
of effort and should be no more than a
Shield RP18 characterized in an earlier report
last resort. Older column types are more
using the same protocol.
variable and may offer a greater chance of
Dr Poole explained that improvements
success,” he said. The use of models such as
in chemically bonded stationary phases
the solvation parameter provides a means
have resulted in new products being much
to select columns with different separation
more alike than in the past. “Large changes
characteristics and to know in advance what
in retention are often due to differences in
chemical basis for a separation is being
the phase ratio, while changes in selectivity
exploited.
(relative retention) are quite small. Even changes in the structure of the bonded ligands seems to have little effect on selectivity when pure silica particles and high bonding densities 6
research round-up
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separation science
driving analytical chemistry forwards
The effect of pressure on small molecule retention using RP-UPLC UK The effect of inlet pressure on the retention of a series of low molecular
THE NEW SEPARATION SCIENCE REPORTS ARE NOW AVAILABLE...
weight acids, bases and neutrals, was investigated at constant temperature in reversed-phase liquid chromatography using a
Enviro Report Organochloride analysis using graphitized carbon black Automated Disposable Pipette Extraction of Pesticides from Fruits and Vegetables
commercial ultra-high-pressure system (Waters UPLC instrument) in the
WWEM 2008 Preview Featured Books
Journal of Chromatography A [1209 (1-2), 195-205 (2008)].
“Changes in retention with pressure, can be considerable for large
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molecules such as proteins, but are they significant for lower molecular
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Interview with Karsten Fjärstedt, GE Healthcare
HPLC instruments (e.g., Waters Acquity instrument) are now capable
SPME in high-throughput drug analysis EDQM Symposium: Pharmaceutical Reference Standards
of solvent delivery at pressures up to 1000 bar,” explained Dr David McCalley, Reader in Separation Science at the School of Life Sciences, October 2008
University of the West of England in Bristol, UK, who conducted the study with graduate research student Morgane Fallas, in conjunction
Food Report
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with Mark Hadley at AstraZeneca in Macclesfield, UK, and Uwe Neue
MSPD-GC-ECD analysis of lindane residues
5
A novel sample treatment for the LC-MS detection of peanut protein in foods Evaluating chondroitin sulfate in dietary supplements
iPod Touch
Screening for mycotoxins with LC-MS/MS
from Waters Corporation in Milford, USA. “Increases in retention with increasing pressure are normally observed, because of changes in the molar volume of the solute in the stationary and the mobile phase. For neutral non-polar molecules such as toluene, increases in retention factor for an increase in the average column pressure of 500 bar were only a few per cent. However, for ionized acidic and basic compounds, increases of more than 50% were observed over the same pressure range,” Dr McCalley said. These selective increases in retention with pressure even caused reversals in the order of elution of peaks for mixtures containing different types of compound. “We believe
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that these previously unobserved and surprising differences arise from differences in the loss of solvation for compounds of various types when they enter the stationary phase,” McCalley remarked. According to him, it is possible that differences in selectivity might be observed between otherwise identical analyses performed on conventional 5 µm and on newer sub-2 µm particles, because of the differences in operating pressure required. Could pressure even be used
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as a tool to change selectivity deliberately? iPod Touch
ce separation scien driving analytical che
separation science — volume 1 issue 1
research round-up
mistry forwards
7
Rapid and direct determination of pesticides in water using anion-exchange chromatography with coulometric detection Brazil A simple, rapid, and low-cost coulometric method for direct detection of glyphosate and aminomethylphosphonic acid (AMPA) in water samples using anion-exchange chromatography and coulometric detection with copper electrode is presented in the Journal of Chromatography A [1208 (1-2), 246-249 (2008)]. Led by Dr Cláudia Coutinho from the Institute for Chemistry at the University of São Paulo, Brazil, conducted the research because most analysis methodologies of glyphosate, a common pesticide in Brazil, apply derivation reactions, which are frequently complex, time-consuming and use expensive reagents. “The main purpose of this work was the development of a new methodology for glyphosate analysis that was direct, simple and low cost, when comparing with traditional ones commonly used for the analysis of this
Computer-assisted solution for multicomponent sample identification
herbicide,” Dr Coutinho said. Under optimized conditions, the limits of detection (LODs) (S/N
Hungary
= 3) were 0.038 μg/mL for glyphosate and 0.24 μg/mL for AMPA,
Identification of multicomponent
without any preconcentration method. The calibration curves were
samples is one of the most common
linear and presented an excellent correlation coefficient. The method
tasks for chromatographers, and one
was successfully applied to the determination of glyphosate and
of the most difficult. Dr Janos Harangi,
AMPA in water samples without any kind of extraction, clean-up or
from the Department of Biochemistry at
preconcentration step. No interferent was found in the water like this
the University of Debrecen in Hungary,
and the recovery was nearly 100%.
developed a method by which the results of
According to Coutinho, glyphosate was directly determined with
chromatographic analysis multicomponent
a cooper electrode, so no derivation reaction was necessary. “This
samples can be stored in a database, such
electrode can be used for the determination of many complexant
as a fingerprint. The database records were
substances. These substances, as well as glyphosate, interact with the
designed to be as independent of the
cooper oxide layer on the electrode surface. This interaction dissolves
analysis environment as possible, for inter-
the cooper oxide layer and displaces the reaction equilibrium, which
laboratory use. In a paper published in
increases the electrode anodic current. The stronger the complexant
Chromatographia [68 (supplement 1), 77-83
is, the bigger the sensitivity of the technique. As glyphosate is a
(2008)], he discussed this computer-assisted
tridentate complexant, an excellent detection limit was obtained, which
solution and the application of this method
is in accordance with many international and national organizations.
is tested by identification of several pine
Besides the possibility of analysis without the necessity of a derivation
species by recognition of their needle oils and
reaction, the developed methodology is fast, simple and uses cheaper
by identification of wine samples from their
instrumentation,” she added.
aroma composition.
In the future, the team will apply this technique to the determination
“The original need arrived from the
of other compounds of environmental and pharmaceutical importance,
food industry to identify changes of
such as bisphosphonates, which showed good results in preliminary
essential oil production from different
tests.
suppliers. Those kinds of samples have hundreds of components, and the manual evaluation of the analysis results was almost impossible,” he explained. The computer
8
research round-up
www.sepscience.com
aided data evaluation is similar to when a chromatographer overlays two (or more) chromatograms to find similarities. “First, the conversion of the chromatogram to a chromatographic fingerprint had to be performed. The conversion means that the retention times are converted to retention indices (Kovats indices) and the peak areas are converted to relative intensities. These data are easy to store and compare, similar to mass spectra. The only difference is that the retention indices may vary. The mathematical comparison of the chromatographic
SPE and GC–microEDC multiresidue analysis of royal jelly
fingerprint counts both variables, and results
Greece
in a method for fast multicomponent sample
Royal jelly, one of the most important products produced by bees, can
identification,” explained Harangi.
be contaminated with pesticides and/or antibiotic residues resulting
The fingerprinting method was tested at
from treatments applied either inside beehives or in the agricultural
a wine company to match the contents of a
environment. A new multiresidue method, published in the Journal
wine bottle with the label. Wine producers
of Chromatography A [1209 (1-2), 17-21 (2008)] was developed and
use several additives during production,
validated for the analysis of nine pesticides in royal jelly.
some of which can modify the taste and
According to lead author, Professor Urania Menkissoglu-Spiroudi from
smell of wine. “Non-expert consumers cannot
the Pesticide Science Laboratory at Aristotle University of Thessaloniki,
distinguish between such modified wines
Greece, EC legislation requires that all active substances used in
and original wines. With the aroma analysis
veterinary medicines for food-producing animals must be assessed
of the wines and use the laboratory made
so that a Maximum Residue Limit (MRL) can be set. “Commission
database of the chromatographic fingerprint
Decision 2002/657/EC introduced the concept of Minimum Required
helped to find the modified wines.”
Performance Limits (MRPLs), which recognizes the need for EU
He continued, “The applied technique is
harmonization of analytical detection limits and corresponds to the
a simple chromatographic analysis with a
concentration at which regulatory laboratories should be able to detect
postrun data evaluation. I believe that this
and confirm the presence of particular substances. As MRLs for royal jelly
method is one of the simplest and rather
have not been established either at EU or national level, the presence
reliable. Possible applications are one-
of any acaricide or pesticide residues could result in a non-compliant
dimensional chromatographic analyses of
product. Consequently, proper analytical methods developed and
any kind of multicomponent samples, such
validated according to Analytical Quality Control (AQC) performance
as perfumes, aromas, essential oils, refinery
criteria established by EC are necessary for control purposes,” Professor
products (distillate fragments), and many
Menkissoglu-Spiroudi explained.
others.”
Solid-phase extraction RP-C18 cartridges were used for sample
The next step is for the method to be
purification and isolation of analytes and the final solution was analysed
tested in cooperation with laboratories in
with GC and micro-electron-capture detection. Four synthetic acaricides
the food industry for possible application in
used by beekeepers (bromopropylate, coumaphos, malathion and
quality control or proof of origin.
τ-fluvalinate), as well as one pyrethroid, two organochlorine, and two organophosphate insecticides were tested. “The method proved to be linear in the range of interest and the coefficient correlation was >0.99846 for each analyte. The accuracy and precision are good enough to make the procedure applicable to routine
separation science — volume 1 issue 1
research round-up
9
use in the analysis of residual levels of the tested pesticides and especially the residues of the synthetic acaricides used for apicultural purposes,” she said. In the future, she believes this method can be used by analytical laboratories in order to determine the presence of acaricide and other pesticide residues in royal jelly samples. “The method has already been used for assessment of residues detected in royal jelly after the application of four registered in apiculture synthetic acaricides,” she said. The next step is to assess the presence of pesticide residues in royal jelly in vivo. “Royal jelly is a rather polar matrix with a high content of water, proteins and lipids so it is possible for pesticide residues to be present,” she concluded.
Affinity partitioning of plasmid DNA with a zinc finger protein Portugal A study, published in the Journal of
in the PEG phase of a PEG 600–DEX 40 ATPS.
Chromatography A [1206 (2), 105-112 (2008)],
This represents a 1322-fold increase in the
investigated the possibility of using a DNA
protein partition coefficient in comparison
binding protein [in this case a zinc finger
with the non-PEGylated protein (Kc = 0.013).
protein (ZnFP)], as an affinity ligand in aqueous
In the presence of pDNA containing a specific
two-phase extraction of plasmid DNA (pDNA).
oligonucleotide recognition sequence, the
“Aqueous two-phase extraction has already
zinc finger moiety of the PEGylated fusion
proved to be very promising for future
protein bound to the plasmid and steered the
utilization in the large-scale preparation of
complex to the PEG-rich phase.
pDNA for DNA vaccines and gene therapy.
“We found that, in systems where isolated
However, it lacks selectivity that could be
ZnFP and pDNA accumulate in different
improved by addition of affinity ligands,” said
phases, after mixing them together the
lead researcher, Dr João Marcos from the
partition of pDNA shifts to the phase where
Department of Chemistry at the University of
the ZnFP accumulates. This was true both for
Minho in Braga, Portugal.
the case where a native and a PEGylated ZnFP
Previously, small molecules have been used
were used. The latter case is more promising as
as affinity ligands in aqueous two-phase
it selectively accumulates pDNA in the phase
extraction of proteins with excellent results.
where less protein accumulates,” Dr Marcos
However it was not known if a similar principle
explained.
could be used for pDNA given its structural
According to him, the results presented in
and size differences. Therefore, in this study
this paper show it is possible to increase the
the affinity isolation of prepurified pDNA
selectivity of aqueous two-phase systems for
from model buffer solutions using native and
pDNA extraction using a DNA binding protein.
poly(ethylene glycol) (PEG) derivatized zinc
“After this proof-of-concept paper we are
finger–GST (glutathione-S-transferase) fusion
now applying this knowledge to design and
protein was examined in PEG–dextran (DEX)
develop a purification process of pDNA based
aqueous two-phase systems (ATPSs).
on aqueous two-phase systems,” he concluded.
In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected 10
research round-up
www.sepscience.com
FD-LC-MS/MS in breast cancer studies
is our opinion that such an FD-LC-MS/MS method could clarify the
Japan
dynamic cellular processes of proteins in
Kazuhiro Imai and colleagues from Musashino
tissues and demonstrate other candidates for
University and the Japanese Foundation
molecular markers in breast cancer diagnosis.”
for Cancer Research (both Tokyo, Japan),
“Previous proteomic studies have reported
have published the results of a proteomics
individual changes in expressed proteins.
study on human breast cancer cell lines
In contrast, our work has demonstrated
using fluorogenic derivatization-liquid
the dynamic flow of signal proteins in both
chromatography/tandem mass spectrometry
normal and cancer cells, and thus highlighted
[Biomedical Chromatography, 22 (11), 1304-
presumptive mechanisms leading to invasion,
1314 (2008)].
metastasis, proliferation and inhibition of
“Previously, we developed fluorogenic
cancer cells, as well as the new biomarker
reagents possessing a benzofurazan skeleton,
candidates. Furthermore, we have suggested
such as NBD-F, which are now available
that tropomyosin-1 could play a key role in
as reagents for amines and amino acids1,”
counteracting tumour progression,” explained
began Imai, before expanding, “The current
Imai. The research team feel that the
article relates to works combining the use of these reagents with the HPLC separation of
FD-LC-MS/MS method is a highly reliable,
fluorescent derivatives.”
reproducible, sensitive and easy-to-handle
He continued, “Although several molecular
method for proteomics analysis and, in fact,
markers for diagnosis and therapy of breast
is superior in terms of understanding the
cancer exist, their versatility is limited because
performance of the dynamic events in tissues.
of the lack of direct information on dynamic
The method should be further applied to
cellular processes of proteins in tissues. To
understanding the complex cellular events in
distinguish and identify minute changes
disease states.
in the expressed proteins between cancer
1
Biomedical Chromatography, 9, 106-109 (1995).
and normal tissue, highly sensitive and reproducible proteomic analysis are required. It
Improved liquid chromatography — Online radioactivity detection for metabolite profiling Belgium “Up until this point, radiotracer technology (14C or 3H) has been the method of choice to study the in vivo disposition of a new drug as it enables the quantitative detection of the parent drug and all of its metabolites in complex matrices. Metabolites are structurally related entities and structure differences can be minor. Therefore, a complete separation between all drug-related compounds is, although necessary for quantification, difficult to achieve,” said Dr Filip Cuyckens from Global Preclinical Development at Johnson & Johnson Pharmaceutical R&D in Beerse, Belgium. In a paper he published in the Journal of Chromatography A [1209 (1-2), 128-135 (2008)], Dr Cuyckens describes the successful combination of very-high-pressure liquid chromatography (VHPLC) with online radioactivity detection (RAD) facilitated by improvements in online radioactivity detection, as well as in column loading and peak capacity. “Many researchers in our field were hoping for an efficient coupling of ultra-performance liquid chromatography (UPLC) separation with online radioactivity detection. The major issue is that the narrow peak widths obtained in separation science — volume 1 issue 1
research round-up
11
UPLC separations appear intrinsically incompatible with the relatively high counting times needed to reach adequate sensitivity in radiochemical detection,” he explained. Traditionally 500 to 1000 μL flow-through cells are used in radioactive detection of drug metabolism samples because the radioactivity concentrations in these (especially in vivo samples) are rather low. According to him, these large cell volumes are detrimental for the peak width and high-resolution separations obtained in UPLC. In order to keep a good resolution, the cell volume needs to be decreased, which in turn is detrimental to sensitivity. “In our laboratory, we developed an efficient combination of
Fast GC–MS pesticide multiresidue analysis of apples Slovakia A fast gas chromatographic–mass
UPLC with a conventional radiodetector. This combination was
spectrometric (GC–MS) method is proposed
made possible by improving the on-line radioactivity detection, as
for pesticide multiresidue analysis of apples
well as increasing the column loading and peak capacity for ultra-
in Chromatographia [68 (supplement 1),
high-pressure separations,” he said.
49-55 (2008)]. Professor Eva Matisová from
In the study, the sensitivity of 14C detection was improved
the Institute of Analytical Chemistry at the
by the use of a variable scintillation flow achieved via a simple
Faculty of Chemical and Food Technology of
modification to the classic online radiochemical detection set-
the Slovak University of Technology, Slovak
up. A modification of the flow-through cell design in which
Republic, used the QuEChERS method for
internal diameter of the tubing was reduced further increased
sample preparation and GC–MS analysis was
the sensitivity and resolution by decreasing peak tailing. The
performed with a PTV, an autoinjector, and a
injection of relatively large volumes was made possible by the use
quadrupole benchtop MS detector.
of columns packed at ultra-high-pressure with 2.2 μm particles.
“Speeding up GC analysis provides
Because of the reduced back pressure using these larger particles,
unquestionable benefits towards conventional
two 3 x 150 mm columns could be coupled, allowing four fold
GC, such as higher laboratory throughput,
larger injection volumes and a 50% improved separation at a
reduced GC operating costs, and better
similar backpressure compared with a standard 2.1 x 150 mm UPLC
analytical precision by making possible
column.
more replicate analyses. Nowadays, fast
“This is the first UPLC ¬ on-line radioactivity detection set-up that
GC can be performed on commercial gas
works in practice for samples with limited radioactivity, not only
chromatographs, which are equipped with
for standards. We are now using the approach in our lab for real
high-speed injection systems, electronic gas
projects and are replacing our existing radio-HPLC systems with
pressure control, rapid oven heating/cooling
radio-UPLC systems. The use of the larger and coupled columns
and fast detection,” Professor Matisová said.
allowing higher loadability and plate numbers is also very useful for
In the study, compounds were separated
other applications; for example, chemical impurity quantification,”
under temperature-programmed conditions
he concluded.
on a narrow-bore diphenyldimethylsiloxane column. In one chromatographic run 61 pesticides of different chemical classes, and triphenyl phosphate as internal standard, were determined in 11 min. Calibration was performed with matrix-matched standard solutions and response to the pesticides was a linear function of concentration in the range 1–500 ng/mL (equivalent to 1–500 μg/kg in real samples). High values of the determination
separation science — volume 1 issue 1
research round-up
13
coefficients (R2; 0.9900–1.0000) were obtained for most of the pesticides. Limits of detection and quantification were determined. According to Matisová, the key outcome was the development and validation of the method for multiresidue analysis of pesticides in nonfatty food (apples) by fast GC-MS with PTV injection in solvent mode and using a narrow-bore capillary column. Quadrupole MS detectors have been most widely utilized in analytical practice. “From the results obtained it can be concluded that the used quadrupole detector is fast enough for proper reconstruction of the analytes peaks and that the method can be used for multiresidue analysis,” she said. The method is applicable to pesticide residues analysis at ultra-trace concentration levels in real samples in non-fatty food. “Our future work will be directed to endocrine disrupting pesticides analysis in environmental matrices,” she added.
Sequential injection methodologies for environmental analyses Brazil Published in Analytical Chimica Acta [628(2), 123-132 (2008)] is an article by Jorge Masini and colleagues (Insituto de Quimica, Universidade de São Paulo, Brazil), describing the implementation of a stepwise solvent elution in sequential injection chromatography (SIC) for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis. “Part of my research work focuses on the development of sequential injection or flow injection methodologies for environmental analyses,” explained Masini, adding “These techniques are suitable for precise liquid handling and control of reaction times, but are limited to only a few analytes per sample injection, such that multi-analyte determinations have been a target for practitioners of flow/sequential injection techniques.” “Coupling low pressure flow systems to monolithic stationary phases has allowed us to improve the capabilities of sequential injection or flow injection techniques toward multi-analyte determinations. We were originally interested in the on-line determination of amino acids that are excreted by marine microalgae in batch cultivation. This was a goal that required multi-analyte determinations and precolumn derivatization, so sequential injection chromatography was our choice to start the method development,” he continued. The concept of sequential injection chromatography was exploited to automate the fluorimetric determination of amino acids after precolumn derivatization with o-phthaldialdehyde in the presence of 2-mercaptoethanol using a reverse-phase monolithic C18 stationary phase. Additional to the on-line precolumn derivatization, the proposed method involved five steps of isocratic elutions allowing the use of a wide range of polarities during the elution. “To our best knowledge, this simple approach had not yet been described in SIC methodologies, which had previously used only a single eluting solution (isocratic), being limited to separate components of simple mixtures, and being especially applied to the determination of pharmaceutical products. In a mix of 18 amino acids, the proposed procedure allowed us to 14
research round-up
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separate 14 analytes with resolutiongreater than1.5,” Masini explained. He concluded, “We believe that the methodology described in the referred article can extend the application of SIC to the separation of complex mixtures with potential application to physiological studies of amino acid metabolism in algae, as well as in environmental studies related to carbon and nitrogen assimilation by phytoplankton. Development and application of SIC procedures to perform on-line monitoring for adsorption/desorption of herbicides and their metabolites onto/from soil particles is another on-going project in our laboratory.”
Simultaneous HPLC and GC analysis of lignans in Forsythia leaves Hungary The biological activities of lignans are of pharmaceutical importance. Among butyrolacton lignans, arctigenin and its glucoside, arctiin occur in Forsythia species in high quantities. Éva Sedlák from the Department of Plant Anatomy at Eötvös Loránd University in Budapest, Hungary conducted a study to find the most suitable extraction method for these lignans, and identify and quantify the lignan constituents present in leaf samples of different Forsythia species and cultivars by highperformance liquid chromatography (HPLC) and gas chromatography, (GC), simultaneously. Published in Chromatographia [68 (supplement 1), 35-41 (2008)] lignans, carboxylic acids and sugars were determined by gas chromatography (GC) with mass selective detection, the lignans also by high-performance liquid chromatography with UV detection in the leaf extracts of four species and four cultivars of Forsythia plant. Three methods were used to optimize the extraction of the main lignan constituents (arctiin, arctigenin), which possess significant pharmaceutical effects. “Our results suggested that the supercritical fluid extraction applying CO2+60% methyl alcohol was the best method for obtaining the highest extraction yield of lignans,” said Sedlák. Regarding the identification and quantification of aglycone lignans, both HPLC and GC-MS proved to be efficient. To quantitate lignan glycosides, HPLC was the method of choice. According to her, from a pharmaceutical point of view the most relevant, arctigenin, was found in the highest quantity in the cultivar ‘Robusta’ of Forsythia ovata. “We want to utilize our optimized extraction and analytical techniques in further basic research and technical applications. Our final goal would be to prepare medicinal product from the highest lignan containing Forsythia ovata species,” she concluded.
separation science — volume 1 issue 1
research round-up
15
Extracting amphetamines from urine using monolithic silica disc-packed spin columns Japan To overcome the limitations of solid-phase
follows: linear curves (drug concentrations of
extraction, Dr Akira Namera and colleagues
0.2–20 μg/mL); correlation coefficients >0.99;
from the Department of Forensic Medicine
detection limit, 0.1 μg/mL.
at Hiroshima University, Japan, developed
“In our previous study, we packed monolithic
a device comprising a spin column packed
silica into a capillary glass tube (i.d. = 0.2 mm)
with octadecyl silane-bonded monolithic
and the extraction device was created by
silica for extracting amphetamines and
connecting a microsyringe to the capillary
methylenedioxyamphetamines from
column. We demonstrated that amphetamines
urine. A paper published in the Journal of
in urine could be extracted using this device,”
Chromatography A [1208 (1-2), 71-75 (2008)]
he explained. However, sample filtration was
states that the proposed method is not only
required to avoid blockage within the tube,
useful for drugs from biological materials but
and thus, only one sample could be obtained
also highly reproducible for the analysis of
through a batch processing cycle. In order to
these drugs in urine.
overcome these problems, a monolithic silica
Liquid-liquid or solid-phase extraction
disc was packed into a spin column, wherein
(SPE) methods are widely used for extracting,
the structure of monolithic silica combined
purifying, and enriching drugs and medicines
the support body and the surface area for
from biological materials. However, these
each unit volume is wide by comparing
methods involve laborious, intensive and
with a particle-type silica. “The handling
expensive preparatory procedures. “GL
procedures such as sample loading, washing
Sciences informed me that monolithic silica
and elution of the target drugs were only
has been investigated as a new type of
exhibited by a centrifugation of the column.
separation material for high-performance
In addition, many samples can be processed
liquid chromatography (HPLC). Although
simultaneously,” he added.
the conventional materials used for SPE
According to him, this spin column has many
were similar to those used for HPLC, we
advantages: its operation procedure is simple,
think monolithic silica has potential for drug
requires a low eluate volume and does not
extraction and purification from biological
involve solvent evaporation. “It is expected
materials,” said Dr Namera.
to take the place of conventional solid-phase
Urine (0.5 mL), buffer (0.4 mL) and methoxyphenamine (internal standard) were
extraction cartridges in many fields,” he concluded.
directly added to the preactivated column. The column was centrifuged (3000 rpm, 5 min) for sample loading and washed. The adsorbed analytes were eluted and analysed by HPLC, without evaporation. The results were as 16
research round-up
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“I can’t believe I published the wrong molecular weight.” “It wasn’t your fault, Max. It was your assumption’s fault.” Calibration-based measuring techniques require you to make assumptions which aren’t always correct. That’s why every major pharmaceutical and biotechnology company, as well as most federal regulatory agencies are switching from relative methods to Wyatt Technology’s absolute measurements. Our DAWN® multi-angle light scattering (MALS) instruments allow you to determine absolute molecular weights and sizes without relying on so-called standards, or measurements made in someone else’s lab. Wyatt instruments measure all of the quantities required for determining absolute molar masses directly. To learn how to end your dependence on reference standards forever, call 805.681.9009 or visit wyatt.com and request our free 28-page Ultimate Guide to Light Scattering. Our booklet is bound to lift your spirits. CORPORATION
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Development of immobilized enzyme reactors for phase I drug metabolism studies Switzerland Cytochrome P450 enzymes (CYP450) are the major drug-metabolizing enzyme systems in
inhibition studies were also reported. In comparison with in-solution assays, in
the human liver responsible for the oxidative
which CYP450 enzymes are known to be
conversion of approximately 90% of marketed
rapidly inactivated, the system was stable
drugs.
for performing ca. 15 experiments in 2 days,
In order to determine the CYP450 isozyme
according to him.
involved in a specific drug metabolism, in vitro
“This technique can be applied in drug
screening is usually performed by incubating
discovery for the evaluation of drug-drug
in-solution human liver microsomes or
interactions and assessment of metabolism
recombinantly expressed human CYP450
profiles of novel chemical entities. Thanks
isozymes with drugs (alone or with a potential
to the possibility to reuse the immobilized
inductor or inhibitor). These incubations
enzyme for several analyses, this strategy is
require relatively large amounts of expensive
cost-effective in comparison with conventional
recombinant enzymes. Prof. Jean-Luc
in vitro tests,” he said. To date, the team is trying
Veuthey and colleagues from the School of
to improve the stability of the IMERs, and is
Pharmaceutical Sciences at the University of
working also on a different approach which
Geneva, Switzerland have developed CYP450-
will be published in the near future.
based immobilized enzyme reactors (IMERs) to perform automated on-line phase I drug metabolism studies. Published in the Journal of Chromatography A [1206 (1), 2-10 (2008)], the major aim of this study was to prepare two IMERs containing CYP2D6 and CYP3A4 for performing automatically phase I drug metabolism. “CYP450 is a very complex enzymatic system constituted by three enzymes embedded in a phospholipidic membrane and requiring NADPH as cofactor. It was, therefore, not possible to use the covalent immobilization procedure already developed in our laboratory with trypsin,” Prof. Veuthey explained. Using an original strategy (NeutrAvidin covalent immobilization on monolithic disc; biotinylation of CYP450; coupling of the biotinylated CYP450 on the NeutrA-disc), it was possible for the team to obtain two discs (2 x 6 mm i.d.) containing immobilized CYP2D6 and CYP3A4 to perform automatically drug metabolism studies by LC-ESI-MS/MS. The method was tested with probe substrates and 18
research round-up
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Bioactive compound fishing with DNA-based bioseparations and chemical analysis China According to a recent article in Biomedical Chromatography [22 (10), 1164-1172 (2008)], Professor Ping Li and researchers from the China Pharmaceutical University, Nanjing, China, have performed a screening and mechanism study of components targeting DNA from the Chinese herb Lonicera japonica by liquid chromatography/mass spectrometry and fluorescence spectroscopy. DNA is the potential molecular target of many antimicrobial, antiviral and antitumour active drugs. The screening and identification of DNA-targeting agents from natural products extracts are attractive, and the interaction of DNA with small molecules has been an intensive topic which has fascinated scientists for decades as it provides insights into rational design of drugs targeting to DNA. “As the biological separation system, DNA can be used to selectively fish for bioactive compounds from multicomponent samples. By ultrafiltration sampling and LC-MS analysis, DNA-bound compounds can be easily identified from their chromatographic fingerprints before and after interaction with DNA,” explained Li, adding “Four flavonoids were fished and identified from Lonicera japonica extract using this method. The binding mechanism of these flavonoids with DNA was then evaluated to be groove binding by fluorescence spectroscopy. For L. japonica, the flavonoids binding to DNA may be one of the mechanisms of its antimicrobial, antiviral or antitumour actions.” “Our future work will extend our bioseparation and chemical analysis strategy to other biological separation systems; for example, target protein, cell etc., for rapid and effective screening of potential bioactive compounds from complex multicomponent samples,” Li concluded.
Sample Preparation Solutions Phenomenex provides complete solutions including SPE tubes & 96-well plates, protein precipitation plates, syringe filters, vials and more. r Quality products r Responsive customer service r Complete technical support r Worldwide availability with fast delivery
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Phenomenex products are available worldwide. Email us at
[email protected].
separation science — volume 1 issue 1
research round-up
19
technical articles on chromatography? updates on recent research studies?
MSPD extraction of carbadox and olaquindox in feed followed by hydrophilic interaction ultra-highpressure liquid chromatographic analysis Lithuania A new method involving matrix solid-phase dispersion
practical advice on routine analysis?
(MSPD) extraction and hydrophilic interaction ultraperformance liquid chromatography (HILIC-UHPLC)
applications of new technology?
with photodiode array detection was developed for the determination of carbadox and olaquindox in feed.
information on product developments? Described in the Journal of Chromatography A [1209 (1-2), market trends and opinions?
83-87 (2008)], the separation of carbadox and olaquindox was achieved within 1 min on the 1.7 μm Acquity UPLC BEH HILIC column using isocratic elution with a mobile phase consisting of
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10 mmol/L ammonium acetate in acetonitrile-water (95:5, v/v) at a flow rate of 0.5 mL/min. “In recent years our group has collaborated closely with the National Food and Veterinary Risk Assessment Institute
separation science driving analytical chemistry forwards
of Lithuania in developing new analytical methods for the determination of veterinary drug residues in food and feed,” said main author, Professor Audrius Padarauskas from the Department of Analytical and Environmental Chemistry at Vilnius University in Lithuania. The institute tasked his
at
group with the development of a simple, fast and reliable method for the routine monitoring of carbadox and
c n e i c s n ratio cl driiving anallyytitica
20
research round-up
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olaquindox in feeds. Optimization of MSPD extraction parameters, such as type of solid sorbent and elution solvent were performed. Optimal conditions selected for MSPD extraction were: 0.25 g of feed sample, 0.5 g of octadecylsilica as solid sorbent and 10 mL of acetonitrile-methanol (8:2, v/v) as eluting solvent. Both analytes provided average recoveries from spiked feed samples ranging from 89.1 to 98.4% with relative standard deviations less than 10%. “In my opinion, the main strength of the present work is the combination of matrix solid-phase dispersion extraction with hydrophilic interaction (HILIC) ultra performance liquid chromatographic analysis. Direct injection, without evaporation/reconstitution, of the extract onto the separation column is therefore possible, which saves time and prevents sample losses. Another significant advantage is that less polar interfering compounds requiring gradient elution by conventional reversed-phase HPLC are eluted early in an isocratic HILIC system, thereby simplifying and accelerating the overall analysis,” Professor Padarauskas explained. He believes this combination should be very promising for the analysis of polar compounds in complex biological matrices such as food, feed, plants etc.
separation science — volume 1 issue 1
research round-up
21
22
feature article — SAX-HPLC analysis of food supplements
www.sepscience.com
Two analytical approaches to the evaluation of chondroitin sulfate in european food supplements Nicola Volpi and Francesca Maccari Department of Biologia Animale, Biological Chemistry Section, University of Modena and Reggio Emilia, Italy
The amount and quality of chondroitin sulfate (CS) from several Czech Republic food supplement/nutraceutical preparations was determined. To quantify CS, two different analytical approaches were validated [11] and applied: specific and sensitive agarose-gel electrophoresis and strong anion exchange (SAX)-high performance liquid chromatography (HPLC) determination of the constituent disaccharides after treatment with specific chondroitin lyases. The CS content in food supplement products were found to conform to the label specifications in only four of the ten analysed samples. Four of the food supplement preparations were found to contain approximately 0-1% CS in comparison with 47, 17, 12 and 6% declared on the label. Two products were found to have approximately 30-45% of the declared CS, and one preparation was found to contain approximately 2% hyaluronic acid. SAXHPLC separation of unsaturated disaccharides for the nutraceutical CS was also used to evaluate its quality and possible origin. The CS contained in eight food supplements resulted to be of bovine or porcine origin, one from cartilagineous fish and in one case it was not possible to determine the origin because of very low CS content. Based on these analytical results, quality of the sampled Czech Republic food supplement formulations was poor and strict regulations for quality control should be mandatory in order to guarantee the manufacture of high-quality products. Furthermore, specific and accurate analytical procedures should be enforced for the control of high-quality products and applied by quality control laboratories to confirm the purity and label claim of CS in raw materials and nutraceuticals.
Introduction Chondroitin sulfate (CS) (Figure 1), a
hand (OA) [5-7]. Moreover, CS alone
and various nutraceutical preparations
very complex, polydisperse, natural
or in combination with glucosamine,
[9, 10], and their purity and quality
glycosaminoglycan (GAG) highly
is utilized as a dietary supplement [7,
were generally found to be
heterogeneous for relative molecular
8]. As a consequence, the number
inconsistent with the specifications
mass, charge density, structure, and
of pharmaceuticals containing CS
claimed on the product labels. In this
biological and pharmacological
has increased, as well as the types of
article, we determine the CS amount,
activities [1, 2], is recommended
formulations, including tablets and
quality and origin in several Czech
by EULAR [3, 4] as a SYSADOA
caplets, capsules, ophthalmic solutions
Republic nutraceutical samples using
[Symptomatic Slow Acting Drug for
and liquid preparations [9-11].
the same analytical methodology
osteoarthritis (OA)] drug in Europe for the treatment of knee, hip and separation science — volume 1 issue 1
Previous studies reported a determination of CS in raw materials
applied to a recent CS evaluation in US food supplements [11].
feature article — SAX-HPLC analysis of food supplements
23
Figure1
6S: ΔUA-[13]-GalNAc-6S, ΔDi-4S:
CH2OR 2 COO H O
H O
ΔUA-2S-[13]-GalNAc-6S, ΔDi-4,6diS:
O
ΔUA-[13]-GalNAc-4S,6S, ΔDi-2,4diS
H H
H
H
H
H H
O
OR 1 O
H OH
ΔUA-[13]-GalNAc-4S, ΔDi-2,6diS:
ΔUA-2S-[13]-GalNAc-4S) were from Seikagaku. High resolution agarose,
NHCOCH3
certified for molecular biology, was
OR 3
from Sigma. All other reagents were of
R1 = R2 = R3 = H: nonsulfated chonroitin R 1 = SO3 and R2 = R3 = H: chondroitin-4-sulfate, CSA R 2 = SO3 and R1 = R3 = H: chondroitin-6-sulfate, CSC R 2 = R3 = SO3 and R 1 = H: chondroitin-2, 6-disulfate, CSD R 1 = R2 = SO3 and R3 = H: chondroitin-4, 6-disulfate, CSE R 1 = R3 = SO3 and R2 = H: chondroitin-2, 4-disulfate, CSB R 1 = R2 = R3 = SO3 : trisulfated chondroitin
analytical grade. Sample preparation: All the used nutraceuticals contained various other ingredients in different amounts, in particular glucosamine, collagen, vitamins and/or MSM (Table 1).
Figure 1. Structures of disaccharides forming chondroitin sulfate.
Stock solutions (10 mg/mL) of all the Materials and Methods
analyses [12], and has a claimed CS
products were carefully prepared.
Materials: Various food supplement
content greater than 98% [12]. CS
No sample pretreatment was
samples in the form of tablets or
fractions having different molecular
performed apart from centrifugation
capsules containing CS in different
mass values for high-performance
at 10,000 RPM for 10 min to remove
formulations and amounts
size-exclusion chromatography
insoluble material, mainly derived
(Table 1) were obtained from the
(HPSEC) analysis, were prepared by
from excipients and salts present in
Czech Republic market. The European
gel-permeation and evaluated by
the preparations, and a proteolytic
Pharmacopeia CS (EPCS) reference
means of analytical ultracentrifugation
treatment to remove possible
standard, manufactured by Bioiberica
[13]. Chondroitinase ABC, chondroitin
interfering proteins. The EPCS
(www.bioiberica.com/) and approved
ABC lyase, from Proteus vulgaris
reference standard was dissolved in
in 2004 as the Chemical Reference
(EC 4.2.2.4), 0.5-2 units/mg, and
water to make authentic CS solution
Substance (CRS) by the European
chondroitinase ACII, chondroitin
(10 mg/mL).
Pharmacopeia Commission was
AC lyase, from Arthrobacter aurescens
Analytical procedures: The
utilized as a standard in these analyses.
#(EC 4.2.2.5), were from Sigma.
analytical techniques used for
Unsaturated CS/DS disaccharides
This CS standard is produced from bovine cartilage, as evaluated by HPLC
(ΔDi-0S: ΔUA-[13]-GalNAc, ΔDi-
the quantitative and qualitative evaluation of CS in food supplements
Table1
24
Sample
Declared CS Content
Formulation
A
250 mg/capsule
Glucosamine, Collagen, Saccharides
B
250 mg/tablet
Glucosamine, MSM
C
25 mg/tablet
Glucosamine, Collagen, MSM
D
200 mg/tablet
Glucosamine, MSM
E
200 mg/tablet
Glucosamine
F
67 mg/capsule
Glucosamine, Collagen, MSM, Vitamin C, Vitamin E
G
200 mg/tablet
Glucosamine, MSM
H
400 mg/tablet
Glucosamine
I
25 mg/tablet
Glucosamine, Collagen, MSM
L
75 mg/tablet
Glucosamine, Collagen
feature article — SAX-HPLC analysis of food supplements
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have been previously published in
Drug Evaluation and Research (CDER),
Results
detail [11]. In particular, agarose-
Center for Veterinary Medicine (CVM)
First, an accurate determination
gel electrophoresis and HPLC
published in May 2001 [18], including
of the theoretical CS content was
separations of the unsaturated CS
specificity, linearity, detection (LOD)
performed by evaluating real weight
disaccharides produced by the action
and quantitation (LOQ) limit, precision,
of tablets/capsules, repeated several
of chondroitinases ABC and ACII were
accuracy, recovery, and robustness
times to have a statistical variation,
used for quantitative purposes [11,
tests (see [11]). Furthermore, HPLC
and to calculate the theoretical
14–17]. These methods have been
of the CS disaccharides was used to
CS percentage in each of the food
validated according to the Guidance
evaluate the possible origin of CS in
supplements. The declared CS
for Industry, Bioanalytical Method
nutraceuticals [11, 12, 19] and HPSEC
percentage was found to be from 1.5
Validation from US Department of
was applied for the determination of
up to 47.2 (Table 2).
Health and Human Services, Food
the CS molecular mass parameters
and Drug Administration, Center for
[11–13].
The CS present in the ten nutraceuticals was separated
Table2
Parameters
Food Supplements A
B
C
D
E
F
G
H
I
L
47.2%
16.9%
1.5%
12.4%
24.1%
12.3%
12.3%
30.7%
1.6%
6.0%
Agarose-gel
0.12%
0.34%
1.54%
0.62%
10.20%
4.87%
10.12%
28.50%
1.36%
0.75%
SAX-HPLC
<0.20%
0.74%
2.71%
0.67%
11.42%
3.00%
12.85%
28.56%
1.09%
1.93%
Mean *
<0.2%
0.9%
2.1%
0.9%
10.7%
3.9%
11.8%
29.1%
1.4%
0.8%
MWn
nd
nd
nd
nd
18,400
nd
12,990
18,980
29,400
191,370
MWw
nd
nd
nd
nd
52,600
nd
26,250
45,160
117,670 726,260
MWz
nd
nd
nd
nd
161,520
nd
76,240
174,650
868,120 nd
Dispersity
nd
nd
nd
nd
2.8577
nd
2.0212
2.3796
4.0029
nd
ΔDi-0s
nd
nd
nd
nd
nd
nd
nd
6.9
7.4
nd
ΔDi-6s
nd
27.1
17.7
38.0
44.5
33.0
21.8
20.9
16.0
20.3
ΔDi-4s
nd
72.9
82.3
62.0
33.0
67.0
78.2
72.2
76.6
79.7
ΔDi-2,6dis
nd
nd
nd
nd
19.4
nd
nd
nd
nd
nd
ΔDi-4,6dis
nd
nd
nd
nd
1.5
nd
nd
nd
nd
nd
ΔDi-2,4dis
nd
nd
nd
nd
1.6
nd
nd
nd
nd
nd
R
nd
1.00
1.00
1.00
1.22
1.00
1.00
0.93
0.94
1.0
4s/6s ratio
nd
2.69
4.65
1.63
0.74
2.03
3.59
3.45
4.79
3.93
Origin
nd
Bovine/ Porcine
Bovine
Cartil.
Bovine
Porcine
Porcine
Porcine
Porcine
Declared CS % CS content %
Molecular mass
Disaccharides
Porcine
Fishes
nd = not detected, * Mean of analyses performed under different experimental conditions, i.e. agarose-gel and HPLC of disaccharides without and in the presence of proteolytic treatment. Table 2. CS content, molecular mass parameters (Mn, number of average molecular weight; Mw, weight average molecular weight; Mz, Z average molecular weight, polydispersity index, Mw/Mn) and unsaturated disaccharide percentages, with the charge density values (as sulfate groups number per disaccharide unit) and the 4-sulfated/6-sulfated ratio (4s/6s ratio as the ratio between the sulfated groups located in position 4 or 6 on N-acetyl-galactosamine), of CS samples from the various Czech Republic nutraceutical samples analysed in this study. The percentage of each disaccharide was calculated by considering the total absorbance %age corresponding to each peak. The data are means of three different analyses.
separation science — volume 1 issue 1
feature article — SAX-HPLC analysis of food supplements
25
Figure2
preparation. SAX-HPLC was also used for the quantitative determination (Table 2) of the CS constituent disaccharides [11]. The relevant results are fairly consistent with the data obtained by electrophoresis. Finally, the molecular mass parameters, in particular Mn, Mw, Mz Figure 2. Agarose-gel electrophoresis of chondroitin sulfate (CS) of several Czech Republic nutraceutical samples (from A to L, see Table 1). CS was separated on an agarose-gel having a thickness of about 4-5 mm and, after migration, the plate was stained with toluidine blue. The migration of the European Standard CS reference standard (St) is also illustrated.
and polydispersity, were determined for just a few of the ten nutraceutical samples (Figure 4 and Table 2) because
and quantified by agarose-gel
disaccharides confirming the absence
of the very low CS content. However,
electrophoresis (Figure 2) without
of iduronic acid (and dermatan sulfate)
this analytical approach indicates the
and in the presence of digestion
as previously reported by agarose-
possible presence of depolymerized
with proteases, and centrifugation
gel electrophoresis. The pattern of
or degraded CS. In fact, the molecular
to remove insoluble material. The
unsaturated disaccharides (illustrated
mass values of nutraceutical CS are
quantitative results obtained using
in Figure 3) for nutraceutical CS
similar to those of CS samples purified
this analytical approach are illustrated
samples is reported in Table 2. As
from porcine or bovine cartilages, and
in Table 2.
can be seen, CS of various origin
cartilagineous fishes [12] confirming
— bovine, porcine and one from
that these polymers have not been
were treated with chondroitinases ABC
cartilagineous fishes [11, 12, 19] —
degraded during the food supplement
and ACII producing virtually the same
have been used for the nutraceutical
preparations.
The food supplement CS samples
It is worth mentioning, that
Figure3
in sample L the presence of Di-6s
approximately 2% high molecular
Absorbance at 232 nm
Sample E
mass hyaluronic acid, was detected by agarose-gel [Figure 5(a)], disaccharide
Di-4s
analysis [Figure 5(b)] and HPSEC Disulfated disaccharides
evaluation [Figure 5(c)].
Di-0s
Discussion In a previous study [11], we performed the quantification and characterization
Absorbance at 232 nm
aADi-4s
of CS present in several nutraceutical Sample G
preparations available as caplets, tablets and capsules in the US market
Di-6s Di-0s
by using the specific and sensitive agarose-gel electrophoresis and HPLC analyses. The electrophoresis and SAX-HPLC methods were validated and confirmed to be applicable to the quantification of CS in finished products even in the presence of
Figure 3. Strong-anion exchange (SAX)-HPLC separation of the disaccharides from the chondroitin sulfate samples of two food supplements (Sample E and G) produced by the action of chondroitinase ABC. ΔDi-0S, ΔHexA-GalNAc; ΔDi-6S, ΔHexA-GalNAc (6-OSO3); ΔDi-4S, ΔHexA-GalNAc (4-OSO3). Various unsaturated disulfated disaccharides are also separated in sample E.
26
feature article — SAX-HPLC analysis of food supplements
various additives and ingredients. The CS content in ten Czech www.sepscience.com
Figure4
Sample E
16000
5.5
results, the quality (in particular the
5.0
CS content), of several Czech Republic
4.5
12000
4.0 3.5
8000 4000 0 0.00
agents. On the basis of these analytical
Log M
Absorbance at 214 nm
20000
6.0
5.00
10.00
15.00
20.00
25.00
min
30.00
clinical studies and CS efficacy have
2.5
been evaluated by using a very pure
2.0 35.00 37.84
Sample H
product having specific properties and physico-chemical characteristics
12000
5.5
as approved by various National
5.0
Institutes of Health [1, 3, 4]. However,
4.5
this is not the first report on the quality
4.0
Log M
Absorbance at 214 nm
16000
3.5
8000 4000
5.00
10.00
15.00
20.00
30.00
25.00
of CS in food supplements. The quality
3.0
of raw material and finished products
2.5
was found to be poor in Korea [10]
2.0 34.99
min
and a great variability in the CS origin was found in 12 products from Japan,
Figure 4. High-performance size-exclusion chromatography (HPSEC) separation of the chondroitin sulfate from two nutraceutical samples (E and H). 100 μg of each food supplement sample was injected in the column. The third grade polynomial curve having the formula y(fx) = – ax3 + bx2 – cx + d performed by CS fractions of different molecular mass is also illustrated.
Republic dietary supplements was
and it is worthy of mention that
3.0
6.0
-163 0.00
dietary supplements was found poor
30-45% of the declared CS.
generally not corresponding to the label claim when declared [20]. This is a key point as the biological and
The pattern of disaccharides to
pharmacological properties are known
or in the presence of a proteolytic
determine the CS origin and the
to vary with the structure, and the oral
treatment to remove proteins possibly
molecular mass parameters to
absorption, and bioavailability and
interfering with the CS content
evaluate the absence of degradation,
pharmacokinetic parameters may be
evaluation, and the results were
were evaluated in samples in which CS
influenced by the different structural
quite similar. In particular, sample A,
was detected. The CS was found to be
characteristics and origin [21, 22], and,
claimed to contain 47% CS by the
of high molecular mass showing that
more importantly, there is a possible
manufacturer, contained almost no CS,
no degradation occurred during the
risk of species specific diseases. In fact,
lower than 0.2%. Sample B was found
food supplement preparations and it
there are strict regulations ensuring
to contain approximately 1% (17%
was evaluated to be of bovine, porcine
that material from mammals used
declared), sample D approximately
or cartilagineous fish origin. One
for drugs are prepared from healthy
1% (12% declared), sample E
sample was not determined becaues
mammals because of the risk of
approximately 11% (24% declared),
of very low CS content.
bovine spongiform encephalopathy
determined without any treatment
sample F approximately 4% (12%
CS, like other natural
(BSE), foot-and-mouth disease,
declared), and sample L approximately
polysaccharides, is derived from
influenza spread in birds, and other
1% (6% declared). In this last sample,
animal sources by extraction and
animal diseases. However, there are
2% hyaluronic acid was also found.
purification processes [1, 2, 12] and it is
no definite regulations on the origin
well known that its structure changes
of ingredients in nutraceuticals. The
to conform to label specifications.
according to the tissue, organ and
origin of the ingredients in natural
As a consequence, four of the food
species [1, 2, 12]. As a consequence,
products is the most important factor
supplement preparations were found
source material, manufacturing
ensuring quality, and thus safety and
to contain approximately 0-1% CS
processes, the presence of
efficacy.
in comparison with 47, 17, 12 and
contaminants, and many other factors
6% declared on the labels, and two
contribute to the overall biological
As the number of products containing
products were found to have only
and pharmacological actions of these
CS increases, stricter and more
Samples C, G, H and I were found
separation science — volume 1 issue 1
CS is widely used as a nutraceutical.
feature article — SAX-HPLC analysis of food supplements
27
Figure5
(2005). 10. J.S. Sim, et al., Food Chem., 101,
Absorbance at 232nm
Di-HA
532–9 (2007). 11. N. Volpi & F. Maccari, Food Anal. Chem., 1, 195-204 (2008). Di-6s
12. N. Volpi, J. Pharm. Sc., 96, 3168-80
Di-6s
(2007). 13. N. Volpi, L. Bolognani, J. Chromatogr., 630:390-6 (1993).
min
14. N. Volpi. Anal. Biochem., 273, 229-39
6.0
Sample L
7000
5.5
(1999).
5.0
15. N. Volpi, F. Maccari, J. Chrom. B, 834,
4.5 5000
4.0 3.5
3000
3.0
1000 -1586 0.00
Log M
Absorbance at 214 nm
9000
2.5
HA 5.00
10.00
15.00
20.00
30.00
25.00
2.0 34.99
min
1-13 (2006). 16. N. Volpi, et al., J. Chrom. B, 820, 131-5 (2005). 17. N. Volpi, F. Maccari, Electrophoresis, 23, 4060-6 (2002). 18. Biopharmaceutics Coordinating
Figure 5. Agarose-gel electrophoresis (A), disaccharide analysis by means of strong-anion exchange (SAX)-HPLC (B), and highperformance size-exclusion chromatography (HPSEC) evaluation (C) of sample L showing the presence of high molecular mass hyaluronic acid. ΔDi-HA is the unsaturated hyaluronic acid disaccharide produced by the action of lyase. HA = hyaluronic acid. CS = chondroitin sulfate.
Committee in the Center for Drug Evaluation and Research (CDER)
accurate evaluation and more strict
References
in cooperation with the Center for
regulation for quality control should
1. N. Volpi, Chondroitin sulfate:
Veterinary Medicine (CVM) at the Food
be required for the production of
structure, role and pharmacological
and Drug Administration. Guidance
high-quality products. Furthermore,
activity. Amsterdam, Boston,
for Industry, Bioanalytical Method
the regulatory requirements should be
Heidelberg, London, New York,
Validation, (May 2001).
implemented on the manufacturers
Oxford, Paris, San Diego, San Francisco,
19. N. Volpi, Carbohydr. Polym., 55, 273-
so that the ingredients and their
Singapore, Sydney, Tokyo, Academic
81(2004).
origin are correctly listed, and specific
Press (2006).
20. S. Sakai, et al., Chem. Pharm. Bull.
and accurate analytical procedures
2. K. Sugahara, et al., Curr. Opin. Struct.
(Tokyo), 55, 299-303 (2007).
should be enforced for the control of
Biol., 13, 612-20 (2003).
21. N. Volpi, Osteoarthritis Cart., 10, 768-
high-quality products and applied by
3. K.M. Jordan, et al., Ann. Rheum. Dis.,
77 (2002).
quality control laboratories to confirm
62, 1145-55 (2003).
22. N. Volpi, Osteoarthritis Cart., 11:433-
the purity and label claim of CS in
4. W. Zhang, et al., Osteoarthritis
41(2003).
raw materials and nutraceuticals. At
Cartilage, 15, 981-1000 (2007).
the moment, the quality of Czech
5. N. Volpi, Curr. Drug. Targets Immune
Republic food supplements is very
Endocr. Metabol. Disord, 4, 119-27
poor with no possibility for the
(2004).
customer to verify the content and
6. N. Volpi, Curr. Med. Chem., 13, 1799-
origin of CS. While we are waiting
810 (2006).
for more strict regulations for quality
7.P. Sarzi-Puttini, et al., Semin. Arthritis.
control, pharmaceutical formulations
Rheum., 35(1 Suppl 1), 1-10 (2005).
approved by local National Institutes
8. T.E. McAlindon, et al., JAMA, 283,
of Health is strongly recommended
1469-75 (2000).
instead of general food supplements.
9. J.S. Sim, J, et al., Chromatogr. B Analyt. Technol. Biomed. Life Sci., 818, 133-9
28
feature article — SAX-HPLC analysis of food supplements
www.sepscience.com
26–28 August Biopolis Science Park, Singapore
Singapore
Separation Science Singapore 2009 is a cross-discipline, cross-culture chromatography meeting aimed at scientists, engineers, business and technical experts from the diverse analytical instrumentation industries. Presentations will cover issues of vital importance to application chromatographers working in drug discovery and development, molecular diagnostics, food and agriculture, traditional Chinese medicine, forensics and security, process analytics and energy science industries. Confirmed speakers include:
Professor fessor Alastair Lew Lewis
Professor ofessor Gert Desmet Desm
Professor P f Ping Pi Li
“Current and Future Approaches to Speed Up HPLC Separations”
“HPLC and Hyphenated Techniques for Analysis of Constituents in Herbal Medicines”
“Trace Pollutant Detection in Challenging Environments”
Milos Novotny, (Indiana University, USA), Philip Marriott, (RMIT, Australia), Edward Browne, (GSK R&D Singapore), Peter Myers, (University of Hian Kee Lee, (National University of Singapore), Yi Chen, (Chinese Academy of Sciences, Beijing, China), C. Bor Fuh, (National Chi Nan University, Taiwan), Y.S. Fung, (Hong Kong University), Yizeng Liang, (Research Center for Modernization of TCM, Changsha, China), Tung-Hu Tsai, (National Yang-Ming University, Taiwan), Yong-Chien Ling, (National Tsing Hua University, Taiwan), Paul Haddad, (University of Tasmania, Australia), Siu Kwan Sze, (Nanyang Technical University, Singapore), Eric Chan, (National University of Singapore), Thomas Walczyk, (National University of Singapore), Manfred Raida, (Singapore). Liverpool, UK),
Call for Papers Submitted contributions are invited on all aspects of separation science pertinent to the theme, and specifically on the following topics:
Bioscience
proteomics, lipidomics, metabolomics, biomarker research, human/equine doping control, forensics
Energy
high-resolution separation methods for petrochemical, hydrocarbon and biodiesel applications
Enviro
air, soil and water testing, portable field analysis, sample preparation protocols, industrial emissions
Food
Pharma
TMC
contaminants, traceability, ingredients testing, flavours and fragrances, marine toxins high-throughput separations, chiral separations, ADME/PKPD studies, biopharmaceutical analysis TCM chemistry, fingerprinting, metabolism, quality control
For all programme enquiries email David Hills.
Confirmed sponsors:
For all delegate enquiries email Jackie Tan.
Deadline for oral contributions: 15 May Deadline for poster contributions: 12 June Instructions for submissions and further information can be found at
www.sepscience.com
Cd The Chrom Doctor
Developing new methods for LC-MS analysis
LC-MS methods employed today must cover
slightly more hydrophobic analytes need to be
a much wider range of sample types and
analysed (Figure 1).
complexities than ever before. Compared
Figure 2 shows basic compounds analysed
with isocratic elution, gradient elution
in positive ion mode. The data allows for
chromatography allows much faster elution
highly specific identification of analytes and
of strongly retained analytes without loss of
is more sensitive than traditional UV methods
resolution of the more polar analytes.
of detection. The total ion current (TIC) gives
Gradient elution chromatography forms the cornerstone for the majority of LC-MS methods
Figure 1
developed to date. The choice of buffer, its
Molecular Weight
concentration and type of organic modifier
100,000
used are important considerations when using MS as the detection system, as does the choice
LC/MS Electrospray
of HPLC column. The LC-MS interface
1000 LC/MS APCI
The basic principle of MS is the production GC/MS EI & CI
of ions which are subsequently separated according to their mass-to-charge ratio
Polar
Non-Polar
(m/z) and detected. The technique provides information that is both wide ranging and
Figure 1: The suitability of different techniques for compounds of different molecular weight.
accurate. Atmospheric pressure ionization (API) has greatly expanded the range of compounds
Figure 2
that can be analysed by LC/MS. The analyte is (A) TIC
brought to the interface via a solvent typically
311
at a rate of 1.0mL/min, depending on column diameter and method requirements. The API interface must separate the analytes from
(B) m/z=311
the solvent, ionize the analyte molecules and maintain a vacuum in the mass detector. The method by which this is done differentiates the API processes. Electrospray (ES) gives the widest range of application, both in terms of molecular mass and analyte polarity.
312
(C) m/z=317
348
LC-MS13 0
3
6
9
200
240
Min
280
320
LC-MS14
340
380
M/Z
Atmospheric pressure chemical ionization (APCI) may be the method of choice where 30
chrom doctor
Figure 2: A comparison of different mass spectral information. www.sepscience.com
Figure 3
Figure 3: A comparison of the sensitivity of an LC-MS separation with different mobile phase additives.
relative abundance of all ions detected.
applications. In general, it helps the LC-MS
Those that co-elute can still be isolated and
sensitivity to have at least 20-30% organic.
quantified by plotting their relative abundance
100% or very high organic content may also
as a function of time (mass chromatograms).
lower the sensitivity, especially where no additive is used. This is primarily because the
Proportion of organic solvent in the
conductivity of organic solvent is too low. A
mobile phase
small percentage of water in the mobile phase
The ratio of aqueous-to-organic solvent
helps the droplet formation.
is particularly important in electrospray ionization. The efficiency of the electrospray
Buffers/additives
process depends on the conductivity and
Buffers serve two purposes in LC-MS:
surface tension of the liquid being nebulized.
to act as a buffer for the chromatographic
When the conductivity and/or the surface
process in the traditional way (i.e. control and
tension are too high (i.e., highly aqueous), it
maintain the pH of the mobile phase in order
is difficult to produce a stable spray and it is
to keep the ionization state of an analyte
difficult to vaporize the droplets formed by
constant), and to adjust the pH of the carrier
the action of the high voltage and nebulizing
solvent (mobile phase) in such a way as to
gas. The percentage of water used should
present the analytes to the MS already in ionic
not be too high because surface tension
form.
of water is much higher than the surface
The most compatible buffers are ammonium
tension of methanol or acetonitrile. Additive
formate, ammonium acetate and ammonium
concentration will have considerable effect
hydroxyde at concentrations of 10 to 50mM.
on conductivity of the nebulized liquid and
The preferred additives are formic and acetic
consequently should be kept low. These
acids (0.01 to 1%v/v) because they improve
factors are most important when working at
protonation of basic samples in positive
high flow rates because there is more solvent
ionization. Other additives occasionally used
to be nebulized and vaporized. One alternative
include trifluoroacetic acid and trialkylamine
is to use a sheath liquid which is highly organic
type bases, but these need to be used at low
(e.g., IPA) to help the spray and vaporization.
concentrations (<0.1% v/v) because they may
However, this involves a more complex setup,
cause ionization suppression. Non-volatile salts,
and may not be suitable for high-throughput
such as phosphates and borates, ion pairing
separation science — volume 1 issue 1
chrom doctor
31
Figure 4
employed was 0.01% TFA. High levels of additive concentration lead to ion suppression that in turn will lead to loss in sensitivity for the method. Balancing UV detection with MS sensitivity requirements In the same study diode array detection was used in series with the MS system. The aim was to use the chromatographic data from the diode array to help identify analytes that either gave rise to poor MS detection or which were present only as minor peaks Figure 4: Comparison of two additives with UV detection.
barely visible in the noise of the total ion chromatogram. Diode array is ideal for this
agents and inorganic acids should be used
purpose because it can operate across a wide
with precaution.
wavelength range. Sensitivity of each additive
Modern orthogonal (off-axis) sources are
system towards diode array detection was,
more robust, and have been designed to
therefore, investigated in the 190-280 nm
operate with nonvolatile buffers/additives and
wavelength region. The chromatograms in
minimal sample clean-up. In order to achieve
Figure 4 show clearly the difficulties that can
the best response in LC/API-MS, mobile phase
arise at low wavelength where an unstable
composition must be such that the solution
baseline can often lead to loss in sensitivity.
and gas phase chemistries are optimized
In this case, TFA was found to be a more
to maximize ionization and eliminate any
attractive additive than formic acid as it
components which will cause ion supression.
gives better UV transparency, especially at the lower wavelengths. A compromise was
Additives for optimum MS sensitivity
made whereby the final method used TFA as
Some additives provide more sensitivity than
the additive in place of formic acid in order
others when used with MS (Figure 3). The
to maximize UV sensitivity, but used at low
additive used can greatly effect the sensitivity
concentrations (i.e. 0.01%), to maximize MS
of the MS ionization source and hence the
sensitivity with TFA.
sensitivity of the method. Careful consideration
Figure 5 shows how the method was used
is therefore required on which additive will
to analyze some in vitro incubation samples.
give the greatest MS sensitivity. A recent study
Note how the UV diode array chromatogram
of volatile additives showed formic acid to give
can be used to help identify regions of the
the greatest sensitivity
total ion chromatogram where sensitivity is
Reducing the concentration of each additive
poor. Identification of metabolites can often
gave significant increase in sensitivity. For
be a time consuming process when the peaks
example, improved sensitivity was observed
in the total ion chromatogram are lost amidst
when the TFA concentration was reduced
the noise of the baseline. On these occasions
from 0.1% to 0.01% TFA. Chromatographic
identification can often be aided by the
performance conditions are shown in
diode array chromatogram where a sensitive
Figure 4. In this example, the additive
LC method may allow for increased sample
32
chrom doctor
www.sepscience.com
Figure 5
LOOK TO TOMORROW
Figure 5: A UV (A) and MS (B) comparison of pethadine and its metabolites.
detection at low UV wavelength as shown for pethidine. In this example, the diode array provides a clear indication for the presence of a metabolite. The lambda max (not shown) can provide further information in support of MS data that can aid in structural elucidation and peak location on the MS total ion chromatogram. As mentioned earlier, low additive concentrations offer the reward of increased sensitivity for LC-MS. This is an important consideration when trying to identify trace quantities of a drug compound or impurity that may normally disappear into the noise of the baseline. The choice of HPLC column used is of key importance as the quality of the bonded phase and underlying silica can strongly influence the concentration of additive required. This Chrom Doctor article was written by Harold Ritchie, Thermo Fisher Scientific, Runcorn, Ceshire, UK.
analytica Anacon India SEPT. 29–OCT. 01, 2009 HYDERABAD analytica Vietnam MARCH 18–20, 2009 HANOI analytica MARCH 23–26, 2010 MUNICH analytica China SEPT. 21–23, 2010 SHANGHAI analytica Anacon India is part of the exhibition network analytica worldwide—the most important marketplace in the world for marketable products and solutions in the analysis, laboratorytechnology and biotechnology sectors. It is a business and networking platform and a driving force behind future trends and innovations. www.analyticaindia.com
separation science — volume 1 issue 1
chrom doctor
33
Tu Technology update
Key
Email the company
Product information
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Additional Information
6500 Series Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS Manufacturer: Agilent Manufacturer’s Description: Using proprietary True Hi-Def TOF technology, Agilent’s 6520 and 6530 bring an unrivaled combination of mass accuracy and resolution, sensitivity, acquisition speed, and in-spectrum dynamic range to research challenges. The Agilent 6530 Accurate-Mass Q-TOF LC/MS with Jet Stream thermal gradient focusing technology increases sensitivity by a further fivefold for even greater analytical confidence. All 6500 Series instruments feature the powerful, compound-centric MassHunter Workstation software with its data mining and qualitative and quantitative data analysis capabilities. Features include typical mass accuracy – sub-1-ppm MS and 2-ppm MS/MS rivals or exceeds that of much more expensive FTMS and orbital trapping instruments; enhanced mass resolution – up to 20,000 resolving power without sacrificing spectral acquisition rates; in-spectrum dynamic range – up to five orders reveal trace-level targets even in the presence of vastly more abundant compounds; spectral acquisition rates – up to 20 MS or 10 MS/MS spectra per second for compatibility with high-throughput, rapid-resolution chromatography; time-of-flight mass range – m/z 20 – 20,000; automated tuning that is unavailable from or unreliable on competitive instruments, Agilent Jet Stream technology provides 5x greater on-column sensitivity in the 6530, MassHunter Workstation software is compound-centric with powerful data mining and data analysis functionality, supplemented by powerful, application-specific software packages that maximize productivity and result quality for specialized analyses.
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Accela High-Speed LC Manufacturer: Thermo Fisher Scientific Manufacturer’s Description: The Thermo Scientific Accela high-speed chromatographic system provides fast, efficient chromatographic separations over an expansive range of flow rates and pressures. The Accela optimizes the performance of sub-two micron particle columns, providing seamless operation spanning conventional LC pressures from short LC columns, up to 15,000 psi for long-column separations of complex bio mixtures. The Accela Pump assures rapid and reproducible transfer of even complex and aggressive gradients. This quaternary pump is capable of handling pressures up to 15,000 psi with a delay volume of only 65 μL, enabling high-speed chromatographic separations. The Accela Autosampler has a specialized diamond-coated, high-pressure valve that can handle the rigors of constant high pressure injections while the unique sampling design enables 30 second injection cycles. This autosampler integrates isothermal injection and separation to provide reproducibility by eliminating external environmental influences to the chromatography. The Accela PDA has been optimized for the detection of high-speed chromatographic separations. The short 1 cm flow cell pathlength is combined with a minimized flow cell volume of 2 μL. The low level of dispersion in this LightPipe flow cell retains peak shape and chromatographic resolution from the column. Accela, coupled with the sub-two micron particle columns, provides fast, controlled separations with high efficiency and resolution, accelerating LC and LC/ MS applications. It offers optimized system delay volumes for fast separations, a flexible sample format that accepts vials or plates, a quaternary pump capable of conventional and ultra-high pressures, a patented LightPipe technology providing maximum sensitivity and resolution and full compatibility with Thermo Scientific mass spectrometers. Atlas 8.2 provides digital instrument communication and control of the Accela pump, PDA detector, and autosampler for injection, data acquisition, and CDS data processing and reporting as an enterprise-wide client/server CDS. Accela data collected is processed by Atlas peak detection, integration, reporting and data presentation.
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Click here ÄKTAready Liquid Chromatography System Manufacturer: GE Healthcare Manufacturer’s Description: GE Healthcare has launched ÄKTAready, a liquid chromatography system designed for process scale-up and production for Phase I-III drug development and full-scale production to GLP and cGMP standards. It has simplified system handling and reduced downtime between products and batches improves cost efficiency and productivity by saving time and expenditure for start-up, labour and consumables. According to GE Healthcare, ÄKTAready operates with ready-to-use, disposable flow paths, eliminating the risk of cross-contamination and the need for cleaning and validation of cleaning procedures. ÄKTAready comprises the chromatography unit, Unicorn software, and a disposable ReadyToProcess Flow Kit including sensors and detection flow cells. Unicorn includes an installation wizard that provides instructions and reports for column installation and ensures correct functionality of the Flow Kit. All ÄKTA systems use the same software, which enables easy process scale-up and quick transfer to ÄKTAprocess for use in full cGMP production. The ÄKTAready system is supported by extensive regulatory product documentation and services, including validation documentation, product documentation with information about materials used, USP Class VI CFR 177 and AOF certificates and a Regulatory Support File (RSF). ÄKTAready is part of GE Healthcare’s portfolio of ReadyToProcess solutions that help increasing the efficiency in biopharmaceutical production. ReadyToProcess enables lean production schemes by eliminating waste activities in daily routines of upstream and downstream processing. Products include the WAVE Bioreactor and WAVE Mixer with the disposable CellBag, chromatography columns as well as normal flow capsules and the ReadyMate disposable aseptic connectors. The ReadyToProcess portfolio provides maximum flexibility, simplifies and speeds up bioprocessing and is designed for scalable and smooth operations from fermentation through to purification. 36
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Automated Liner Exchange – ALEX Manufacturer: Gerstel Manufacturer’s Description: Matrix material from samples can contaminate a GC-liner, leading to peak broadening and loss of analytes through adsorption in the liner within a few GC runs. Under such conditions, the analyst is forced to perform frequent liner replacement, an operation which normally requires manual intervention. Running large series of samples will represent a serious challenge. The Gerstel Automated Liner EXchance (ALEX) technology automates the task of replacing liners in the Gerstel Cooled Injection System CIS 4. The ALEX system enables fully automated GC analysis of samples with a high matrix load as well as ‘dirty’ extracts, which can be directly injected into the CIS 4 using the Gerstel MultiPurpose Sampler MPS. Specially designed trays can hold up to 14 or 98 CIS 4 liners in individually sealed, contamination-free storage compartments. In connection with the MPS, the ALEX system enables automated CIS 4 liner exchange at user defined intervals in a sequence. The Gerstel MAESTRO-Software provides integrated control of the MPS, ALEX system and CIS 4. In combination with the Agilent ChemStation, a single method and sequence table control the entire system including GC and MS.
Multichannel nanoelectrospray emitters Manufacturer: Parteq Innovations Manufacturer’s Description: Researchers at Queen’s University in Ontario, Canada, have developed a multichannel nanoelectrospray (MCN) emitter that uses a micro-structured silica fibre as a ‘shower head’ to split the fluidic flow. Electrospray ionization (ESI) has become the preferred method of coupling liquid separation techniques to a mass spectrometry (MS) and pulled-glass capillaries are widely employed to improve electrospray performance at nL/min flow rates. While effective for stabilizing low flow rates, pulled-tip emitters have technical limitations such as susceptibility to clogging, limited range of possible flow rates and poor tip-to-tip reproducibility. This new class of ESI-MS emitters offers end-users the same signal sensitivities at 1000-300 nL/min flow rates they have come expect from currently available high-performance emitters, but with significant improvements in tip robustness. By virtue of their multichannel construction, MCN emitters last longer and produce almost no fluidic backpressure. Resistant to clogging, MCN emitters allow the user to plug in the emitter, begin the measurement and walk away. The MCN emitters can be purchased in packages of 5 or 20.
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Lux Chiral HPLC/SFC columns Manufacturer: Phenomenex Manufacturer’s Description: Lux is a line of polysaccharide-based columns for the identification and resolution of enantiomers. Lux columns are offered with two chiral stationary phases (CSPs), both of which use coated derivatized cellulose as the chiral selector. The two phases combine to create a dependable screening set with a wide range of selectivity. Lux Cellulose-1 uses cellulose tris (3, 5-dimethylphenylcarbamate) as the chiral selector and has been demonstrated to provide better resolution than the current market-leading column in a number of applications. LuxCellulose-2 introduces a new chiral selector, cellulose tris (3-chloro-4-methylphenylcarbamate), providing a unique stationary phase. This column offers complementary selectivity to Lux Cellulose-1. These two phases combine to create a dependable screening set with a wide range of selectivity. Lux columns are offered in 3 μm and 5 μm particle sizes, packed for analytical-scale use or Axia-packed for preparative applications. Enantiomers of chiral compounds may have different pharmacological effects in biological systems. Phenomenex states that the demand for chiral separations is on the rise with FDA mandates that enantiomers of all chiral drugs (in development) must be screened separately for their pharmacodynamic and pharmacokinetic properties. Other applications include toxicology, flavour analysis, and chemical and pesticide analysis.
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technology update
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UltiMate 3000 Rapid Separation LC (RSLC) system Manufacturer: Dionex Manufacturer’s Description: The UltiMate 3000 Rapid Separation LC (RSLC) system provides ultrafast LC separations using higher flow rates for increased throughput and small particle sizes for resolving peaks efficiently. Its combination of high flow rate, high pressure and ultrafast data collection rate facilitates high peak capacity in short run times. Even separations of 10 peaks in 10 seconds are easily achieved. RSLC system highlights include ultrafast separations at flow rates up to 5 mL/min and pressure up to 800 bar (11,600 psi) at the same time, in-line split-loop injections for 15 seconds, no-sample loss injections, reduced backpressure with high temperature column compartment operating at up to 110 °C and high data collection rate UV detectors (100 Hz), with variable wavelength, multiple wavelength or diode array (with full spectral scan). It also features Acclaim RSLC columns (2.2 μm) in various column formats, convenient method speed-up with automated calculation and validation tools, significantly reduced solvent consumption (typically by 70% or more) and instant results with Chromeleon dynamic data processing. As well as supporting UHPLC methods, the UltiMate 3000 RSLC runs conventional LC methods with its flexible components. The RS Wellplate Autosampler’s patentpending injection valve can robustly inject 100 μL at 800 bar pressure. It works with a multitude of different sample formats, supporting a maximum of 1,167 samples. Column compartments are available with integrated column switching valves to use up to 12 columns (of up to 30 cm length) at the same time. UV detectors come with a selection of micro and analytical flow cells, available in stainless steel or PEEK. Integration of any module of the UltiMate 3000 product line into the RSLC system is possible. Each component stacks and operates together seamlessly, with full support by Chromeleon chromatography data system.
separation science — volume 1 issue 1
Technology update
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