journal separation science
TRANSCRIPT
Volume 1 / Issue 7
June 2009www.sepscience.com
Getting the most out of your � ash chromatography
Understanding ionization techniques in mass spectrometry
Analysing biomarkers of oxidative stress
separationdriving analytical chemistry forwardsscience
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.
www.parker.com/lcmsTel: 00800 27 27 53 74
Click here
to find out m
ore in
formatio
n
on our range of g
as genera
tors
for LC/M
S
Together, we can reduce nitrogen costs for LC/MS. Pure and simple
contentsVolume 1 / Issue 7
June 2009www.sepscience.com
Getting the most out of your flash chromatography
Understanding ionization techniques in mass spectrometry
Analysing biomarkers of oxidative stress
separationdriving analytical chemistry forwardsscience
Understanding ionization techniques in mass spectrometry
Michal Holcapek
16
feature
separationdriving analytical chemistry forwardsscience
Volume 1 / Issue 7June 2009
research round-up
Improving performance of simulated moving bed chromatography by fractionation and feed-back of outlet streams
Comparing sample injection techniques for the CZE analysis of water-soluble vitamins
Fluorescence HPLC assay for the monitoring of L-asparaginase activity and L-asparagine depletion in children su� ering from acute lymphoblastic leukaemia
Improving detection of biomarkers for congestive heart failure with nanoLC-FT-ICR-MS
Analysing biomarkers of oxidative stress
Amino acid a� nity chromatography shows promise in plasmid DNA studies
Chemotaxonomic studies to quantify iso� avones in clover
Rr
regulars
06
06
08
10
11
12
An
Tu
application notesForensic Toxicology Analysis of Non-derivatized Drugs in Urine by Automated Solid Phase Microextraction (SPME) GCxGC-TOFMS, Leco
On-Line Two-Dimensional Separation of Intact Proteins, Dionex
Multiplexed Quantitative Peptide Assays for Protein Biomarkers of Cardiovascular Disease in Human Plasma, Applied Biosystems
HPLC and UHPLC Methods for Melamine, Grace Davison Discovery Sciences.
Carbohydrates, Organic Acids and Alcohols in Wine, Bischo�
Analysis of Pesticide Residues in Apple using Agilent SampliQ QuEChERS European Standard EN Kits by LC/MS/MS Detection, Agilent
technology update An overview of recent technology advances in separation science and instrumentation.
34
38
Separation Science is published by Eclipse Business Media Ltd, 70 Hospital street, Nantwich,
Cheshire, CW5 5RP, 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, 70
Hospital street, Nantwich, Cheshire, CW5 5RP, 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.
12
16
Mr meeting reportA Meeting to mark the o� cial retirement of Dr CK Lim.
24
Cd chrom doctorTechniques for optimizing normal- and reversed-phase � ash puri� cation
28
www.sepscience.com
scienti� c advisory
councilPeter Myers
– Chief Scienti� c O� cer
David Barrow
University of Cardi� , UK
Zongwei Cai
Hong Kong Baptist University
Yi Chen
Chinese Academy of Sciences,
Beijing, China
Gert Desmet
Vrije Universiteit Brussel, Belgium
C. Bor Fuh
National Chi Nan University, Taiwan
Y.S. Fung
Hong Kong University
Xindu Geng
Northwest University, Xi’an, China
Luigi Mondello
University of Messina, Italy
Paul Haddad
University of Tasmania, Australia
Hian Kee Lee
National University of Singapore,
Singapore
Melissa Hanna-Brown
P� zer, UK
Tuulia Hyötyläinen
University of Helsinki, Finland
Gongke Li
Sun Yat-Sen University, Guangzhou,
China
Yong-Chien Ling
National Tsing Hua University,
Taiwan
Klara Valko,
GSK, UK
Jean-Luc Veuthey
University of Geneva, Switzerland
Claudio Villani
Universita’ degli Studi di Roma “La
Sapienza”, Italy
Cheing- Tong Yan
Center of Environmental Safety and
Hygene, Taiwan
Edward Browne
GSK, Singapore
contactsDean Graimes
Publishing Director
+44 208 398 0209
Stephanie Painter
Associate Publisher
+44 1634 855 296
Kevin McGeehan
Associate Publisher
+44 208 398 1750
Karen High� eld
Financial Controller
Bo Zhang
Technical Editor
David Hills
Scienti� c Director
+44 1270 629496
Marita Kritzinger
Assistant Editor
+44 1270 629496
Professor Peter Myers
Chief Scienti� c O� cer
+44 151 601 2020
Will O’Keefe
Graphic Designer
rationdriving analytical chemistry forwardsdriving analytical chemistry forwardsscienccationdriving analytical chemistry forwardsscienc
separationdriving analytical chemistry forwardsscience
technical articles on chromatography and related technologies?
updates on recent research studies?
practical advice on routine analysis?
applications of new technology?
information on commercial productdevelopments?
market trends and opinions?
Register Now for your 20% Early Bird Discount Conference Highlights
Singapore
www.sepscienceasia.com
FoodEnviro
Day One:
Pat SandraAdvances in Separation Sciences Deriven by the Metabolomics and Proteomics Quest for Biomarkers
Manfred RaidaMultidimensional Gel-free Protein Separation Approaches for In-depth Analysis of Complex Proteomes
Y.S. Fung
Eric Chun Yong Chan
Yi ChenNew Approaches to Online Anti-salt Stacking for Direct Capillary Electrophoresis of Biosamples
Thomas WalczykElement Separation at the Microscale for High-Precision Isotopic Analysis of Biological Samples
Emily HilderOrganic Polymer Monoliths as Temperature Stable Stationary Phases for High Temperature Separations of Intact Proteins and Peptides
Andrew JennerGC-MS Analysis of Lipid Oxidation and Cholesterol Metabolism
C. Bor FuhImmunoassays Using Functional Magnetic Nanopaticles for Biochemical Analysis
Gurmil GendehOptimization Strategies in LC-MS/MS Front-End Separations to Achieve High Throughput, High Resolution and High Selectivity
Zhan ZhaoqiTools for Structural Elucidation & Metabolomics using IT-TOF Technology
Bioscience
sponsors:
For all delegate enquiries email [email protected]
26–28 AugustBiopolis Science Park, Singapore
Day Two:
Gert DesmetCurrent and Future Approaches to Speed Up HPLC Separations
John DolanPractical Tools for the Selection of Equivalent and Orthogonal HPLC Columns
Ronald E. MajorsHPLC Column Technology: Smaller and Faster
Phil NethercoteThe applictaion of Quality by Design Principles to Analytical Method Development, Validation and Transfer.
Sanjay GargThe Role of Analytical Science and Techniques in Early Phase Drug Discovery and Registration for Clinical Studies.
Vinayak NadigerPharmaceutical Analysis in Regulated Environments: Perspectives, Challenges and Insights
Phil DeLandThe Next Frontier: Doubling UHPLC Throughput for Pharmaceutical Applications
Edward BrowneBiomarker Analysis for Preclinical Pharmaceutical R&D
Anne GohOnline Solid Phase Extraction-LC-MS in DMPK Applications
Ping LiHPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines
Yizeng LiangSeparation Science for the Quality Control of Traditional Chinese Medicine
Tung-Hu TsaiMethods and Strategy of Microdialysis for Pharmacokinetics in Herbal Medicine
Day Three:
Alastair LewisTrace Pollutant Detection in Challenging Environments
Hian-Kee LeeSolvent-Minimized Sample Preparation for Separation Science
Siu Kwan SzeAn Advanced Proteomic Approach to the Discovery of Microbial Enzymes for
Gongke LiMolecularly Imprinted Polymers for Trace Analysis of Complicated Samples
Paul Haddad
Terrorist Explosives by Analysis of Inorganic Residues
Philip MarriottHeadspace Analysis of Plant Materials by Using Comprehensive Two-Dimensional Gas Chromatography: Selected Examples
Bahruddin SaadDetermination of Biogenic Amines in Food: Conventional and Nonconventional Approaches
Peter Gorst-AllmanComprehensive Two-Dimensional Gas Chromatography Time of Flight Mass Spectrometry (GCxGC-TOFMS) for Environmental Forensic Investigations in Developing Countries
Robert ShellieOne- and Two- dimensional GC-MS for Hop Metabolics
Jessie TongMultidimensional Gas Chromatographic Analyses of Flavours and Fragrances
Mike KindermanNew Developments for the Analysis of Inorganic and Organic Contaminants
Hui-Lai ChinPesticides Analysis by GPC-GCMS
Yuki HashiMelamine Analysis – New Approaches
Pharma TCM
Shawn StanleyUsing Narrow Bore Monolithic LC Columns to Enhance Analytical Sensitivity in Equine Doping Control
www.sepscience.com
Key
Email the author
Product information
Comment
RrResearchround-up
Germany
Professor Andreas Seidel-Morgenstern from
the Max Planck Institute for Dynamics of
Complex Technical Systems in Magdeburg,
Germany, has several years experience of
working with simulated moving bed (SMB)
chromatography, based on exploiting the
elegant principle of simulated moving
countercurrent movement between
stationary and mobile phases. Having seen
the potential for further improvement, a
study was conducted on a novel concept
that combines non-permanent product
withdrawal at one or both outlet ports
(leading periodically to a ‘product’ and a
‘non-product’ fraction), with an internal
recycle and re-feeding of the ‘non-product’
fraction in alternation to the original feed
mixture.
“After studying the possibilities to
systematically vary feed concentrations,
we studied the concept of partial product
withdrawal and optimized feedback. The
idea was inspired from reaction engineering
concepts based on recycling not sufficiently
converted reactants,” Professor Seidel-
Morgenstern said about the research,
published in the Journal of Chromatography A
[1207 (1-2), 55-71 (2008)].
Using simulation studies for linear and
non-linear isotherms, it was shown that in
terms of process performance and product
recovery, this fractionation and feedback
approach (FF-SMB) is superior to both the
conventional SMB process as well as to a
previously reported fractionation and discard
strategy. “The key finding is the fact that the
productivity of SMB chromatography can
be increased significantly, if the product
streams are collected at the outlets only
within certain fractions of the shift times, and
if the not sufficiently separated fractions are
recycled. This concept allows the processing
altogether of more feed and, thus, allows
higher productivities to be achieved,” he
added.
The results will be tested in the near future
for various applications. “If the theoretical
predictions can be confirmed, conventional
processes might be modified in order to
apply the new FF-SMB concept. For this, just
small modifications have to be made in the
hardware,” he concluded.
Improving performance of simulated moving bed chromatography
by fractionation and feed-back of outlet streams
6 research round-up www.sepscience.com
Chromeleon® 7 is the revolutionary new
chromatography data system from Dionex.
It provides rich, intelligent functionality with
the exclusive benefit of Operational Simplicity™.
Innovative eWorkflows enable anyone to run
complex procedures perfectly with just a few
clicks, and the SmartPeaks™ integration assistant
delivers the baselines you want in seconds.
From Samples to Results. Quickly and Easily.
Learn more at www.dionex.com/chromeleon7
Chromeleon 7
Simply Intelligent
Chromeleon is a registered trademark of Dionex Corporation. PIN 989
NEW!
Comparing sample injection techniques for the CZE analysis of water-soluble vitamins
ChinaA new online concentration method, namely pressure-
assisted field-enhanced sample injection (PA-FESI), was
developed and compared with FESI for the analysis of
water-soluble vitamins by CZE with UV detection and
described in the Journal of Separation Science [32 (7),
1011-1017 (2009)].
Main author, Dr Li Jia from the Key Laboratory of
Laser Life Science & Institute of Laser Life Science at the
South China Normal University in Guangzhou, China,
explained that the relatively low detection sensitivity
with conventional absorbance detectors in CE limits its
application in the analysis of low levels of components
in real-world samples. “In order to enhance the detection
sensitivity in CE, on-line sample concentration technique
represents an effective and versatile way,” Dr Jia added.
As part of the study, conventional sample stacking,
field-enhanced sample stacking (FESI), and two pressure-
assisted-FESI (PA-FESI) methods with and without a water
plug were developed as on-line sample concentration
methods to stack trace amounts of water-soluble
vitamins in CZE and compared. “In comparison with FESI,
PA-FESI with a water plug can achieve an increase in the
injection amounts of analytes, resulting in improvements
of concentration efficiency. FESI and PA-FESI were
suitable for the stacking of the analytes with high
electrophoretic mobilities,” Jia said. Conventional sample
stacking was a universal on-line concentration method
although the concentration efficiency for the vitamins
was less than 10. Using the PA-FESI with a water plug
method, the LODs of the vitamins were in the range from
0.1 to 0.2 ng/mL.
“The developed CZE method in combination with
PA-FESI with a water plug technique was applied to the
analysis of water-soluble vitamins in corns. The successful
evaluation of trace amounts of some water-soluble
vitamins contents (NA and FA) in corns proved that the
developed PA-FESI with a water plug method in CZE can
be applied to complex real-world samples,” he concluded.
8 research round-up www.sepscience.com
My IC saves lots oftime and moneyAnd yours can too when you chooseMetrohm® Ion Chromatography systems.
Their advanced technology and time-savingfeatures mean you work less yet producemore. Let them fully automate your routinesample preparations, including ultra-filtrationand dilutions.
www.professional-ic.com
U.S.A. 800-727-6768Canada 866-260-6069www.metrohmusa.com
Visit us at the ACHEMA 2009:Laboratory analysis: Hall 5.1 booth F39/G43 Process analysis: Hall 10.2 booth L15
Ins_My_IC_A4e_ACHEMA_09
Fluorescence HPLC assay for the monitoring of l-asparaginase activity and l-asparagine depletion in children suffering from acute lymphoblastic leukaemia
Australia
A novel assay for the determination of l-asparaginase activity in
human plasma is described in Biomedical Chromatography [32(2)
152-159] that is based on the HPLC quantitation of l-aspartic acid
produced during enzyme incubation.
Lead author, Dr Christa Nath from the Department of Biochemistry
at the Children’s Hospital in Westmead, Australia, explained that as
originally planned in the study protocol, l-asparaginase was to be
administered every 3 days as per the BFM-95 protocol in Germany.
“However, logistical difficulties arose when drugs needed to be
administered on the weekend. At our institution we administer
l-asparaginase every 3 or 4 days, the latter to avoid administration
on weekends. We wanted to develop the assay to measure
l-asparaginase and l-asparagine to compare l-asparaginase activity
and l-asparagine depletion at 3 versus 4 days after the dose. We
wished to confirm whether there was still therapeutic l-asparaginase
activity and depleted l-asparagine at 4 days after the dose,” Dr Nath
said.
For her, the most significant outcomes of the study were that
while l-asparaginase activity at 96 h postdose was less than the
activity at 72 postdose, there was no difference in l-asparagine levels
suggesting therapeutic equivalence. “We are currently collecting data
to perform a population pharmacokinetic and pharmacodynamic
analysis of l-asparaginase. This analysis will help us to better
understand the factors that affect the decline of l-asparaginase
activity with time, how and why individuals differ, and how these
differences affect l-asparagine concentrations,” Nath added.
This method may also be used to identify those patients with
reduced enzyme activity and reduced clinical efficacy, possibly as a
result of immunological reactions, and who may be candidates for
PEG-asparaginase. It also has an application for patients receiving
PEG-asparaginase. “By measuring l-asparagine levels after the
PEG-asparaginase dose, it is possible to follow the recovery of
l-asparagine levels, which will provide some indication of whether or
not another dose is required, because this information is not clear in
current dosing guidelines,” she concluded.
10 research round-up www.sepscience.com
11research round-upseparation science — volume 1 issue 7
Improving detection of biomarkers for congestive heart failure with nanoLC-FT-ICR-MS
USA
A paper in the Journal of Chromatography B [877 (10), 948-954 (2009)] demonstrates methods that augment the
detection of Brain (B-type) Natriuretic Peptide (BNP-32) by LC-MS. BNP-32 is a biomarker for congestive heart failure
and is measured using ELISA based assays in order to rapidly diagnose and monitor disease progression. The lack of
molecular specifi city aff orded by these assays has recently come into question as emerging studies indicate there are
potentially multiple heterogeneous forms of BNP in circulation with immunoreactive capabilities.
The study was led by Dr David Muddiman from the Department of Chemistry at North Carolina State University in
Raleigh, USA, who explained that FT-ICR mass spectrometry can aff ord unparalleled molecular specifi city, but alone
lacks the sensitivity essential for detection of low abundant biomarkers. “To increase the sensitivity and provide low
limits of detection, it is vital to develop and optimize front-end technologies, in particular, online separations. In our
most recent publication, we detail a comparison of two online sample trapping/clean-up confi gurations and a variety
of packing materials to facilitate detection of BNP-32, a clinically relevant cardiac biomarker, using nanoLC-FT-ICR-MS,”
Dr Muddiman said.
Using BNP-32 tryptic peptides, the team’s eff orts revealed that the continuous, vented column confi guration off ered
superior chromatographic performance relative to the discontinuous confi guration. “The former confi guration
utilizes a continuous bed of stationary phase segmented by a venting tee to direct fl ow during loading and elution.
Using packing material in this manner eliminates dead volume during the gradient elution, and consequently limits
diff usional peak broadening. A novel aspect of this work was the inclusion of a dummy column within the vented
confi guration to provide more rapid pressure stabilization and enhance the chromatographic performance for
tryptic peptides. Collectively, these results provide a benchmark for separation and digestion conditions of BNP-32,”
Muddiman added.
He continues that these fi ndings have resulted in the adoption of the continuous, vented column confi guration
within all their research endeavours utilizing online separations. “To date, this confi guration has been applied to
separations of cell lysates, plasma protein digests, and glycans cleaved from glycoproteins in plasma. For future
studies involving analysis of BNP-32 our fi ndings will serve to facilitate detection and identifi cation of the cardiac
biomarker and its alternative forms,” he concluded.
Amino acid affi nity chromatography shows promise in plasmid DNA studies
Portugal
In a study published in Biomedical Chromatography [23 (2), 160-165 (2009)], focused on the development and
establishment of new chromatographic strategies to purify pDNA, it was possible to implement a new affi nity
chromatography approach, named as amino acids-DNA affi nity chromatography, to purify pDNA. Two diff erent
strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described
to purify supercoiled (sc) isoforms. According to main author, Dr João Queiroz from the Centro de Investigação em
Ciências da Saúde at Universidade da Beira Interior in Covilhã, Portugal, this proves that well-defi ned binding/elution
conditions are crucial to enhance purifi cation performance, resulting in an improvement of the fi nal plasmid yields
and transfection effi ciency.
“In the past few years the main fi eld of our research has been the development of new chromatographic strategies
to effi ciently purify plasmids,” Dr Queiroz said. Gene therapy and DNA vaccination have emerged as two potentially
revolutionary DNA-based therapies. New products have to be developed and the improvement of biotechnological
processes will represent a great advantage in implementing these platforms in the biopharmaceutical industries.
Analysing biomarkers of oxidative stressUSAA robust and rapid high-pressure liquid
chromatography–electrochemical detection (HPLC–ECD)
method was developed and validated for the accurate
determination of ascorbic acid and uric acid in human
plasma. The study was led by Dr Xingnan Li and Professor
Adrian Franke, Director of the Analytical Laboratory
Shared Resource at the Natural Products & Cancer
Biology Program in the Cancer Research Center of Hawaii,
USA, and documented in the Journal of Chromatography B
[877 (10), 853-856 (2009)].
“Ascorbic acid (AA) and dehydroascorbic acid (DHAA)
are important biomarkers for evaluating oxidative stress.
During our ongoing research on oxidative stress factors
and their diagnostic value in disease risk assessment, we
intended to establish a fast, accurate and robust assay for
AA and DHAA analyses,” Professor Franke explained.
The team established an optimized, validated, robust
and rapid HPLC assay with electrochemical detection
(ECD), specifi cally by coulometry, using homogentisic
acid as a stable internal standard for the simultaneous
determination of AA and UA. “DHAA is not responsive
by ECD and is calculated by subtracting native AA from
total AA. The latter is determined after reducing native
DHAA to AA using dithiothreitol. This method was
further improved by applying ultra-HPLC techniques
using 1.9 μm Hypersil Gold C18 columns which
increased sensitivity, lowered detection limit and solvent
consumption twofold, and reduced analysis times over 4
times to 2.5 min. Both methods showed good precision
and accuracy,” Franke said.
After comparing various methods recommended for
clinical and translational research the team presented an
optimized, validated and robust HPLC–ECD method for
the quantifi cation of serum ascorbic, dehydroascorbic,
and uric acid using homogentisic acid as internal
standard. “We also reported on a highly effi cient and
robust UHPLC method for fast turn-around ascorbate
and urate analyses. Our results provide fast, accurate,
and robust method for AA, DHAA and UA analyses from
serum or plasma for clinical and other research,” he
concluded.
Analysing biomarkers of
12 research round-up www.sepscience.com
“The clinical application of plasmid DNA (pDNA) obliges to its recovery and manipulation
with a high level of purity and biological activity. However, because of the biosynthesis of
pDNA in recombinant organisms such as Escherichia coli, the primary pDNA recovery occurs
in a highly impure extract,” he explained. The original idea on the basis of his research was the
application of amino acids to exploit an affinity-based and specific interaction with the pDNA
in order to efficiently purify this biomolecule from all the impurities. This idea was supported
by the natural occurrence of interactions between proteins and nucleic acids molecules, at
biological level.
“Histidine and arginine have been used as amino acid ligands and their ability to isolate
the active sc pDNA proved the presence of specific interactions of pDNA with both amino
acid-based matrices,” he said. The potential of histidine affinity chromatography to efficiently
separate sc pDNA from host impurities was further demonstrated in a study which showed
that only sc pDNA interacted with the histidine ligand, whereas the other isoforms of pDNA
and contaminants did not.
“The pDNA obtained with this histidine affinity approach was of high quality and
contamination with gDNA and endotoxins was within acceptable levels, while neither
RNA nor proteins were detected. Transfection experiments using this purified pDNA also
confirmed that it could efficiently drive gene expression in eukaryotic cells,” he added.
According to Queiroz, the relatively low yield and the high salt concentrations required for
pDNA elution using this strategy highlighted the need for further improvements. “The studies
that employed arginine matrices to purify pDNA revealed the presence of specific interactions
with plasmid molecules, and importantly a significant recognition of the sc isoform. The
multiple interactions that arginine-based matrix was able to promote, allowed the differential
recognition of the biomolecules present in E. coli lysates, representing an important insight to
pDNA purification process. Using this simplified sc pDNA purification process, the majority of
the contaminants were removed, a 79% yield was achieved, sc pDNA was purified under mild
conditions, and showed to be extremely efficient (62%) on cells transfection. Hence,
arginine-chromatography proved to be an interesting option as a late-stage plasmid
purification step,” he explained.
In his view, amino acids-based affinity chromatography represents a particularly promising
approach, because it combines the selectivity of a biological interaction with the simplicity
of a single small molecule as chromatographic ligand. However, the low capacity of available
supports for pDNA still remains to be solved, as well as the low diffusivity of pDNA samples
resulting from their molecular weight. This will require further efforts in order to design more
suitable ligands and supports, in a similar way to the developments already seen for protein
purification matrices.
“Hopefully, some of the findings concerning the chromatographic interactions between
pDNA and amino acids can be applied in future bioseparation methods both for preparative
and analytical purposes. For the moment, it will be intended to apply this purification
strategy in the purification of a specific plasmid with genetic information for p53 expression.
In this particular gene-based treatment, it is expected to recover a delivery system capable
of inserting the p53 gene into tumour cells, thereby inducing cell death. Other research
groups are investing in this field and several studies are in progress. It is considered of crucial
relevance to proceed with this research with the global aim to find an efficient process to
obtain a highly pure pDNA product, efficient on cancer therapies,” he explained.
14 research round-up www.sepscience.com
Chemotaxonomic studies to quantify isoflavones in clover
Poland
A recent paper in the Journal of Separation Science [32 (7), 965-972 (2009)] detailed chemotaxonomic studies
undertaken to establish the qualitative profile and actual amounts of the pharmacologically active isoflavone
aglycones genistein, daidzein, formononetin and biochanin A in aerial parts of thirteen Trifolium L. (clover) species,
native to Poland. A newly elaborated micropreparative technique, developed by Dr Grazyna Zgórka from the
Department of Pharmacognosy with Medicinal Plant Unit at the Medical University of Lublin in Poland,— SPE on
BakerBond octadecyl, cyclohexyl, and phenyl cartridges — was used in combination with ultrasound-assisted
extraction for isolation of isoflavone aglycones from hydrolysed samples.
“For many years I have been working on phytoestrogens, that nowadays attract considerable scientific research
because of health benefits related to menopausal problems and some biodegenerative diseases such as breast and
prostate cancer, osteoporosis, as well as cardiovascular disease, combined with atheromatosis,” Dr Zgórka explained.
Trifolium L. (clover) is an important genus of the Leguminosae (Fabaceae) family containing phytoestrogenic
isoflavone constituents. Isoflavone glycosides and malonylglycosides are metabolized in the mammalian and
human small intestine, releasing pharmacologically active aglycones. Effective extraction techniques, followed by
appropriate hydrolytic procedures are needed to determine the real, total concentration of non-glycosidic isoflavone
phytoestrogens in plant samples examined. “A knowledge on verified, factual amounts of isoflavone aglycones is
undoubtedly of special importance in the establishment of the proper dosage of clover extracts, while performing
in vivo experiments or clinical studies, and for further accurate evaluation of pharmacological activity of these
constituents,” Zgórka added.
The key findings of her work comprised the elaboration of a new micropreparative technique — solid-phase
extraction (SPE) on BakerBond octadecyl, cyclohexyl and phenyl cartridges, in combination with
ultrasound-assisted extraction (UAE), for isolation of isoflavone aglycones from clover samples. “I
documented very high recoveries (> 96%) for four isoflavones using these
micropreparative techniques. To obtain the most reliable qualitative
and quantitative results, I employed photodiode-array (PDA) and
fluorescence (FL) detection, coupled with reversed-phase LC. Using
optimized preparative and detection techniques, I aimed to establish
the clear chemotaxonomic differences, combined with flower colour
variability, within thirteen clover species. Clovers with pink or purple-red
flower corolla possessed the highest concentration levels of isoflavones
(e.g., 3300 µg/g dry wt in Trifolium medium), while in three yellow
flowering clovers (T. aureum, T. dubium and T. campestre) these compounds
were not detected at all,” she added.
Dr Zgórka believes the elaboration of effective extraction techniques (UAE
and SPE), followed by acidic hydrolysis and RP-LC coupled with simultaneous
DAD and FL detection, may be important for both phytochemical
studies, concerning accurate and precise determination of
isoflavone phytoestrogenic compounds in plant material, and
chemotaxonomical investigation of some species belonging
to the Leguminosae (Fabaceae) family. “The method may be
useful in the selection process of new plant sources rich in
active isoflavone bioconstituents,” she concluded.
16 research round-up www.sepscience.com
The TSK-GEL® HILIC portfolio, a selection of stable, silica based HILIC phases, offers:
TSKgel Amide-80 - the standard for glycan analysis
TSKgel NH2-100 - new, robust amino bonded phase
smallparticlesizesforhighcolumnefficiency
virtual absence of bleeding, ideal for MS detection
Discover TSK-GEL® HILIC solutions for HPLC
Visit Tosoh Bioscience at HPLC 2009 in Dresden, booth 44 and talk to our experts.
TOSOH BIOSCIENCEZettachring 6, 70567 Stuttgart, GermanyPhone: +49 (0)7 11-1 32 57-0, Fax: +49 (0)7 11-1 32 57-89 [email protected] www.tosohbioscience.com
TOSOH BIOSCIENCE
18 feature article — Ionization techniques in MS www.sepscience.com
Understanding ionization techniques in mass spectrometryMichal Holcapek, University of Pardubice, Czech Republic
Ionization is the process whereby neutral molecules are converted
into charged species (i.e., ions). The conventional electron ionization
technique can only be used for low-to-moderate molecular weight volatile
compounds, while soft ionization techniques are suitable for the ionization
of a wide range of molecules, regardless of their polarity and molecular
weights, and are capable of creating both positively or negatively
charged ions. Soft ionization techniques, especially atmospheric pressure
ionization techniques (e.g., ESI, APCI and APPI), are well suited for HPLC/
MS coupling. This article on LC/MS describes electron ionization (EI),
chemical ionization (CI) and electrospray ionization (ESI).
19feature article — Ionization techniques in MSseparation science — volume 1 issue 7
identifi cation of the peaks
eluting from the GC column, a
process simplifi ed by the
availability of extensive EI
spectral libraries.
In EI a rhenium or tungsten
fi lament heated to high temperature
produces thermal electrons, which
are accelerated towards an anode by
establishing a potential diff erence.
Large libraries of EI mass spectra
are used for fast identifi cation of
unknown analytes. The high energy
electrons interact with neutral
molecules in the ion source, violently
ejecting valence electrons, a process
represented as:
M + e- M+. + 2e-
and is applicable only for volatile
compounds because it is a gas-phase
ionization technique. EI is considered
a “hard” ionization technique
because the excess internal energy
deposited in the analytes during
the ionization process may lead to
extensive fragmentation and even
to the total absence of the molecular
ion M+. for ~10% of volatile organic
compounds.
EI is mostly coupled to gas
chromatography. This pair is
particularly well suited for several
reasons:
• GCandEIoperateinsimilar
concentration and polarity
ranges,
• EIfragmentationallows
An enormous number of compounds
can be found in nature or are
prepared synthetically. They can
diff er in molecular weights, polarity,
volatility, thermal stability and many
other physicochemical properties.
Because of this variety of properties,
there is no perfect analyser, and
thus mass spectrometers employ
a number of diff erent ionization
techniques. In fact, there are
commercial instruments available
with interchangeable ionizers so
that the ionization technique can be
tailored to the analyte.
Electron ionization (EI)Electron ionization (EI) was the fi rst
ionization technique used in MS,
20 feature article — Ionization techniques in MS www.sepscience.com
Chemical ionization (CI)Chemical ionization (CI) is the soft
ionization technique used in
GC/MS. It is a modifi cation of EI,
whereby high energy electrons are
fi rst used to ionize a reacting gas,
which goes on to ionize the analyte
molecules. Fragment ions intensities
are still higher compared with
API techniques used for HPLC/MS
coupling nowadays.
• Thereactiongasisintroducedin
the ionization chamber at a
pressure of approximately
50-100 Pa (~3 x 10-3 to 7 x 10-3
atm).
• Becauseoftheexcessofthis
reactant gas in the source, the
accelerated electrons collide fi rst
with the reaction gas.
• Theresultingreactiongasions
then react with the remaining
neutral molecules of reaction gas
by a series of ion-molecule
reactions.
• Thesecomplexreactionswill
fi nally achieve a relatively
steady-state composition of
reaction ions (e.g., CH5, C2H5 and
C3H5 for methane) which can
fi nally ionize the analyte by ion-
In principle, EI has rather high
vacuum requirements (about
10-5 Pa) to protect the heated
fi lament, which would burn at
elevated pressures, and to avoid
unwanted ion-molecule reactions
within the ion source. The molecular
and fragment ions formed in the ion
source are forwarded to the mass
analysed region by the repeller
electrode and a system of other
focusing and acceleration electrodes
referred to as ion optics.
Particle beam interface
HPLC/MS is mostly coupled to
atmospheric pressure ionization
techniques. Occasionally, however,
EI is used with HPLC separations
of moderately polar molecules in
the 200-800 Da range (e.g., certain
drugs and pesticides) in order to take
advantage of EI spectral libraries.
Such a measurement employs a
particle beam interface to produce
the gas-phase molecules required
for EI.
A particle beam interface transfers
molecules to a gas-phase (Figure 2).
The HPLC effl uent is:
• Nebulizedbyastreamofhelium
(1 and 2)
• Furtherevaporatedinan
evaporation chamber heated to
about 50-80 °C
• Separatedinatwostage
molecular jet separator (4).
• Thelightermobilephaseand
helium nebulizing gas are
selectively pumped off (3)
by vacuum pumps while heavier
analyte molecules continue to
the EI ion source (5).
Figure 2
Figure 2: Graphical reprsentation of a particle beam interface.
© CHROMEDIA
4
5
32
1
Figure 1
© CHROMEDIA
Ions
Ionisation chamber
Anode trap
Heated filament
Electronbeam
Repellerplate
Sample
MoleculesTo
analyser
Figure 1: The ionization principle of the EI technique.
21feature article — Ionization techniques in MSseparation science — volume 1 issue 7
molecule reactions.
CI is used in either polarity mode
and generates mainly even-electron
ions, for example [M+H]+ in the
positive-ion mode or [M-H]- in
the negative-ion mode plus some
fragment ions. Understanding this
ionization mechanism is useful for
the explanation of APCI and other
modern soft ionization techniques.
Electrospray ionization (ESI)ESI is the softest ionization technique
and is well suited for biomolecules,
organometallic compounds, non-
covalent complexes, gas phase
reactivity studies, etc.
The introduction of electrospray
has proven to be a great tool in
biochemistry, allowing the mass
spectrometric characterization and
sequencing of peptides, proteins
and other biopolymers of great
importance to human life and
medicine. In an ESI source:
• Thecolumneffluentisdirected
through a stainless steel capillary.
• Ahighvoltageisappliedtothe
© CHROMEDIA
Ions
Solvent spray
Heated nitrogen drying gas
Dielectric capillary entrance
Nebulizer gas
Figure 4
Fig 4: A set up with Nitrogen as ‘drying gas’ .
Figure 3
© CHROMEDIA
Mobile phase
Capillary- 3 kV
Spray of fine droplets
Taylor cone
Ion evaporation
Desolvation gas
Solvent evaporated from droplets Coulombexplosion
Fig 3: The desolvation gas speeds up the evaporation and explosions.
capillary (ca. 3-5 kV), which is
kept in a coaxial flow of nitrogen
nebulizing gas (Figures 3 and 4).
• Creatingafineaerosolofvery
small droplets, each of which
carries many excess charges at its
surface.
• Thedropletsizeisfurther
diminished in the ion source
region with counter-flow of
heated drying gas due to solvent
evaporation from the droplet
surface (Figure 5).
• Whenthechargedensityatthe
droplet surface reaches a critical
value (the Rayleigh limit), a so
called Coulombic explosion
occurs and several even smaller
droplets are formed, each
carrying some fraction of the
original droplet’s surface charge.
• Theprocessofsolvent
evaporation, droplet contraction
and Coulombic explosions is
repeated until the molecular
adducts are released from the
final droplet.
If a positive voltage is applied to
the capillary, then the droplets will
carry positive charges and finally
positive ions are formed, such as
[M+H]+and[M+Na]+ adducts.
In the negative-ion mode, the base
peak is typically the [M-H]- ion.
Because many mass analysers (and
ion detectors) have an effective
upper m/z limit, ESI is notable for
the formation of multiply charged
ions, which significantly extends
the mass range up to and beyond
100,000 Daltons. This is of obvious
importance to measurement of
biopolymers.
An example: Let´s consider the
simple example of a protein with
22 feature article — Ionization techniques in MS www.sepscience.com
amolecularweightMW=60,000
Da. The molecular adduct with for
example60protonswillprovide
a signal at m/z=(60,000+60)/60
=1001.Inpractice,weobservea
distribution of multiply protonated
molecules, each with a diff erent
number of charges, as shown in the
figure.Wecaneasilydeterminethe
MWforunknownproteinsusing
simple algebra:
1. First, we assume that the ion
at m/z 1049.8 carries an unknown
number of charges x, which is
related to its m/z by the equation:
1049.8=(MW+x)/x.
2. Likewise, the neighboring ion
at m/z 991.5 carries an unknown
number (x+1) of charges, giving
the relationship:
991.5=(MW+x+1)/(x+1).
The solution of these two
equations with two unknowns will
yield both the charge numbers of
the ions and the molecular weight of
the protein.
This article was written by Michal
Holčapek, Professor of Analytical
Chemistry, University of Pardubice,
Czech Republic.
Publication of this article was made
possible through collaboration with
Chromedia.
Figure 6
Rela
tive
inte
nsity
(%)
800 1000 1200 1400 1600m/z
100
80
60
40
20892.4
939.2
991.51049.8
1115.5
1189.6
1274
1372.5 1486.6
20+
19+
18+17+
16+
15+
14+
13+12+
Figure 6: ESI spectrum of multiply charged protein (Lysozyme).
Figure 5
© C
HRO
MED
IA
Evaporation Analyte ion ejected
Fig 5: In ESI the droplet size decreases and charged + ions are formed.
Special Subscription Off er“We are a worldwide community
of experts with a mission. We cooperate to off er you
Chromedia, a fast growing database of peer reviewed
information with tutorials and solutions for the day-to-day
questions in your lab at aff ordable cost.”
Being Separation Science readers, you and your
organisation can subscribe to Chromedia at an
attractive rate.
Want to see more of Chromedia? Click the image!
Recommended Further Reading
Click titles for more information
The Mass Spectrometry Topic Circle
Analysis of PAH’s in foods
LCMS of pesticides
LCMS of lipids
Chromedia is pleased to off er its members a FREE
download of GCXGC:
GCXGC, a 144 page PDF by multidimensional GC expert
Jan Beens.
7feature article — Ionization techniques in MSseparation science — volume 1 issue 7
The Mass Spectrometry Topic Circle
Analysis of PAH’s in foods
LCMS of pesticides
LCMS of lipids
RECENRECENF
4–6 November
09.e
u TOPICS focused oBioanalytical TecApplications in are
Residues and conAuthenticity, trac
afa2
00
Authenticity, tracFlavours and odouProcessing and paMycotoxins, mariAllergensGenetically modifNanoparticlesN l f d t
ww
.ra Novel foods, nutr
QA/QC and chem
OTHER EVENTS• A workshop focused
research within the 7programmes in collab
ww p g
Centre AS CR• Vendor seminars intr
advanced food contr• A Seminar on issues a
food: US perspective
SPECIAL OPPORA i f • An opportunity for yothe main program
• IAEAC and RAFA 200• The best poster pres
poster award
DEADLINES: August 1, 2009 Rg , 009July 31, 2009 AAugust 31, 2009 AOctober 15, 2009 LaDecember 6, 2009 M
sp
Symposium chair PSymposium co-chair PSymposium co chair P
4th International Symposium on
T ADVANCES IN T ADVANCES IN FOOD ANALYSISr, 2009 Prague, Czech Republic
on Recent Advances in Analytical & hnologies and Emerging Food-Related eas:
taminantsceability, fraudceability, fraudursackaging contaminants ne and plant toxins
fied organisms (GMO’s)
iti l l t i f dritional supplements, organic foodsmometrics in food analysis
S: on opportunities for young scientists to take part in
7th Framework Programme and other related EC boration with Joint Research Centre and Technology J gy
roducing recent instrumentation and strategies for oland approaches to address chemical contaminants in
e
RTUNITIES FOR YOUNG SCIENTISTS: i i l ib i i hi oung scientists to present an oral contribution within
09 Student Travel Grants sentation honoured with the IAEAC Roland W. Frei
Reduced registration fee deadlinegAbstract submission deadline (oral presentation)Abstract submission deadline (poster presentation)ast minute poster deadline
Manuscript’s submission deadline for publication in pecial issue of Analytica Chimica Acta
rof. Dr. Jana Hajslova (ICT, Prague)rof. Dr. Michel Nielen (RIKILT, Wageningen)rof. Dr. Michel Nielen (RIKILT, Wageningen)
MrMeeting report
Applications in Clinical Biochemistry 2009 A Meeting to Mark the Official Retirement of Dr CK Lim
Chang Kee Lim, latterly of Birkbeck College, University
of London, has been a leader in the application of
separation science in biochemistry for many years. A
capacity audience at Guy’s Hospital, London met to pay
tribute to him and his achievements, and to discuss
current trends in the use of separation science in clinical
chemistry. Sponsorship from ABS, Agilent, ESA, Jasco,
Jaytee Biosciences, Thermo Scientific and Waters is
gratefully acknowledged.
In opening the meeting, David Perrett (Queen Mary,
University of London) recounted some milestones in
Dr Lim’s career. A BSc in Chemistry and Zoology was
followed by a PhD from Westfield College, University of
London. In 1973 Chang Kee went to work with Professor
Charles H. Gray at King’s College Hospital Medical School.
There he began to work on porphyrin metabolism, which
he continued at the Clinical Research Centre (CRC),
A joint meeting of the Royal Society of Chemistry, Separation Science Group and the
Association for Clinical Biochemistry Southern Region
Northwick Park, 1976-1991. With the closure of the CRC,
Chang Kee moved to the MRC Toxicology Unit, firstly at
Carshalton and then in Leicester.
In 2000 Chang Kee moved back to London, still under
the auspices of the MRC, to establish a BioAnalytical
Science Unit at Birkbeck College. Although retired,
Chang Kee continues to edit the journal Biomedical
Chromatography that he helped establish in 1986. Prof.
Perrett also recalled the meeting ‘High Pressure Liquid
Chromatography in Clinical Chemistry’ Chang Kee
organized at King’s College Hospital in 1975, and the
ensuing book ‘HPLC of Small Molecules’ (IRL Press, 1986)
that was a classic in its day.
Porphyrins 40 Years OnThe session Chair, Dennis Wright (Northwick Park
Hospital) recounted the valediction ‘look at the
chemistry’ that guided him in his early PhD studies on
porphyrins with Chang Kee. Porphyrins have been Chang
Kee’s abiding interest, and in the opening lecture he
emphasised the contribution of HPLC and of LC-MS/MS
to the study of porphyrins and the porphyrias.
Two topics stood out. First, it had been claimed in
a paper published in the Proceedings of the National
Academy of Sciences, USA, in 2007 that a porphomethene
inhibitor of uroporphyrinogen decarboxylase, namely
uroporphomethene (m/z 835), was the cause of
porphyria cutanea tarda. However, not only was the LC
retention of ‘uroporphomethene’ incompatible with
what would be expected of a porphomethene, but also
fragmentation characteristic of polyethylene glycol (m/z
835 with product ions at 791, 747, 703 and 659; that is
to say, successive loss of OCH2CH2) was present. This was
wrongly interpreted as loss of CO2 from the porphyrin Chang Kee Lim and wife, Soo.
24 meeting report www.sepscience.com
carboxyl groups.
On a related theme, 2-vinyl 4,6,7-tripropionic acid
porphyrin (harderoporphyrin) had been reported
in the Harderian glands of rodents. However, work
using a neutral extraction procedure and LC-MS/MS
showed that ‘harderoporphyrin’ was protoporphyrin
monoxyloside, protoporphyrin itself and protoporphyrin
monoglucoside being minor components of the gland.
Again, understanding the underlying chemistry of the
analytical procedure was vital to a proper understanding
of the natural products under study.
On a lighter note Chang Kee outlined work undertaken
early in his ‘retirement’ in conjunction with the Centre
for Ornithology, Birmingham and the Natural History
Museum on the origin, evolution and functions of
eggshell colours and patterning. Porphyrins again
– it seems that the colouration of sparrowhawk and
emu eggshells, for example, is due to the presence of
biliverdin and protoporphyin in different proportions.
Vitamin D, Purines, and Drugs of Abuse
Sandra Rainbow (Northwick Park Hospital) pointed
out that vitamin D research was very active, with some
1,000 papers being published in 2007 alone. LC-MS/MS
was rapidly becoming the method of choice for clinical
25-OH-D2 and 25-OH-D3 analysis. Derivatization was
not required and there were advantages of selectivity
over immunoassays. However, an internationally agreed
reference material was lacking and measurement of
1,25-dihydroxyvitamin D remained challenging. Sandra
also drew attention to work using chiral LC-MS/MS that
demonstrated the presence of both 25-OH-D3 and of
epi-25-OH-D3 in patient samples.
Tony Marinaki (GSTS Pathology) outlined work in
screening for inborn errors of purine and pyrimidine
metabolism. For purine analysis a 250 x 3 mm i.d. Waters
Spherisorb ODS1 column with gradient elution gave
a 33 min analysis time. Use of a bridged ethyl hybrid
(BEH) C18 column (Waters Acquity) (1.7 µm aps) gave
improved speed of analysis, resolution, and sensitivity,
but with a higher column back pressure. However, more
recent work had suggested that Supelco Ascentis Express
Halo columns (2.7 µm particles) could give analogous or
better results at lower back pressures.
Finally, Richard Evers (King’s College Hospital) discussed
problems and pitfalls in clinical drugs of abuse screening.
LC-MS/MS offered lowered consumables costs and the
ability to identify unequivocally a range of underivatized
analytes. However, sequential analysis limited
sample throughput, the potential for ion suppression
necessitated great care in method validation, and sample
hydrolysis was still needed in the event that sensitivity
towards morphine and buprenorphine glucuronides was
poor or non-existent. The potential for use of ion trap and
accurate mass technology in overcoming these problems
was just beginning to be developed.
From Tswett to MetabonomicsAfter lunch, David Perrett began by paying tribute
to another pioneer in the application of analytical
methods to clinical problems, Professor Charles Enrique
Dent (1911–1976). Using the then new technique
of 2-dimensional paper chromatography, David
felt that Dent had probably discovered/developed
major insights into more diseases (including Fanconi
syndrome, Hartnup disease, argininosuccinic aciduria,
homocystinuria, cystinuria, and xanthinuria) than anyone
else. David then discussed his own recent research
using capillary electrophoresis with low-wavelength UV
detection in which some 80 compounds can be detected
in urine in 10 minutes. He also took the prize for the
oldest sample discussed during the day with a slide of
the CE-DAD analysis of organic compounds in an extract
of an Egyptian mummy (site of sampling not stated)!
25meeting reportseparation science — volume 1 issue 7
David’s talk neatly picked up the theme initiated
by Chang Kee Lim earlier in the day – it is not simply
‘hypothesis driven’ research (with which funding
bodies seem sometimes to be obsessed) that advances
knowledge, but more often than not it is either the
application of new techniques to old problems, or
the critical appraisal of existing data that leads to key
developments.
Having discussed aspects of ICP-MS operation,
including GC- and LC-ICP-MS, Andrew Taylor (Royal
Surrey County Hospital) gave a brief overview of
metallomics (chemical speciation, dynamics and kinetics
of trace elements in biological systems). Applications
discussed included isotope measurement to help identify
sources of lead exposure, the use of 65Cu administration
as an additional diagnostic test in Wilson’s disease,
diff erentiation of arsenic species in urine, and recent
work aimed at unravelling the complexities of selenium
metabolism. An interesting aspect was the use of
selenium isotope patterns to help identify selenium-
containing species in complex chromatograms. Potential
pitfalls such as ‘polyatomic’ (e.g., 40Ar2+ on 80Se+) and
‘isobaric’ (e.g., 64Ni+ on 64Zn+) interferences were given
due prominence. A clinical problem of gadolinium used
as contrast medium was discussed – 156Gd2+ has the same
m/z as 78Se+.
Edwin Carr (St Helier Hospital, Carshalton) next
discussed catecholamine measurement using HPLC with
electrochemical detection (ED). Nowadays the use of ED
in clinical chemistry is virtually confi ned to diagnosis of
phaeochromocytoma, with urine being the specimen of
choice unless the patient has renal failure. There is almost
universal acceptance that, at present, metanephrines
analysis is superior to other means of diagnosing this
condition, yet the evidence for this is still ignored by
many.
The fi nal talk returned to the ‘omics’ theme with Paul
Thomas (University of Loughborough) discussing volatile
profi ling using GC-MS and other techniques. After
describing investigations into breath volatiles in, for
example, chronic obstructive pulmonary disease, Paul
outlined work to develop a hand-held ‘sniff er’ device
based on ion mobility MS to seek out people trapped as
a result of an earthquake, for example. It seems that not
only do specially trained sniff er dogs have a very short
working period (20 min or so) before needing prolonged
rest, but also suff er high mortality (up to 70 % was
quoted), hence there is a real need for such a device.
All-in-all the day was a fi tting tribute to Chang Kee Lim
and his contribution to the application of separation
science in biochemistry.
This report was compiled and written by
Bob Flanagan
26 meeting report www.sepscience.com
Register Now for your 20% Early Bird Discount Conference Highlights
Singapore
www.sepscienceasia.com
FoodEnviro
Day One:
Pat SandraAdvances in Separation Sciences Deriven by the Metabolomics and Proteomics Quest for Biomarkers
Manfred RaidaMultidimensional Gel-free Protein Separation Approaches for In-depth Analysis of Complex Proteomes
Y.S. Fung
Eric Chun Yong Chan
Yi ChenNew Approaches to Online Anti-salt Stacking for Direct Capillary Electrophoresis of Biosamples
Thomas WalczykElement Separation at the Microscale for High-Precision Isotopic Analysis of Biological Samples
Emily HilderOrganic Polymer Monoliths as Temperature Stable Stationary Phases for High Temperature Separations of Intact Proteins and Peptides
Andrew JennerGC-MS Analysis of Lipid Oxidation and Cholesterol Metabolism
C. Bor FuhImmunoassays Using Functional Magnetic Nanopaticles for Biochemical Analysis
Gurmil GendehOptimization Strategies in LC-MS/MS Front-End Separations to Achieve High Throughput, High Resolution and High Selectivity
Zhan ZhaoqiTools for Structural Elucidation & Metabolomics using IT-TOF Technology
Bioscience
sponsors:
For all delegate enquiries email [email protected]
26–28 AugustBiopolis Science Park, Singapore
Day Two:
Gert DesmetCurrent and Future Approaches to Speed Up HPLC Separations
John DolanPractical Tools for the Selection of Equivalent and Orthogonal HPLC Columns
Ronald E. MajorsHPLC Column Technology: Smaller and Faster
Phil NethercoteThe applictaion of Quality by Design Principles to Analytical Method Development, Validation and Transfer.
Sanjay GargThe Role of Analytical Science and Techniques in Early Phase Drug Discovery and Registration for Clinical Studies.
Vinayak NadigerPharmaceutical Analysis in Regulated Environments: Perspectives, Challenges and Insights
Phil DeLandThe Next Frontier: Doubling UHPLC Throughput for Pharmaceutical Applications
Edward BrowneBiomarker Analysis for Preclinical Pharmaceutical R&D
Anne GohOnline Solid Phase Extraction-LC-MS in DMPK Applications
Ping LiHPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines
Yizeng LiangSeparation Science for the Quality Control of Traditional Chinese Medicine
Tung-Hu TsaiMethods and Strategy of Microdialysis for Pharmacokinetics in Herbal Medicine
Day Three:
Alastair LewisTrace Pollutant Detection in Challenging Environments
Hian-Kee LeeSolvent-Minimized Sample Preparation for Separation Science
Siu Kwan SzeAn Advanced Proteomic Approach to the Discovery of Microbial Enzymes for
Gongke LiMolecularly Imprinted Polymers for Trace Analysis of Complicated Samples
Paul Haddad
Terrorist Explosives by Analysis of Inorganic Residues
Philip MarriottHeadspace Analysis of Plant Materials by Using Comprehensive Two-Dimensional Gas Chromatography: Selected Examples
Bahruddin SaadDetermination of Biogenic Amines in Food: Conventional and Nonconventional Approaches
Peter Gorst-AllmanComprehensive Two-Dimensional Gas Chromatography Time of Flight Mass Spectrometry (GCxGC-TOFMS) for Environmental Forensic Investigations in Developing Countries
Robert ShellieOne- and Two- dimensional GC-MS for Hop Metabolics
Jessie TongMultidimensional Gas Chromatographic Analyses of Flavours and Fragrances
Mike KindermanNew Developments for the Analysis of Inorganic and Organic Contaminants
Hui-Lai ChinPesticides Analysis by GPC-GCMS
Yuki HashiMelamine Analysis – New Approaches
Pharma TCM
Shawn StanleyUsing Narrow Bore Monolithic LC Columns to Enhance Analytical Sensitivity in Equine Doping Control
CdThe Chrom
Doctor
Techniques for optimizing normal- and reversed-phase flash purification
Traditional flash purification techniques can
prove challenging and time-consuming
for chemists, and many organic synthesis
laboratories are now looking to new
methods to achieve better separations.
This article will examine the parameters
for achieving effective results from flash
chromatography and will discuss method
development examples to illustrate how to
get the most out of flash chromatography.
Setting purification goalsTo effectively use flash chromatography
chemists need to define purification goals.
1. Purity (how pure do I need my product?)
2. Yield (how much product do I need to
isolate?)
3. Speed/throughput (how fast do I need
the pure product?)
Understanding these goals will help with
creating an efficient purification method.
Purification modesFlash chromatography, like other liquid
chromatography techniques can be
performed in several different modes, the
primary modes being:
1. Normal-phase (polar stationary phase,
non-polar mobile phase)
2. Reversed-phase (non-polar stationary
phase, polar mobile phase)
Flash chromatography is a technique used by organic and natural product chemists to quickly purify chemical mixtures. In basic terms, the chemical mixture is passed through a purification cartridge containing a stationary phase (adsorbent) using solvents (mobile phase) to carry them through the cartridge. Compounds within the reaction mixture will interact with the stationary phase and separate.
Elution modesWithin the purification modes there are also
elution modes, which are selected based on
sample complexity. Simple separations can
typically be performed using isocratic elution
while more complex mixtures might require
gradient elution.
In isocratic elution, the mobile phase
strength and selectivity is kept constant
throughout the purification. With gradient
elution the solvent strength increases linearly
or in programmed steps throughout the
purification. Typically, modern flash systems
allow several solvents to be programmed
into a gradient providing a selectivity change
as well as an increase in solvent strength
to improve compound solubility or reduce
compound-silica interactions.
Method developmentFlash purification method development can
be very quick but several criteria must be
evaluated:
1. Solubility – in which solvents is the
sample mixture soluble? This will help
decide on the purification mode. If
soluble in non-polar solvents, normal-
phase is usually the best option, if
soluble in alcohols or water, reversed-
phase is typically best.
2. Chemistry – the target product’s
28 chrom doctor www.sepscience.com
functional groups can play an important
role in method development. For
example, an organic soluble
N-heterocycle may be
chromatographically problematic on
silica requiring a mobile phase of DCM/
MeOH/NH3 in normal-phase. However,
an amine-functionalized silica or
basic alumina will likely provide a better
separation using simpler, more
eco-friendly solvents such as heptanes/
EtOAc.
Once solubility and compound chemistry
have helped decide the chromatographic
mode, chromatographic method
development can begin.
Normal-phase method development: Thin
layer chromatography (TLC) is always a good
method development tool for normal-phase
chromatography. With TLC, it is very easy to
scout various solvent mixtures in parallel to
find the optimal separation conditions.
Compound retention on TLC, measured as Rf
(retention factor), is inversely proportional to
the number of column volumes (CV) required
to isocratically elute the same compound
with the same solvent on a flash cartridge, so
CV = 1/Rf. Retention is influenced primarily
by solvent strength whereas selectivity
(ΔCV), the relative retention difference
between any pair of eluting compounds,
is influenced by the solvent type. Solvent
pairings should be based on compound
solubility and solvent miscibility. Refer to
Table 1 for typical normal-phase solvents and
their relative strength and selectivity class.
To ensure accurate method transfer, TLC
plates should be coated with the same silica
as that packed in the flash cartridge. This
is because of differences in silica surface
chemistry between silica manufacturers,
primarily silanol type and ratio.
With silica there are three silanol types,
single (one OH on a single Si), geminal (two
OH groups on a single Si), and bridged
(H-bonded) [1]. The ratio of these three
silanols influences compound retention and
selectivity so developing a method on a
brand X TLC plates and transferring brand Y
flash cartridge may not provide the desired
or expected separation.
When the TLC plate elution studies have
finished, the Rf data need to analysed and
converted in CV. If the compound of interest
elutes between Rf 0.15 and Rf 0.35 (2.9-6.7
CV) and the ΔCV with its nearest eluting
neighbour is two or more, isocratic elution
should work well. However, gradient elution
is now state of the art and can produce
improved separations compared with
isocratic elution, see Figure 1.
Reversed-phase method development:
Method development in reversed-phase
is slightly different than normal-phase.
Although reversed-phase TLC plates
are commercially available, often the
hydrophobic media can have physical issues
when using aqueous mobile phases. These
issues include surface wetting and physical
stability on the solid backing material.
As most synthetic chemistry labs have
Table 1
Solvent Selectivity Class Strength
Methanol II 0.95
Ethanol II 0.88
IPA II 0.82
Acetonitrile VIb 0.65
Ethyl acetate VIa 0.58
THF III 0.57
Acetone VIa 0.56
DCM V 0.42
Chloroform VIII 0.40
Ether I 0.38
Toluene VII 0.29
Cyclohexane ---- 0.04
Hexane ---- 0.01
Heptane ---- 0.01
Iso-octane ---- 0.01
Table 1: Solvent selectivity class and solvent strength table
29chrom doctorseparation science — volume 1 issue 7
access to an HPLC, a better method
development technique is HPLC. Developing
methods using this approach allows for
gradient adjustment and loading studies to
be conducted. The same HPLC methods can
be used on fl ash systems with an increase
in fl ow rate and sample load. The fl ow and
load increases are based on the ratio of fl ash
media mass to method development column.
Making reversed-phase workThe key to getting reversed-phase fl ash
to provide retention and separation is
equilibration. Commercially available
fl ash cartridges are manufactured and
shipped dry. With normal-phase, although
equilibration is preferred, it is not required
because solvent easily passes through the
media’s pores. With reversed-phase, however,
the hydrophobic bonded phase impedes
wetting by aqueous solutions, signifi cantly
reducing the available surface for sample
partitioning.
Three steps to equilibration: Equilibrating
reversed-phase fl ash cartridges requires only
three steps.
1. 100% of the water-miscible organic
solvent (CH3CN, CH3OH, THF, etc.) for
5 CV
2. 50:50 organic/water (including any pH
adjusters) for 5 CV
3. Initial elution conditions for 5 CV
With proper equilibration, the reversed-
phase surface is “wetted” and the maximum
surface area for partitioning will be available.
Adjusting mobile phase pH: This is a very
important step to optimize resolution and
sample load. Because this is purifi cation and
not analysis, inorganic buff ers or modifi ers
should not be used. Instead, volatile buff ers,
acids and bases should be considered.
The key to increasing sample load in
reversed-phase is compound retention.
If trying to purify organic amines, the
mobile phase should have its pH adjusted
into the alkaline range to deprotonate the
compounds. Likewise, acidic samples should
be purifi ed under acidic conditions.
For basic compounds, adjust the mobile
phase pH to two units above the compound’s
pKa (if possible). Typical pH modifi ers include
MEA, DEA, TEA, and NH3 in methanol.
For acidic compounds, adjust the
mobile phase pH to two units below the
compound’s pKa (if possible). Typical pH
modifi ers include formic acid, acetic acid,
and TFA.
Examples of reversed-phase purifi cations
in pH-adjusted solvent systems are shown in
Figures 2 and 3.
ConclusionFlash chromatography can be a time-
consuming process; however, the techniques
discussed will help improve fl ash purifi cation
results by increasing compound load,
resolution and throughput. Through
Figure 1
AUAU
AU
Fractions
Fractions
Fractions
% S
tron
g%
Str
ong
% S
tron
g
1
0
2
3
1
0
2
3
1
0
2
3
1 5
1
1
5
0
6
12
18
24
30
36
42
48
0
7
14
21
28
35
42
49
56
0
4
8
12
16
20
24
28
Figure 1: A comparison of elution modes shows that using gradient elution can radically improve a separation creating more resolution and increased sample load potential. Top = isocratic, middle = linear gradient, bottom = step gradient.
30 chrom doctor www.sepscience.com
MORE SEPARATING POWERIntroducing a C18 with Extra Resolving Power
ACE C18-ARHPLC Columns
A C18 bonded phase with unique selectivity
Hydrophobic and aromatic “mixed mode” interaction Ultra low bleed for UV & LC/MS compatibility Excellent temperature and pH stability Stable in 100% aqueous mobile phasesGuaranteed reproducibility
Further Information:To receive your free copy of the newACE C18-AR technical brochurecontact your local distributor or requestyour copy at: www.ace-hplc.com or viaemail at: [email protected]
Choose ACE. Superior HPLC Columns.Be an ACE Chromatographer
®
ACE is a registered trademark ofAdvanced Chromatography Technologies
®
®
defi ning clear purifi cation goals, selecting
the right purifi cation and elution modes and
eff ectively developing the method, chemists
can achieve better separations with higher
purity and yield in signifi cantly less time.
Reference[1] U.D. Neue, HPLC Columns Theory, Technology, and
Practice, Wiley-VCH: New York, 1997, p.171
Figure 2
AU
Fractions
% S
tron
g
1 5 10
1
2
3
00
20
40
60
80
1001 2 3
Figure 2: Purifi cation of organic amines by reversed-phase. Cartridge, Biotage SNAP KP-C18-HS 12 g; Sample, (1) Dimethylamino antipyrine, (2) Phenylbenzyl amine, (3) Amitriptyline; Load, 120 mg; Solvents, (A) DI H2O with 0.1% TEA, (B) CH3CN 0.1% TEA; Gradient, 10% B for 12 mL, 10-100% B in 120 mL, 100% B for 24 mL; Flow rate, 12 mL/min.
Figure 3
AU
Fractions
% S
tron
g
1 5
1
2
3
00
20
40
60
80
1001 2 3
Figure 3: Purifi cation of organic acids by reversed-phase. Cartridge, Biotage SNAP KP-C18-HS 12 g; Sample, (1) Homovanillic acid, (2) Nicotinic acid, (3) 3-Nitrobenzoic acid; Load, 120 mg; Mobile phase, Acetonitrile/H2O/TFA (3:2:0.05), Flow rate, 8 mL/min.
32 chrom doctor www.sepscience.com
June 28 – July 2, 2009
34th International Symposium on High-Performance Liquid Phase Separations and Related TechniquesChairman: Prof. Dr. Christian Huber, Paris-Lodron University Salzburg
Separation Science
ArbeitsKreis
SCIENTIFIC PROGRAMME:
Advances in Liquid Phase Separation Technology Multidimensional and Hyphenated Techniques Fundamental Aspects of Separations Industrial Aspects of Separations Clinical and Pharmaceutical Analysis Life Sciences Food and Environmental Analysis
VENDOR EXHIBITION:
More than 50 companies on display
Contact: Gesellschaft Deutscher Chemiker e.V. Congress Team E-mail: [email protected]
ww
.hp
lc20
09.c
om
International Congress Center Dresden · Germany
is
Register now online on
www.hplc2009.com
AnApplication
notes
34 application notes www.sepscience.com
Forensic Toxicology Analysis of Non-derivatized Drugs in Urine by Automated Solid Phase Microextraction (SPME) GCxGC-TOFMS
Company: Leco
Summary: Comprehensive two dimensional gas
chromatography in combination with time-of-� ight mass
spectrometry detection (GCxGC-TOFMS) was used for
drugs of abuse analysis in urine without time consuming sample derivatization.
Methamphetamine, cocaine, diacetylmorphine, codeine, oxycodone, ecstasy,
acetylcodeine, monoacetylmorphine, hydrocodone, and LSD were identi� ed in this
research. This application presents experimental data from the forensics analysis
conducted by automated solid phase microextraction (SPME)-GCxGC-TOFMS.
Click here to view the application
On-Line Two-Dimensional Separation of Intact Proteins
Company: Dionex
Summary: While 2-D-electrophoresis is a powerful tool for protein separation,
it is di� cult to automate, and has limited utility for the analysis of many classes
of proteins. Multidimensional liquid chromatography of peptides, exempli� ed
by shotgun peptide analysis, is highly automated, but requires the proteolytic
digestion of the sample, which greatly increases sample complexity and therefore
the required resolution of subsequent separations. Furthermore, much of the
information on di� erential post-translational modi� cation and isoform expression is lost. The use of Dionex
ProPac and Dionex PepSwift polystyrene divinylbenzne (PS-DVB) columns allow improved resolution, as well
as rapid and automated separation of intact proteins from complex samples.
Click here to view the application
Forensic Toxicology Analysis of Non-derivatizedDrugs in Urine by Automated Solid PhaseMicroextraction (SPME) GCxGC-TOFMSJohn Heim • LECO Corporation; Saint Joseph, Michigan USA
Key Words: Non-Derivatized Drugs, Automated SPME, GCxGC-TOFMS
1. Introduction
2. Application Objectives
3. Experimental Conditions
4. Results and Discussion
Comprehensive two dimensional gas chromatography incombination with time-of-flight mass spectrometrydetection (GCxGC-TOFMS) was used for drugs of abuseanalysis in urine without time consuming samplederivatization. Methamphetamine, cocaine, diacetyl-morphine, codeine, oxycodone, ecstasy, acetylcodeine,monoacetylmorphine, hydrocodone, and LSD wereidentified in this research.
This application presents experimental data from theforensics analysis conducted by automated solid phasemicroextraction (SPME)-GCxGC-TOFMS. A multiple drugstandard mixture prepared from Sigma-Aldrich standardswas spiked at concentrations from 10 to 1000 ppb into8mL aliquots of urine. Hexachlorobenzene (HCB) wasadded as an internal standard at a concentration of500 ng/mL. Automated solid phase microextraction(SPME) sample preparation was conducted on the non-derivatized spiked urine standards using a GerstelMPS2 autosampler equipped with a SPME prepstationfollowed by thermal desorption of the extracted samplein the GC injection port.
The data from this research will show the identification oftargeted analytes in very complex sample matrices. Theuse of automated SPME applied to non-derivatized urinesamples coupled with comprehensive two dimensionalchromatography and time–of–flight mass spectrometrydetection demonstrates this is a favorable technique forqualitative and quantitative analysis for drug screening inurine.
• Demonstrate the detectability of non-derivatized drugsin complex sample matrices such as urine byautomated SPME combined with GCxGC-TOFMS.
• Show calibration linearity capabilities over the range of10 to 1000 ng/mL for the forensic application of drugscreening in urine.
• Illustrate the feasibility of this analysis for non-derivatized drugs in urine by SPME-GCxGC-TOFMSdescribing the advantages of multidimensionalchromatography (GCxGC) and time-of-flight massspectrometry (TOFMS).
Aliquots of urine spiked with a drug standard mixture atconcentrations from 10, 50, 250, 500, and 1000 ng/mLwere prepared and analyzed without derivatization.Hexachlorobenzene was added to each sample as aninternal standard (ISTD) at 500 ng/mL prior to extraction.Each sample was placed in a 10 mL glass SPMEautosampler vial and sealed. Automated SPME extractionwas conducted using the Gerstel MPS2 Prepstation.
GCxGC-TOFMS results were generated with a LECOPegasus 4D GCxGC-TOFMS equipped with a GerstelMPS2 autosampler and a SPME prepstation. The Pegasus4D instrument was equipped with an Agilent 7890 gaschromatograph featuring a LECO quad jet dual stagethermal modulator and secondary oven. LECOChromaTOF software was used for all acquisition controland data processing. The autosampler was a single railCTC Combi Pal equipped with SPME sample agitator/prepstation and SPME fiber conditioning station.Automated sample extraction was performed using a50/30 µm DVB/Carboxen™/PDMS Stable Flex SPME fiber.The SPME method was developed in the ChromaTOFsoftware autosampler methods section using the CTCCombi PAL option. The sample agitator was set to ON at aspeed of 200 rpm and extraction temperature of 37 C. Thesolid phase microextraction time was set for 30 minutesand sample desorption time in the GC injection port was 2minutes. The fiber was then conditioned in the fiberbakeout station at 270 C for 40 minutes prior to concurrentsample extraction for the next analysis.
A 30 m x 0.25 mm x 0.25 µm film thickness, Rxi-5ms,(Restek Corp) GC capillary column was used as the primarycolumn for the analysis. In the GCxGC configuration, asecond column 1.5 m x 0.18 mm id. x 0.18 µm filmthickness Rtx-200, (Restek Corp) was placed inside theLECO secondary GC oven after the thermal modulator.Helium carrier gas flow rate was set to 1.5 mL/min at acorrected constant flow via pressure ramps. The primarycolumn was programmed with an initial temperature of40 C for 2 minutes and ramped at 6 C/minute to 290 C for10 minutes. The secondary column temperature programwas set to an initial temperature of 50 C for 2 minutes andthen ramped at 6 C/minute to 300 C with a 10 minutehold time. The thermal modulator was set to +25 Crelative to the primary oven and a modulation time of 5seconds was used. The MS mass range was 45-550 m/zwith an acquisition rate of 200 spectra per second. The ionsource chamber was set to 230 C and the detector voltagewas 1650V with an electron energy of -70eV.
Three major points will be discussed including thedetectability of non-derivatized drugs in the complexsample matrix urine. The calibration linearity over therange of 10 to 1000 ng/mL will be shown along with thefeasibility of this analysis for non-derivatized drugs in urineby SPME-GCxGC-TOFMS.
®
®
o
o
o o o
o
o o
o
o
®
Life
Sci
ence
and
Che
mic
al A
naly
sis
Solu
tions
35application notes separation science — volume 1 issue 7
Multiplexed Quantitative Peptide Assays for Protein Biomarkers of Cardiovascular Disease in Human Plasma
Company: Applied Biosystems
Summary: As the study of protein biomarkers increases in importance, extensive
lists of candidate markers are being developed based on protein pathway
information and discovery-based proteomics experiments. Even larger numbers
of candidate genomic markers exist from DNA transcriptional pro� ling based on
microarray-based gene expression studies or other genomic information. This
application note demonstrates the speci� city, sensitivity and reproducibility of this
MRM based strategy in human plasma.
Click here to view the application
Overview
As the study of protein biomarkersincreases in importance, extensive listsof candidate markers are beingdeveloped based on protein pathwayinformation and discovery-basedproteomics experiments. Even largernumbers of candidate genomicmarkers exist from DNAtranscriptional profiling based onmicroarray-based gene expressionstudies or other genomic information.
While these genomic markers can bevalidated at the RNA level using realtime PCR assays such as TaqMan® kits,and at the protein level using Westernblots, validation at the protein levelrequires specific antibodies to eachprotein which is a very time consumingand expensive task if hundreds ofmarkers are of interest. A much higherthroughput and more universal strategyis needed to narrow the huge numberof candidate biomarkers that are beinggenerated by both genomic andproteomics today at the protein level.The MIDAS™ workflow combinedwith multiplexed high throughputquantitative MRM assays on aMIDAS™ TRAQ system addressesthese challenges.
Many technical challenges exist in thepreliminary validation of putativeprotein biomarkers, such as thedetection of low abundance proteinsin complex tissue or biological fluids,
as well as high-throughput, highprecision quantitation. Validation ofthese markers for clinical use willrequire analysis of extensive candidatemarker panels in thousands of clinicalserum and plasma specimens. In thisstudy, an assay was developed usingmultiple reaction monitoring (MRM)and MIDAS™ workflow on theMIDAS™ TRAQ system to detect andquantitate selected tryptic peptides,where each peptide represents aspecific candidate protein biomarkeridentified for cardiovascular disease.
This application note demonstrates the specificity, sensitivity andreproducibility of this MRM basedstrategy in human plasma.
A number of strategies wereemployed to develop high qualityMRM methods to obtain the bestsensitivity and dynamic range forpeptides from a panel of putativeprotein biomarkers for cardiovasculardisease. For candidate proteinspreviously detected in proteomicsexperiments, MRMs were designedbased on peptide MS/MS spectra.For the remaining candidates,MRMs were developed either by insilico design, based on gene orprotein sequences, or by targeted,direct detection in plasma using theMIDAS™ workflow.
Multiplexed Quantitative Peptide Assays for Protein
Biomarkers of Cardiovascular Disease in Human Plasma
Application Note LC/MS
www.appliedbiosystems.com
Figure 1. Dynamic range of protein abundance in plasma is extremely large1 (inset, blue). This multiple reactionmonitoring (MRM) multiplexed assay profiles 53 plasma proteins (137 MRM transitions) over the first 5 orders ofabundance with minimal sample preparation.
HPLC and UHPLC Methods for Melamine
Company: Grace Davison
Discovery Sciences
Summary: Grace has
developed MS-compatible
LC methods for melamine
using an HILIC media
platform that can be applied
to both traditional HPLC
as well as UHPLC systems. Melamine was analysed
with a standard HPLC system using a 5 μm particle
HILIC phase packed into a 250 x 4.6 mm column.
The 1.5 μm version of this phase was then packed
into a high throughput format conducive to UHPLC
and fast LC systems. Both methods deliver excellent
linearity and use isocratic elution for fast analysis
without the need for re-equilibration.
Click here to view the application
Grace Davison Discovery Sciences
HPLC and UHPLC Methods for Melamine
Milk, infant formula and other dairy products were recently found contaminated with melamine, following an earlier melamine contamination outbreak in pet food in 2007.
Toxicology studies show ingestion of melamine in large quantities may lead to reproductive damage or bladder cancer due to the formation of bladder/kidney stones. At lower levels, melamine and cyanuric acid are absorbed into the bloodstream. Together, they concentrate and form melamine cyanurate in the urine-filled renal microtubules. Cystallization blocks and damages the renal cells that line the tubes, causing the kidneys to malfunction.
After 2007 and more recent melamine contamination outbreaks, there is an urgent need for analytical methods that can identify and quantify melamine in food. Current melamine detection methods involve LC-MS and GC-MS. GC-MS methods require derivatization, and LC-MS methods generally use gradient conditions that require column clean up and re-equilibration.
Grace has developed MS-compatible LC methods for Melamine using an HILIC media platform that can be applied to both traditional HPLC as well as UHPLC systems. Melamine was analyzed with a standard HPLC system using a 5µm particle HILIC phase packed into a 250 x 4.6mm column. The 1.5µm version of this phase was then packed into a high throughput format conducive to UHPLC and fast LC systems. Both methods deliver excellent linearity and use isocratic elution for fast analysis without the need for re-equilibration.
1
0 2.5 5 7.5 10 12.5 15 17.5 20 Min.0
0.5
1.0
1.52.0mV(x100)
N N
NH2
NH2H
2N N
N N
OH
OHHO N
Melamine
Cyanuric Acid
HPLC Method for Melamine
HPLC Column: Alltima™ HP HILIC, 5µm, 4.6 x 250mm (Part No. 86466)Mobile Phase: Acetonitrile:10mM Ammonium Acetate in Water (95:5)Flow Rate: 1mL/minDetection: UV@240nmColumn Temperature: 30ºCInjection: 40µg/mL x 20µL
0 20 40 60 10080 1200
2
4
6
Peak
Are
a (x
106 m
AU x
sec
.)
Sample Weight (µg)
8
10
16
12
14
1
0 1 2 3 4 5 Min.
-1
123
mAU
0
45
UHPLC Method for Melamine
UHPLC Column: VisionHT™ HILIC, 1.5µm, 2 x 50mm (Part No. 5141919)Mobile Phase: Acetonitrile:10mM Ammonia Acetate in Water (95:5)Flow Rate: 0.2mL/minDetection: UV@240nmColumn Temperature: 30ºCInjection: 50µg/mL x 0.5µL
Compared to the conventional HPLC method, the UHPLC method is 4 times faster. With the use of 1.5μm particles, optimal linear velocities extend over a wider range. Therefore, it is possible to maintain efficiency and resolution while running samples at faster flow rates.
0 0.5 1 1.5 2 2.5 3 Min.
0
10
20
30
40
UHPLC Column: VisionHT™ HILIC, 1.5µm, 2 x 50mm (Part No. 5141919)Mobile Phase: Acetonitrile:Water(20mM Ammonium Formate) (90:10)Flow Rate: 0.2mL/minDetection: UV@240nmColumn Temperature: 30ºCInjection: 50µg/mL x 0.1µL
0 200 400 600
Sample Weight (ng)
Area
(mAU
x s
ec.)
1000800 12000
200
400
600
800
1000
1200
1400
y = 146800x + 87120R2 = 0.998
9 injections in parallel shows good reproducibilityConc.
Inj. (µL)
Weight(ng)
PeakArea
50µg/mL 0.1 5 7.3
50µg/mL 0.5 25 32
50µg/mL 1 50 63
50µg/mL 5 250 318
50µg/mL 10 500 641
50µg/mL 20 1000 1293
y = 1.2924x - 1.8252R2 = 1.000
This HPLC analytical method for melamine fulfills the FDA requirements using a HILIC column and an ionizable mobile phase compatible with mass spec. Low UV detections offers excellent linearity between 40ng and 100µg.
This method exhibits excellent linear response between 5ng and 1000ng for accurate quantitation.
36 technology update www.sepscience.com
Carbohydrates, Organic Acids and Alcohols in Wine
Company: Bischo�
Summary: Bischo� o� ers an application booklet desribing a series of separations
using its ProntoSIL family of columns. Applications include Carbohydrates, Organic
Acids and Alcohols in Wine, Fast Analysis of a pharmaceutical Test standard with
Multiwavelength Detection, Fast Analysis of Parabens II with Multiwavelength
Detection, Aromatic Hydrocarbons according to prEN 12916, Ergosterol in Grass
and Acetaldehyde in Mineral water.
Click here to view the application
Carbohydrates, Organic Acids and Alcohols in Wine
Carbohydrate H+
Part. Number: 00253776Dimension: 300 x 7.8 mmEluent: 1.25 mM H2SO4Flow: 0.6 ml/minDetection: RITemperature: 45 °CInjection: 50 µlSample: Wine Complett Standard
no. 1
51
etha
nol
~
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 min
22 mV
citric
tarta
ricgl
ucos
em
alic
fruct
ose
succ
inic lac
ticgl
ycer
ol
acet
ic
2,3-b
utan
diol
Analysis of Pesticide Residues in Apple using Agilent SampliQ QuEChERS European Standard EN Kits by LC/MS/MS Detection
Company: Agilent
Summary: This application note describes the use of a quick, easy, cheap, e� ective,
rugged, and safe (QuEChERS) sample preparation approach described in the
European Committee Standard (EN) for extraction and cleanup of 16 multiple
class pesticide residues of interest in apple. The method employed involves initial
extraction in an aqueous/acetonitrile system, an extraction/partitioning step
after the addition of salt, and then a cleanup step utilizing dispersive solid phase
extraction (dispersive SPE).
Click here to view the application
Analysis of Pesticide Residues inApple using Agilent SampliQQuEChERS European Standard EN Kitsby LC/MS/MS Detection
Abstract
This application note describes the use of a quick, easy, cheap, effective, rugged, and
safe (QuEChERS) sample preparation approach described in the European Committee
Standard (EN) for extraction and cleanup of 16 multiple class pesticide residues of
interest in apple. The method employed involves initial extraction in an aqueous/ace-
tonitrile system, an extraction/partitioning step after the addition of salt, and then a
cleanup step utilizing dispersive solid phase extraction (dispersive SPE). The two dif-
ferent dispersive SPE clean-up approaches (1 mL and 6 mL sample volume) are evalu-
ated simultaneously after sample extraction. The target pesticides in the apple
extracts are then determined by liquid chromatography coupled to an electrospray ion-
ization tandem mass spectrometer (LC-ESI-MS/MS) operating in positive ion multiple
reaction monitoring (MRM) mode. The method is validated in terms of recovery and
reproducibility. The 5 ng/g of limits of quantitation (LOQ) for pesticides in apple estab-
lished in this application is well below their regulatory maximum residue limits
(MRLs). The spiking levels for the recovery experiments are 10, 50, and 200 ng/g.
Excluding pymetrozine, recoveries of the pesticides ranged between 73 and 111%
(87% on average), and RSDs below 20% (5.8% on average).
Authors
Limian Zhao, David Schultz, and
Joan Stevens
Agilent Technologies, Inc.
2850 Centerville Road
Wilmington, DE 19809
USA
Application NoteFood Safety
separationdriving analytical chemistry forwardsscience
Get your FREE subscription to
Separation Scienceonline. . .
technical articles on chromatography?
updates on recent research studies?
practical advice on routine analysis?
applications of new technology?
information on product developments?
market trends and opinions?Volume 1 / Issue 3
March 2009
www.sepscience.com
Coupling capillary columns in gas
chromatography
Analytical trends in iso�avone
studies
Minimizing downtime in QA
pharmaceutical laboratories
separation driving analytical chemistry forwardsscience
Volume 1 / Issue 1
Febraury 2009
www.sepscienceasia.comwww.sepscienceasia.com
Volume 1 / Issue 1
separation driving analytical chemistry forwardsscience
Volume 1 / Issue 2
February 2009
www.sepscienceasia.com
Exploiting particle size to reduce
acetonitrile consumption
Multiresidue analysis using SBSE
and GC-MS/MS
Chromatographic methods for
Con�rming biological activity
markers in fruit
Asia Paci�c
separation driving analytical chemistry forwardsscience
Volume 1 / Issue 2
April 2009
www.sepscienceasia.com
固相微萃取-高效液相色谱分析羟烷基喹诺酮
固相萃取和气相色谱与三级四极杆质谱联用测
定熟食品中痕量食品衍生的有害物质
液相色谱填料尺寸对降低
溶剂消耗的影响
中国版
Volume 1 / Issue 5
May 2009
www.sepscience.com
A liquid chromatographer’s
introduction to mass spectrometry
Analysing synthetic polymers with
solvent enhanced light scattering
Minimizing decomposition of
components during GC analysis
separation driving analytical chemistry forwardsscience
Volume 1 / Issue 5
May 2009
www.sepscienceasia.com
RP-HPLC determination
of anti-malarials
A liquid chromatographer’s
introduction to mass spectrometry
Minimizing decomposition of
components during GC analysis
separation driving analytical chemistry forwardsscience
37Technology update separation science — volume 1 issue 3
New Kromasil® Eternity™
Designed for long life
How old can a column get? It’s natural to think it depends on how wide the pH variations are or how high the temperature gets. But even under such tough conditions, Kromasil® Eternity™ lives longer than other columns.
It’s all about being designed for long-term survival.
Kromasil® Eternity™ has an organic/inorganic interfacial gradient cover ing the silica, ensuring long life for the column. Check out our website for more information and continuous updates. www.kromasil.com
has
Kromasil®
Eka_Ad_longlife_190x272.indd 1 09-06-02 10.19.03
TuTechnology
update
38 technology update www.sepscience.com
Key
Email the company
Product information
Applications
Additional information
Oligonucleotide separation technology columns
Manufacturer: Waters
Manufacturer’s description: Oligonucleotide Separation Technology (OST) columns contain
second-generation hybrid-silica BEH Technology particles functionalized with C18. The
separation of detritylated synthetic oligonucleotide samples is based on the well-established
method of ion-pair, reversed-phase chromatography.
This chemistry delivers exceptional sample resolution and superior column life. In addition,
Waters manufacturing and quality control testing procedures help ensure consistent batch-
to-batch and column-to-column performance regardless of application demands.
The availability of 1.7 μm UPLC technology or 2.5 μm HPLC particles in various column
dimensions gives you the flexibility to meet various lab-scale isolation or analysis needs.
Features include:
• Scalable reversed-phase HPLC columns for lab-scale purifications
• Increased sample through-put with maintained component resolution with UPLC
Technology
• LC/UV and LC/MS methods for enhanced quantitative and qualitative analyses
• Exceptional column life using Waters patented Ethylene-Bridged (BEH Technology) Hybrid `
particles
39technology update separation science — volume 1 issue 7
separationdriving analytical chemistry forwardsscience
www.sepscience.com
Nitrogen generator
Manufacturer: Parker Hannifi n
Manufacturer’s description: The Parker Balston membrane nitrogen generators can produce up to 75 lpm of pure LC/
MS grade nitrogen at pressures up to 8 bar. Generators are complete systems engineered to transform standard
compressed air into a safe regulated nitrogen supply with minimal operator attention, according to the company.
Typical applications include LC/MS, LC/MS/MS, nebuliser gases for APCI and ESI, ELSD, Turbo Vaps and chemical
solvent evaporation. The membrane nitrogen generators have been tried and tested by all the major LC/MS
manufacturers.
Features
• Recommended and used by all major LC/MS manufacturers
• No electrical requirement, no noise and no moving parts
• Can supply up to 3 LC/MS from one generator
• Utilises Parker’s propriety membrane technology
• Phthalate-free, no organic vapours
• Pliminate inconvenient nitrogen dewars from the laboratory
separation driving analytical chemistry forwardsscience
separationdriving analytical chemistry forwardsscience
separation driving analytical chemistry forwardssciencedriving analytical chemistry forwardsseparation
THE NEW SEPARATION SCIENCE REPORTS ARE NOW
AVAILABLE...
separation scienceseparation sciencescienceseparationseparationiPod Touch
Recommend your colleague(s) for a free
subscription to Separation Science Reports and enter a
1of 5draw to win
Keep up to date with the latest analytical methods and
chromatographic applications in the Pharmaceutical, Environmental
or Food science Industries by taking a FREE subscription to the
Separation Science Reports.
October 2008
Enviro ReportOrganochloride analysis using graphitized carbon black
Automated Disposable Pipette Extraction of Pesticides from Fruits and Vegetables
WWEM 2008 Preview
Featured Books
Pharma ReportCharacterization of drug metabolites in epidemiological studies
Interview with Karsten Fjärstedt, GE Healthcare
SPME in high-throughput drug analysis
EDQM Symposium: Pharmaceutical Reference Standards
October 2008
November 2008
Food ReportMSPD-GC-ECD analysis of lindane residues
A novel sample treatment for the LC-MS detection of peanut protein in foods
Evaluating chondroitin sulfate in dietary supplements
Screening for mycotoxins with LC-MS/MS
Please turn the pages using the control panel below
Recommend your colleague(s) for a free
subscription to Separation Science and enter a draw to win
1of 5
iPod Touch
40 technology update www.sepscience.com
Robotic Soliprep – new automation possibilities in ion chromatography
Manufacturer: Metrohm
Manufacturer’s description: Pressure to increase output while reducing
costs requires laboratory automation wherever possible. Metrohm’s
new 815 Robotic Soliprep meets such demands as it allows to
completely automate preparation and analysis of liquid and solid
samples in ion chromatography. The 815 Robotic Soliprep provides
maximum fl exibility as it can be customized on demand to meet
individual customers’ specifi c requirements, according to Metrohm.
The 815 Robotic Soliprep is a state-of-the-art sample processor,
which reliably transfers both liquid and solid samples to an ion
chromatograph. A broad range of working steps such as pulverizing,
homogenizing, extracting, fi ltering or diluting can be safely entrusted
to the 815 Robotic Soliprep and included in the analysis method,
the company states. Due to the incorporation of inline sample
preparation into the analysis system, manual intervention is no
longer required during the determination. The sample rack of the 815
Robotic Soliprep is equipped with various inserts, sample beakers,
syringe fi lters and needles and can be customized on demand to
meet individual customers’ specifi cations. Combined with Metrohm’s
advanced ICs the 815 Robotic Soliprep opens up numerous new
applications and application fi elds. Applications include, to mention
just a few, the analysis of ingredients and contaminants in tablets,
animal feed, sediments or food.
41technology update separation science — volume 1 issue 7
7100 Capillary Electrophoresis system
Manufacturer: Agilent Technologies
Manufacturer’s description: Agilent Technologies has introduced its next generation Agilent 7100 Capillary
Electrophoresis (CE) system, which it states provides at least 10 times more sensitivity than any other commercial CE
instrument.
CE currently attracts attention because the technique uses very small amounts of solvent. The new 7100 system also
requires 25% less bench space, weighs 30% less than its predecessor and uses less power, according to Agilent. The
superior sensitivity is a result of a new detector used in combination with proprietary extended light path capillaries
or a high-sensitivity cell.
The Agilent 7100 offers a wide selection of detectors for flexibility and sensitivity, and the new instrument is
reverse-compatible to the previous Agilent CE platform, so existing methods can continue to be used.
Agilent confirms the instrument performs the full range of CE separation techniques, including capillary
electrochromatography for fast separation of closely related compounds. Its standard replenishment system provides
high-throughput for unattended operation, and has been improved to use less buffer for the replenishment function.
The Agilent 7100 CE system was designed to enhance productivity, reliability and ease of use. The new, rugged
internal pressure system and improved capillary cooler supports higher currents and/or larger capillary diameters
to increase throughput and also to enable a wider range of applications. In addition, the system comes with a new
Chemstation software with an easy-to-use graphical user interface and an improved method setup that minimizes
start-up and training time.
The system’s modular architecture allows fast, easy access to electrodes, prepunchers, electronics and tubing to
facilitate routine maintenance and servicing. The quick-change, self-aligning capillary cartridge can be changed out
in seconds. It is compatible with all
commercially available capillaries.
The Agilent 7100 CE provides
plug-and-play connectivity to Agilent
mass spectrometers (MS), combining the
short analysis time and high separation
efficiency of CE with the molecular
weight and structural information of MS.
These include the single quadrupole,
time-of-flight, ion trap, triple quadrupole,
ICP and quadrupole time-of-flight MS
systems.
www.sepscience.com
Varian 385-LC ELSD
Manufacturer: Varian
Manufacturer’s description: Evaporative light scattering detectors are ideal for detecting analytes with no UV
chromophore as they do not rely on the optical properties of a compound. The Varian 385-LC Evaporative Light
Scattering Detector is the latest high performance ELS detector from Varian, with almost 20 years’ experience in the
design and manufacture of evaporative light scattering detectors.
The Varian 385-LC Evaporative Light Scattering Detector is the only ELSD that delivers sub-ambient operation,
for unrivalled detection of thermally labile analytes, according to the company. Designed for the analysis of all
compounds, the Varian385-LC delivers evaporation down to 10°C, providing maximum sensitivity for compounds with
significant volatility below 30°C. Programmable control of gas flow and temperature during an injection eliminates
solvent gradient effects and maximizes response for improved accuracy. To detect everything you inject with high
sensitivity, the Varian 385-LC is the ELSD of choice for all applications.
Key Benefits
• High sensitivity provides superb responses for all compounds, down to low nanogram levels.
• Sub-ambient operation using a Peltier cooled evaporation tube delivers temperatures down to 10°C, preventing
degradation of the thermally labile compounds that other ELSDs cannot detect.
• Real-time control during an injection via programmable Dimension Software maintains maximum sensitivity
throughout the run.
• Real-time gas programming eliminates solvent enhancement effects during gradient elution, for excellent
quantification.
• Low dispersion and high-speed data output rates are the perfect match
for Fast LC applications.
• Superb reproducibility below 2% gives reliable and accurate results.
• Multi-vendor software control and data acquisition using Varian’s
Galaxie chromatography data system, and other vendors’ interfaces,
eliminates the need for an analog to digital converter.
• Rapid heating and cooling of the evaporator tube minimizes
equilibration time and increases sample throughput.
• Full DMSO transparency ensures that responses from early eluting
compounds are not hidden.
• Fully integrated with the Varian 920-LC analytical HPLC system for the
complete chromatographic solution.42 technology update
analytica Anacon India is part of the exhibition network analytica worldwide—the most important marketplacein the world for marketable products and solutions in the analysis, laboratory-technology and biotechnologysectors. It is a business and networking platform and a driving force behind future trends and innovations.
www.analyticaindia.com
analytica Anacon India SEPT. 29–OCT. 01, 2009HYDERABAD
LOOK TOTOMORROW
analytica MARCH 23–26, 2010MUNICH
analytica VietnamSPRING 2011
analytica ChinaSEPT. 15–17, 2010SHANGHAI
AnaInd_NetAd_210x297_E.qxd 03.04.2009 10:10 Uhr Seite 1
44 technology update www.sepscience.com
Axima MALDI-TOF MS
Manufacturer: Shimadzu
Manufacturer’s description: Axima Confidence offers powerful MALDI-TOF performance for reliable mass information
and MS/MS derived structural detail, according to Shimadzu. Its linear mode allows the interrogation of high
molecular weight samples, whilst reflectron mode, incorporating the patented curved-field reflectron (CFR), provides
the high resolution and mass accuracy necessary for successful proteomics and life science experiments. It is a high
sensitivity system using a variable repetition rate 50 Hz N2 laser and a variety of target formats to meet all sample
throughput requirements.
The company states it is an affordable robust option for all laboratories requiring routine manual or automated
analysis of a wide variety of sample classes, with excellent sensitivity achieved using near-axis laser irradiation and
advanced ion optics for enhanced ion transmission.
MS/MS may be easily performed using a seamless approach – ions of interest can be isolated using a precursor
ion selection device, incorporated as standard, and data-rich fragment ion spectra quickly and simply acquired.The
newly improved curved field reflectron design augments the low mass fragment region providing useful additional
information.
Key Performance Features
• Near-axis laser irradiation with a 50Hz variable repetition rate N2 laser
• Patented improved curved field reflectron
• Mass range 1-500kDa in linear mode; 1-80kDa in reflectron mode
• Mass resolution 5000 FWHM (ACTH 18-39) in linear mode; 15000 FWHM
(ACTH 7-38) in reflectron mode
• Sensitivity 500 amol (Glu-fibrinopeptide) in reflectron mode
• Mass accuracy <10ppm in reflectron mode with internal calibration
• Precursor ion resolution >200 FWHM
• Seamless MS/MS utilising PSD
• Flexible target formats - ranging from 48 well microscope slide format
targets to 384 well microtitre plate targets
45Technology update separation science — volume 1 issue 7
IntelliTarget
Manufacturer: ACD Labs
Manufacturer’s description: New LC/MS and GC/MS deconvolution software makes confirming the presence of known
compounds in messy samples faster and easier. The new software enables the target analysis of complicated LC/MS
and GC/MS datasets, without the tedious and difficult manual interpretation. ACD/IntelliTarget quickly confirms the
presence or absence of known compounds in samples, even at low concentrations, or when obscured by the presence
of other substances.
ACD/IntelliTarget quickly and accurately deconvolutes LC/MS and GC/MS data and presents a concise report
indicating whether your compounds of interest are ‘Found’ or ‘Not Found’. Specify just one compound or a list of
hundreds, either by formula or by monoisotopic mass. ACD/IntelliXtract then extracts and identifies the ionized
molecule and adducts for each component, even when compounds are present at low concentration, or co-eluting
with other species. ACD/IntelliTarget is an essential tool to ease the workload of experienced spectroscopists, and for
inexperienced users who may find the manual interpretation of data to be overwhelming.
ACD/IntelliTarget is intended for screening, monitoring, and focusing analytical efforts in many fields of study,
including forensic science, environmental monitoring, water analysis, food safety, and drug discovery. It is compatible
with data formats of most major instrument vendors, and can be automated to screen larger batches of samples, or to
monitor samples on-the-fly.
ACE C18-AR - a C18 bonded HPLC column offering alternate selectivity
Manufacturer: Advanced Chromatography Technologies
Manufacturer’s description: ACE C18-AR is a unique C18 bonded HPLC column from Advanced Chromatography
Technologies, which has been developed to provide alternate selectivity to all existing C18 bonded phases. The phase
combines the hydrophobic characteristics of a C18 phase with the enhanced aromatic selectivity of a phenyl phase,
enabling the benefits of both interactions to be fully exploited. According to the company the phase also exhibits
ultra low bleed to ensure LC/MS compatibility and demonstrates hight temperature and pH stability. Additionally, the
ACE C18-AR is suitable for use in highly aqueous (up to 100%) mobile phases to enable the retention and separation
of polar compounds. At launch, materials are available in 3 µm, 5 µm and 10 µm particle sizes and a full range of
column dimensions from high throughput through to preparative scale.
sepa on scienc
technical articles on chromatography and related technologies?
updates on recent research studies?
practical advice on routine analysis?
applications of new technology?
information on commercial product developments?
market trends and opinions?
... then get your FREE subscription to
separationdriving analytical chemistry forwardsscience
dsencon scaration
driving analylyl ticticti al chemistcal chemistc ral chemistral chemist y ry r fofof rwawaw rdsrdsrscienceenal chemistrry ritarasepa atioatioaratio
iPod Touch
Recommend your colleague(s) for a free
subscription to Separation Science and enter a draw to win
1of 5
cecece
Volume 1 / Issue 3
March 2009
www.sepscience.com
Coupling capillary columns in gas
chromatography
Analytical trends in iso�avone
studies
Minimizing downtime in QA
pharmaceutical laboratories
separation driving analytical chemistry forwardsscience
Volume 1 / Issue 1
Febraury 2009
www.sepscienceasia.com
液相色谱-质谱联用新方法的建立
微波辅助溶剂萃取与气相色谱-质谱
联用分析太子参中的挥发物
微芯片电泳用于生物医学
分析
separation driving analytical chemistry forwardsscience
Volume 1 / Issue 2
February 2009
www.sepscienceasia.com
Exploiting particle size to reduce
acetonitrile consumption
Multiresidue analysis using SBSE
and GC-MS/MS
Chromatographic methods for
Con�rming biological activity
markers in fruit
Asia Paci�c
separation driving analytical chemistry forwardsscience
Volume 1 / Issue 2
April 2009
www.sepscienceasia.com
固相微萃取-高效液相色谱分析羟烷基喹诺酮
固相萃取和气相色谱与三级四极杆质谱联用测
定熟食品中痕量食品衍生的有害物质
液相色谱填料尺寸对降低
溶剂消耗的影响
中国版
Volume 1 / Issue 5
May 2009
www.sepscience.com
A liquid chromatographer’s
introduction to mass spectrometry
Analysing synthetic polymers with
solvent enhanced light scattering
Minimizing decomposition of
components during GC analysis
separation driving analytical chemistry forwardsscience
Volume 1 / Issue 5
May 2009
www.sepscienceasia.com
RP-HPLC determination
of anti-malarials
A liquid chromatographer’s
introduction to mass spectrometry
Minimizing decomposition of
components during GC analysis
separation driving analytical chemistry forwardsscience