separation science
driving analytical chemistry forwards
Mercury and arsenic speciation analysis by IC-ICP/MS
Comparing TLC methods for constructing medicinal plant fingerprints Determining carcinogenic aromatic amines in cigarette smoke by LC-MS/MS
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separation science
driving analytical chemistry forwards
separation science
driving analytical chemistry forwards
Mercury and arsenic speciation analysis by IC-ICP/MS
contents
Comparing TLC methods for constructing medicinal plant fingerprints Determining carcinogenic aromatic amines in cigarette smoke by LC-MS/MS
Volume 1 / Issue 6 May 2009
www.sepscience.com
Rr
Volume 1 / Issue 6 May 2009
features
06
research round-up 06 A CTAB-based method for the proteomic analysis of wine spoilage microrganisms
08 Rapid and sensitive determination of
16 Mercury and arsenic speciation analysis by IC-ICP/MS Laura Reyes, H.M. Skip Kingston, Andrea Wille, Jürgen Knöll and Alfred Steinbach
six carcinogenic aromatic amines in cigarette smoke by LC-MS/MS
09 HPLC assays for desmethoxyyangonin,
methysticin, kavain and their microsomal metabolites
10 Thermodynamic study of salbutamol interaction with an immobilized β2-adrenoceptor by HPLC
12 Pharmaceutical quality control by
22 A comparison of TLC methods for constructing medicinal plant fingerprints Łukasz Cieśla and Monika Waksmundzka-Hajnos
micellar electrokinetic chromatography
13 Reversed-phase chiral LC–MS/MS
quantitation of reboxetine enantiomers in rat plasma and brain
14 Preparation and ion chromatographic
properties of a new core-shell chromatographic support Al2O3/SiO2-10
15 Detection of Salmonella enteriditis from egg using immunomagnetic beads
regulars
An
Tu
28
application notes High Speed, Ultra-High Sensitivity, and Robustness Needed for the Quantitation of Pharmaceuticals in Blood Plasma, Agilent. Hormones Norgestrel and Ethynyl Estradiol on Allure C18, Restek. Artemisinin in Artemisia Annua Leaf, Camag. Automating Calculations for Rapid Seed Oil Quality Control and Authenticity, Waters.
30
technology update An overview of recent technology advances in separation science and instrumentation.
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.
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technical articles on chromatography and related technologies?
Zongwei Cai Hong Kong Baptist University
Tuulia Hyötyläinen University of Helsinki, Finland
updates on recent research studies?
Yi Chen Chinese Academy of Sciences, Beijing, China
Gongke Li Sun Yat-Sen University, Guangzhou, China
Gert Desmet Vrije Universiteit Brussel, Belgium
Yong-Chien Ling National Tsing Hua University, Taiwan
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c n e i c s n ratio
C. Bor Fuh National Chi Nan University, Taiwan Klara Valko, GSK, UK Y.S. Fung Hong Kong University Jean-Luc Veuthey University of Geneva, Switzerland Xindu Geng Northwest University, Xi’an, China Claudio Villani Universita’ degli Studi di Roma “La Luigi Mondello Sapienza”, Italy University of Messina, Italy Cheing- Tong Yan Paul Haddad Center of Environmental Safety and University of Tasmania, Australia Hygene, Taiwan Hian Kee Lee National University of Singapore, Singapore
Edward Browne GSK, Singapore
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One- and Two- dimensional GC-MS for Hop Metabolics
Current and Future Approaches to Speed Up HPLC Separations
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The applictaion of Quality by Design Principles to Analytical Method Development, Validation and Transfer.
Sanjay Garg
The Role of Analytical Science and Techniques in Early Phase Drug Discovery and Registration for Clinical Studies
Anne Goh
Online Solid Phase Extraction-LC-MS in DMPK Applications
Edward Browne
Biomarker Analysis for Preclinical Pharmaceutical R&D
Shawn Stanley
Using Narrow Bore Monolithic LC Columns to Enhance Analytical Sensitivity in Equine Doping Control
Ping Li
HPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines
Yizeng Liang
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A CTAB-based method for the proteomic analysis of wine spoilage micro-organisms Italy According to a paper in the Journal of Chromatography B [877 (10), 887-891 (2009)] a protocol based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) and its salt-dependent solubility was developed.
Comment
Dr Alessandra Bossi from the Department of Science and Technology at the University of Verona in Italy explained that the contamination of nucleic acid and polysaccharides is a real problem when mapping the proteome of microrganisms by 2D electrophoresis. “Polysaccharides are complex proteins, giving rise to high molecular weight aggregates that do not migrate in gel; plasmids and nucleic acid could also bind to proteins and, due to their external skeleton exposing phosphoric groups, such complexes result in the apparent acidification of the proteins and in their defocusing. Both polysaccharides and nucleic acid cause severe streaks and smears on the gels and pose problems in terms of resolution of the maps. Thus, a protocol for an efficient removal was really needed,” Dr Bossi said. The team proposed a solution to the problem of efficient removal of nucleic acid and polysaccharide residues from the total protein extract of a microrganism prior to proteomic analysis by setting up a protocol based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) and its peculiar solubility properties, which are dependent on the concentration of NaCl in the solution. “CTAB is routinely exploited in molecular biology and genetic for nucleic acid purification and we propose it as treatment before performing two-dimensional electrophoresis of the total protein extracts. Results showed a far superiority of the CTAB protocol, respect the available and already described procedures. More than 500 protein spots were counted on minigels (7 cm length) after the CTAB treatment, which is almost a hundred more than what obtained with other methods, tested for comparison,” Bossi added. She believes the technique proved to be simple and very efficient. “The protocol was applied to several microrganism and to yeasts and the results were always remarkable. The protocol is nowadays employed for mapping other microroganisms, such as Prevotella species and to the treatment of cells from superior plants. We think that this technique is beneficial for the broad community that devotes its scientific interests to proteome research,” she concluded.
6
research round-up
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Rapid and sensitive determination of six carcinogenic aromatic amines in cigarette smoke by LC-MS/MS India Aromatic amines are one of the sources of
of six aromatic amines (aniline, benzidine,
carcinogenicity in cigarette and tobacco smoke, and
o-toluidine, 1-naphthylamine, 2-naphthylamine and
accurate quantification of these chemicals is needed to
4-aminobiphenyl) using gradient LC-ESI-MS/MS and
assess public health risk. As documented in the Journal
determined them from cigarette smoke. All aromatic
of Chromatography A [1216 (15), 3059-3063 (2009)], a
amines were well resolved in 8 minutes using these
validated rapid, sensitive and analyte-specific liquid
gradient conditions. “We also studied the degradation
chromatography/electrospray ionization tandem mass
of benzidine present in cigarette smoke extract. The
spectrometric (LC/MS/MS) method has been developed
separation and detection technique is a robust one and
for the simultaneous determination of six aromatic
will be useful for chromatographers for determination
amines in mainstream cigarette smoke using research
of these chemicals from other sources when present at
reference cigarette 2R4F.
trace levels. We also introduced a recovery calculation
Dr Bidhan Ray from the Department of Chemistry
technique using the standard addition method, without
at Jadavpur University in India, explained that these
using isotopic-labelled standards,” Ray explained.
aromatic amines possess different adverse effect
In this work liquid-liquid extraction was used for the
on human health and are also considered as potent
extraction of aromatic amines from cigarette smoke
genotoxic compounds. “Accurate quantification of trace
followed by liquid chromatographic separation and
levels of these aromatic amines is a challenging task. Our
tandem mass spectrometric detection. “Determination
main aim was to develop a rapid and compound-specific
of all other toxic volatiles from cigarette smoke has been
determination method for aromatic amines, so that
performed and will be published soon. At present we are
the technique can be used for quantification of other
studying the biological effects of the chemicals present
chemicals when present at trace level,” Dr Ray added.
in cigarette smoke on humans in an attempt to uncover
The team developed and optimized the separation
ways of reducing or eliminating them,” he concluded.
8
research round-up
www.sepscience.com
HPLC assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolites Australia Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones – desmethoxyyangonin, methysticin and kavain – in rat liver microsomes using diazepam as an internal standard; liquid-liquid extraction was used for sample preparation and analysis was performed on a Shimadzu 10A high-performance liquid chromatography system. The study was published in Biomedical Chromatography [23 (1),81-91 (2009)] and authored by Professor Iqbal Ramzan, Dean of the Faculty of Pharmacy at the University of Sydney, Australia, who explained that kava had interested him since childhood. With a background in Pharmacokinetics, Professor Ramzan wanted to contribute to the knowledge about kava, which entailed developing an analytical technique to measure its active constituents. “We are able to assay kava, some of its metabolites to determine the structure of the metabolites,” he said. His fundamental work is designed to see why kava is hepatotoxic. “Using this assay we can see if the heptotoxicity is due to kava itself, its metabolites or both. Also it will allow us to see which specific kava component or kavalactone is the offending species in terms of liver damage,” he added.
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research round-up
9
Thermodynamic study of salbutamol interaction with an immobilized β2-adrenoceptor by HPLC China Investigation of the interaction between drugs and receptors is very important in revealing the biologic basis and mechanism of the drug, and designing new bioactive compounds. A study published in the Journal of Chromatography B [877 (10), 911-918 (2009)], purified the β2-adrenoceptor (β2-AR) from rabbit lung tissues and immobilized it on a surface of macro-pore silica gel through covalent bonds to prepare the stationary phase. Dr Xinfeng Zhao from the Department of Pharmacology at the Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education at Xi’an Jiaotong University in China explained that the technology for screening bioactive compounds of innovative and efficient drugs is a hot topic in pharmaceutical fields. “Traditional Chinese medicine (TCM) and its complex prescription compounds have a complete theoretical system and long clinical history, and have unique advantages in the treatment of cardiovascular diseases. In recent years, TCM have become a major source to develop innovative new drugs. However, they also have the disadvantages of complicated composition and flexible application. Therefore, screening of their bioactive ingredients has become the bottleneck of the entire research process,” Dr Zhao said. Zhao added: “It is well known that drugs have pharmaceutical activities by recognizing and then binding to membrane receptors, so as to activate some cellular signal transmission cascade.” Based on this theory, if these receptor could be bonded onto the surface of the stationary phase, a fast, effective and stable affinity chromatographic method could be established for the screening of active compounds in TCM by taking advantage of the receptor’s unique recognition to drugs and the high separating capacity of liquid chromatography. “The results showed that the association constant of terbutaline with β2-AR in the biochromatographic column was 9.37 × 10-6 M, and the binding sites were 9.76 × 104 M. Salbutamol has two binding sites with β2-AR in the column, for which the association constants were 1.11 × 104 M and 1.34 × 103 M. All the above results demonstrated that the receptor biochromatographic model was a new technology for online investigation of the interactions between receptor and drug. More importantly, our results showed that thermodynamic theory could be used for investigating the binding mechanism of the drug and receptor,” he said. The team believes the results indicate that receptor biochromatography had the properties of recognizing, combing and separating its ligands. “It could be used for screening bioactive compounds in TCM [Chinese Science Bulletin, 2008, 53(6): 842-847]. In our future work, we will combine different receptor columns online by column switching technology to screen bioactive compounds in complex samples such as TCM,” he concluded.
10
research round-up
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Pharmaceutical quality control by micellar electrokinetic chromatography Greece According to the Journal of Pharmaceutical and Biomedical Analysis [49 (2), 201-206 (2009)], a micellar electrokinetic chromatography (MEKC) method has been developed and validated for the determination of nimesulide related compounds in pharmaceutical formulations. “This paper was part of our continuous research cooperation with the Greek pharmaceutical industry aiming to develop advantageous analytical methods for the quality control of formulations during development and production stages. We are mainly focused on the development of reliable and high-throughput methods for the analysis of samples produced from assay, purity, dissolution and in-process tests. On this basis, we developed, validated and applied the first micellar electrokinetic chromatography (MEKC) method for the separation and quantitation of the process and degradation impurities of the non-steroidal anti-inflammatory drug Nimesulide,” explained Dr Paraskevas Tzanavaras from the Laboratory of Analytical Chemistry at the Aristotelian University of Thessaloniki, Greece. After careful investigation of the key variables that affected the behaviour of the target compounds under MEKC conditions, the team was able to separate and quantify the active ingredient and its six related substances within 20 minutes. “The validation of the method in terms of the most critical parameters according to international guidelines (specificity, linearity, accuracy and precision) proved its potential usefulness and efficacy for its intended purposes,” said Dr Tzanavaras. Tzanavaras believes the results confirmed the potential of capillary electrophoretic techniques to the quality control of pharmaceutical formulations. “If properly developed and validated, these techniques offer adequate analytical figures of merit, comparable reliability and performance to traditional and well-established HPLC protocols and dramatic reduction in organic waste generation. We intend to continue our research activities towards this direction,” Tzanavaras concluded.
12
research round-up
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Reversed-phase chiral LC–MS/MS quantitation of reboxetine enantiomers in rat plasma and brain UK A sensitive liquid chromatography–mass spectrometry (LC–MS) method has been developed for stereoselective determination of reboxetine in rat plasma and brain homogenate (LLOQ, 50 pg/mL). Dr Paul Turnpenny from the Discovery Bioanalytical Group at Pfizer Global Research and Development in Sandwich, UK, explained the research was performed to characterize CNS and systemic exposure levels for the potent pharmaceutical agent reboxetine administered in specific receptor occupancy investigations. Stereo-selectivity was required because the two enantiomers are known to exhibit differences in their receptor potency. “Using two different biological matrices and working with low analyte concentrations, the primary focus of the work was to optimize the sensitivity and robustness of the assay. MS detection, as opposed to UV/fluorescence detection in previously described methods, was anticipated to offer the best chance of this,” Dr Turnpenny said. Published in the Journal of Pharmaceutical and Biomedical Analysis [49 (1), 133-139 (2009)] the study’s most significant outcomes include the chiral recognition of the stationary phase α1 acid glycoprotein which enabled good separation to be achieved, although the interaction was extremely sensitive to mobile phase pH and needed adequate buffering (12.5 mM ammonium carbonate). The study also revealed a pH below 6.6 caused co-elution of the enantiomers and above pH 7 excessive analyte retention and peak tailing. The pKa of reboxetine is 8.3, therefore at the assay pH of 6.7 (1.6 units lower) the analyte was still predominantly ionized and only a low percentage of organic modifier (15%) was needed to elute. “I found that the composition of flow entering the MS source was hugely influential upon the detection sensitivity. By introducing a secondary postcolumn organic modifier to aid the desolvation and ionization process, the sensitivity was improved up to 10-20 fold and gave a much smoother response. I also found that column washing was absolutely essential to maintain good chromatography. A small amount of acetic acid (0.05%) helped remove protein debris and enabled >80 sequential injections to be performed with no problem and upwards of 300 in total on the same column,” he added. He believes that for complicated multi-analyte or chiral separations in which the eluting mobile phase contains a low percentage of organic solvent or incompatible pH to MS detection, postcolumn adjustment of the flow can significantly improve both the sensitivity and consistency of the response and is relatively easy to setup. “The stereospecific effects of reboxetine, currently administered in the racemic form, are still of considerable interest to my company and it could be the analytical technique will be utilized in further research to establish an improvement in its clinical use,” he concluded.
separation science — volume 1 issue 6
research round-up
13
Preparation and ion chromatographic properties of a new core-shell chromatographic support Al2O3/SiO2-10 China A new stationary phase Al2O3/SiO2-10 was prepared and characterized by XPS, XRD, SEM and surface analysis and documented in the Journal of Chromatography A [1216 (15), 3054-3058 (2009)]. Main author, Dr Shengxiang Jiang from the Key Laboratory for Natural Medicine of Gansu Province at the Chinese Academy of Sciences in Lanzhou, China, explained, “Some inorganic oxide supports, such as zirconia, titania and alumina have been found to offer some special chromatographic properties. Of the above-mentioned oxides, alumina is the most extensively studied material and it has many desirable ion-exchange properties; however, until now it has not been broadly used.” He believes the main reason is that these alumina supports provide low ion exchange capacities, low resistance to strong acidic and basic conditions, and often result in poorly defined peak shapes and zones in chromatographic applications. “To improve the chromatographic characteristics of alumina-based supports, we employed a layer-bylayer self-assembly technique to develop a new core-shell chromatographic support Al2O3/SiO2-10,” Jiang added. The team found that the core-shell chromatographic support Al2O3/SiO2-10 had a large surface area, and very uniform diameter and surface. “Compared with other alumina supports, this core-shell support had excellent column efficiency, well-defined chromatographic peaks and favourable retention times for the separation of inorganic anions,” he explained. The core-shell chromatographic support appeared to combine the advantages of both core and shell materials, and provided better chromatographic properties. “In my opinion, further research into these core-shell chromatographic materials may result in many excellent supports for the HPLC separation, including fast or multiaction chromatographic supports,” he said. In the future, Jiang plans to choose new shell materials to make further advances in core-shell chromatographic supports; and develop functionalized core-shell chromatographic supports after chemical modifications.
14
research round-up
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Detection of Salmonella enteriditis from egg using immunomagnetic beads USA The types of chemical linkages used to bind antibodies
As far as which bead/antibody interaction provided
to magnetic beads to form immunomagnetic beads
better detection, those results varied among the three
(IMB) were compared in the capture and detection
S. enteriditis strains tested. We concluded that antigenic
of Salmonella enteriditis from egg white, egg yolk
differences among those strains contributed to different
and whole egg in a paper in Food Analytical Methods
degrees of interaction with the immunomagnetic
[2 (1), 14-22 (2009)]. According to Sue Reed from the
beads,” she said. Second, they found that the presence
Eastern Regional Research Center at the United States
of egg yolk resulted in lower signal intensities for all
Department of Agriculture, USA, there are many different
types of beads. “This suggested that egg yolk may have
types of immunomagnetic beads on the market today.
an adverse effect on the immunological interactions
“We were interested to study what differences may
between the bacteria and the immunomagnetic beads,”
exist among different beads with regard to the linking
she added.
chemistry by which antibodies are attached. We chose
Reed believed that based on the results of this work,
two common types of beads having streptavidin/
they have realized that the capture of a target organism
biotin interactions and covalent attachment through
by immunomagnetic beads may vary depending on
carboxyl groups. In addition, we included a commercially
the strain being studied. “In subsequent work, we have
prepared immunomagnetic bead for comparison. We
continued to study the differences in detection among
also chose to use Salmonella enteriditis infected eggs
immunomagnetic beads utilizing different antibody
as a real-world model with which to study the capture
conjugating chemistries. We have expanded our research
abilities of these immunomagnetic beads,” Reed
to include tosylactivated beads, as well as different sizes
explained.
of these beads. Perhaps a different target organism and/
The team’s findings were twofold. Both the
or food matrix will yield more definitive findings,” she
streptavidin-coated and carboxylated beads yielded
concluded.
higher relative signals than the commercially prepared bead. “That is attributed to a much higher background signal associated with the commercially prepared beads.
separation science — volume 1 issue 6
research round-up
15
16
feature article — Speciation analysis with IC-ICP/MS
www.sepscience.com
Mercury and arsenic speciation analysis by IC-ICP/MS Laura Reyes,1 H.M. Skip Kingston,1 Andrea Wille,2 Jürgen Knöll3 and Alfred Steinbach2 Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania, USA Metrohm International Headquarters, Herisau, Switzerland 3 Philipps Universität Marburg, Germany
1
2
By means of IC-ICP/MS, different valence states of arsenic and mercury in the form of inorganic and organic species can be sensitively and unambiguously identified in one single run. Owing to the absence of interconversions during sample preparation, the determination of arsenic species in biological and environmental matrices is straightforward and can be performed by traditional speciation analysis down to the sub-ppb level. In contrast, species transformations of mercury during sample preparation require the use of specific isotope dilution mass spectrometry (SIDMS). EPA Method 6800 was applied to evaluate and efficiently compensate for potential errors during measurement.
Introduction
conductivity detection.
Arsenic
Ion chromatography (IC) with
Especially in view of the zero
Arsenic is ubiquitously found in a
conductivity detection has been
tolerance policy concerning
high number of minerals and its
successfully used to determine
chromium, arsenic and selenium
use as a weed killer and rat poison
anionic and cationic substances as
compounds in drinking water
illustrates its high toxicity. Inorganic
well as polar compounds such as
and mercury in food samples,
arsenical derivatives are considered
amines or organic acids. However,
ICP/MS detectors have gained
to be carcinogenic and possibly
in environmental samples a higher
increasing importance. IC-ICP/MS
teratogenic. Therefore, the EPA
sensitivity and selectivity are
can distinguish between different
proposes a maximum allowable
required to test for potentially toxic
oxidation states and chemical forms
drinking water concentration of
substances with low maximum
of a given element. This approach
10 ppb. In environmental and
contaminant levels (MCL).
is called speciation analysis. From
biological samples, more than 20
The coupling of IC with multi-
the toxicological point of view,
arsenic species have been identified.
dimensional detectors such as
individual concentrations of
Depending on their binding
electrospray ionization mass
element-containing species are far
characteristics, they have different
spectrometers (ESI-MS) or
more significant than total element
toxicities and chemical behaviours.
inductively coupled plasma
concentrations as different valence
Based on structural data, IC-ICP/MS
mass spectrometers (ICP/MS)
states of an element often have
allows separation and unambiguous
solves even complex separation
completely different properties.
identification of different arsenic
problems, simultaneously achieving
For example, while chromium(III)
species in inorganic and organic
high sensitivity and selectivity.
is an essential trace element
forms.
Additionally, these hyphenated
for mammals as it is involved in
techniques allow unambiguous
glucose metabolization, all forms of
Mercury
peak identification and are less
hexavalent chromium are regarded
Mercury is found in several forms,
prone to matrix interferences than
as highly toxic and carcinogenic.
particularly as elemental (Hg0),
Figure 1
separation science — volume 1 issue 6
feature article — Speciation analysis with IC-ICP/MS
17
inorganic (Hg2+), or alkylated
death, even in small quantities.
dose for methylmercury (Rf ) of
mercury (CH3Hg ). Of the most
According to the US Food and Drug
0.1 µg/kg of body weight per day,
common mercury species found in
Administration (FDA), the major
while the World Health Organization
the environment, methylmercury is
exposure pathway to methylmercury
(WHO) has set a tolerable dose of
considered the most toxic species.
in humans and wildlife is through
1.6 µg/kg of body weight per week [1].
It is classified as a neurotoxin that
consumption of contaminated fish.
However, mercury species are
rapidly bioaccumulates and can
The US Environmental Protection
prone to interconversion. Mercury
cause major health problems or
Agency (EPA) stipulates a reference
shows a pronounced transformation
+
from inorganic mercury (Hg2+) to the
Table 1
biologically active and highly toxic
Instrumental operation conditions for the determination of the arsenic species
methylmercury (methylation) and
via IC-ICP/MS
vice versa (demethylation). Similarly,
Column
Metrosep A Supp 15 – 150
the extraction techniques used for
Injection volume
10 µL
separation and preconcentration
Column temperature
Ambient
tend to alter the original distribution
Eluent
8 mmol/L NH4NO3, pH = 8.3
of mercury species, which affects the
Elution
Isocratic
legal defensibility of the data.
Flow rate
0.7 mL/min
Speciated isotope dilution mass
ICP/MS
Without reaction or collision mode
spectrometry (SIDMS) has been
m/z
75
developed to correct for species conversions. According to US EPA
Table 2
Method 6800 [2] each species is
Instrumental operation conditions for the determination of mercury species
labelled with a different isotope-
via SIDMS IC–ICP/MS.
enriched spike in the corresponding
RF power
1475 W
form. By measuring the isotope
Plasma gas flow
Ar, 115 L/min
ratio of both the unspiked and
Auxiliary gas flow
Ar, 1 L/min
spiked sample and knowing the
Nebulizer
Quartz, concentric
isotopic ratio of the addition,
Spray chamber
Quartz
interconversions between the
Sample and skimmer cones
Ni, 1.1 and 0.8 mm, respectively
species become traceable and can be corrected.
Measurement parameters of ICP/MS
This article deals with the
Monitoring isotopes
199
Acquisition mode
Time-resolved analysis
determination of organic and
Integration time per mass
0.20 s
inorganic arsenic and mercury
Replicates
1
compounds by means of IC-ICP/MS.
Total analysis time
300 s
Arsenic species (monoisotopic) are
Hg,
Hg,
200
201
Hg and
202
Hg
not prone to interconversion and
Separation conditions Column
DVB-C18 column, 150 × 4.6 mm, 2 µm
are thus determined by traditional
Injection volume
100 µL
speciation analysis. Several
Column temperature
Ambient
established extraction techniques
Eluent
50 mmol/L pyridine,
used for mercury speciation in
5% (v/v) methanol, pH 3,
biological samples are evaluated by
0.5% (w/v) cysteine,
applying both internal SIDMS and
Elution
Isocratic
external calibration.
Flow rate
1 mL/min
18
feature article — Speciation analysis with IC-ICP/MS
www.sepscience.com
Experimental
or analysis procedure.
enzymatic digestion with protease
External calibration: Separation
Extraction methods: The extraction
XIV. The methods are summarized
of mercury species was automated
methods to be evaluated were
in Table 3 [3]. The reference material
using the 858 Professional Sample
based on literature-referenced
used for the comparison of sample
Processor and the 850 Professional
methods such as alkaline extraction
preparation methods was Tuna Fish
IC (both Metrohm AG, Herisau,
with potassium hydroxide (KOH)
Tissue Certified Reference Material
Switzerland) coupled to an ICP/
or tetramethyl ammonium
(ERM-CE464) supplied by IRMM
MS model HP 4500 (Agilent
hydroxide (TMAH) solution; acid
(Geel, Belgium), which is certified for
Technologies, Palo Alto, California,
leaching with hydrochloric acid
total mercury and methylmercury
USA and Yokogawa Analytical
(HCl), nitric acid (HNO3) or glacial
content.
System Inc., Tokyo, Japan). For the
acetic acid (CH3COOH); extraction
Reagents, standard solutions and
determination of arsenic species,
with l-cysteine hydrochloride and
eluents: All reagents used in this
the IC system was coupled to an Agilent 7500ce ICP/MS. The
Figure 1
conditions used during the study are detailed in Tables 1 and 2. Each sample was analysed three times. Quantitation was based on peak areas by external calibration using the arsenic isotope m/z 75 and the most abundant mercury isotope m/z 202. Quantitation using the mercury isotopes m/z 199, 200 and 201 yielded similar results. For the determination of total mercury concentrations, the digested and extracted solutions were analysed by ICP/MS. SIDMS: To correct for mutual
Figure 1: Separation and detection of arsenite, dimethylarsenate, monomethylarsenate and arsenate.
Figure 2
interconversion, Hg2+ and CH3Hg+ compounds were quantified by EPA 6800 protocol specifications, spiking the sample before the extraction with 199Hg2+ and CH3200Hg+ and application of SIDMS equations [2]. The isotopic species reagents and calculation software in a SPC-M Mercury Speciation Kit was provided by Applied Isotope Technologies (AIT, Sunnyvale, California, USA). This double-spike approach allowed tracking of any artifact stemming from methylation/demethylation reactions that might have occurred during the sample preparation and/ separation science — volume 1 issue 6
Figure 2: IC-ICP/MS chromatogram for 10 µg/L Hg2+ and CH3Hg+. Chromatograms obtained at different masses were shifted for clarity. Instrumental operation conditions are given in Table 2.
feature article — Speciation analysis with IC-ICP/MS
19
work were of the highest purity
prepared with deionized water with
species. However, ASB interference
grade (puriss. p.a.). Analytical
a specific resistance higher than
can be overcome by changing the
reagent grade HNO3, HCl, TMAH,
18 MΩ•cm.
chromatographic parameters. Mercury: Figure 2 shows the
potassium hydroxide, optimagrade methanol and HPLC-grade
Results and Discussion
chromatogram of the separation
glacial acetic acid were purchased
Arsenic: IC-ICP/MS allows the
of the divalent mercury ion from
from Fisher Scientific (Pittsburgh,
separation and unambiguous
methylmercury [3] on a polymer-
Pennsylvania, USA). Reagent grade
identification of different arsenic
based C18 reversed-phase column.
l-cysteine, l-cysteine
species in inorganic and organic
Separation was achieved in less than
hydrate, ammonium phosphate
forms. Figure 1 displays the peaks
five minutes and the retention times
dibasic, pyridine and protease XIV
of a 10 µg/L standard solution
were 1.87 ± 0.02 and 2.98 ± 0.03
were obtained from Sigma Aldrich
containing monomethylarsenate,
minutes. Linear calibration curves for
(St. Louis, Missouri, USA). The arsenic
dimethylarsenate, arsenite, arsenate
Hg2+ and CH3Hg+ were obtained in
standard solutions were purchased
and arsenobetaine (ASB). Under the
the range from 1–20 µg/L. Detection
from Fluka (Sigma Aldrich, Buchs,
given conditions (Table 1), ASB is not
limits were 0.46 ± 0.02 and 0.78
Switzerland). All solutions were
separated from the trivalent arsenic
± 0.08 µg/L for Hg2+ and CH3Hg+,
hydrochloride
Table 3
Concentrations of mercury species (in mg/kg Hg) determined in Tuna Fish Tissue Reference Material (ERM-CE464) by external calibration and by EPA Method 6800 (SIDMS). The values are means of ± 95% confidence limits (n = 3). The percentage recoveries of total Hg and CH3Hg+ are indicated in parentheses. Extraction procedure
External calibration Hg
2+
0.12b Extraction technique, extraction reagent
a
EPA Method 6800 (SIDMS)
CH3Hg
Sum of species
Hg
5.12 ± 0.16a
5.24 ± 0.10a
0.12b
+
Temp. Time [°C] [min]
2+
mg/kg Hg
CH3Hg+
Sum of species
5.12 ± 0.16a
5.24 ± 0.10a
mg/kg Hg
A
Sonication/water bath, 25% (w/v) KOH in methanol
70
180
0.06 ± 0.02b
5.05 ± 0.13 (99 ± 3)
5.11 ± 0.13 (98 ± 3)
0.07 ± 0.02b
5.22 ± 0.31 (102 ± 6)
5.29 ± 0.31 (101± 6)
B
Sonication/water bath, 25% (w/v) TMAH in methanol
70
180
0.12 ± 0.03b
5.05 ± 0.18 (99 ± 4)
5.17 ± 0.18 (99 ± 3)
0.07 ± 0.03b
5.20 ± 0.18 (102 ± 4)
5.27 ±0.18 (101 ± 6)
C
Microwave, 5% (w/v) TMAH in methanol
180
20
0.18 ± 0.05b
4.88 ± 0.17 (95 ± 3)
5.06 ± 0.18 (97 ± 3)
0.30 ± 0.07b
5.18 ± 0.13 (101 ± 3)
5.48 ± 0.15 (105 ± 3)
D
Sonication bath, 5 mol/L HCl
25
5
0.07 ± 0.02b
4.29 ± 0.39 (84 ± 8)
4.36 ± 0.39 (83 ± 7)
0.13 ± 0.05b
5.11 ± 0.38 (100 ± 7
5.24 ± 0.38 (100 ± 7)
E
Microwave, 4 mol/L HNO3 (EPA 3200)
180
20
0.06 ± 0.04b
3.94 ± 0.12 (77 ± 2)
4.00 ± 0.13 (76 ± 2)
0.11 ± 0.07b
5.60 ± 0.33 (109 ± 6)
5.71 ± 0.34 (109 ± 6)
F
Microwave, glacial CH3COOH
165
10
0.35 ± 0.08b
3.29 ± 0.14 (64 ± 3)
3.64 ± 0.16 (69 ± 3)
0.27 ± 0.12b
5.12 ± 0.19 (100 ± 4)
5.39 ± 0.22 (103 ± 4)
G
Water bath, 1% L-cysteine hydrochloride
60
120
0.45 ± 0.10b
4.87 ± 0.20 (95 ± 4)
5.32 ± 0.22 (102 ± 4)
1.05 ± 0.14b
5.08 ± 0.25 (99 ± 5)
6.13 v 0.29 (117 ± 5)
H
Hybdridization oven, Enzymatic digestion with protease XIV
37
120
0.16 ± 0.07b
4.42 ± 0.14 (86 ± 3)
4.58 ± 0.16 (87 ± 3)
0.07 ± 0.02b
5.09 ± 0.24 (99 ± 5)
5.29 ± 0.31 (100 ± 5)
certified methyl mercury and total mercury content in Tuna Fish Tissue Certified Reference Material (ERM-CE464) supplied by IRMM (Geel, Belgium) inorganic mercury concentration was calculated as the difference between certified total mercury and methylmercury concentrations.
b
20
feature article — Speciation analysis with IC-ICP/MS
www.sepscience.com
Table 4
respectively.
Extraction procedure
Mean degree of transformation [%]
Table 3 shows the accuracy of the
Methylation
Demethylation
extraction procedures tested by
A
5±3
6±1
both external calibration analysis
B
6±2
4±1
and SIDMS using the Tuna Fish
C
3±2
6±2
Tissue Certified Reference Material
D
5±3
3±1
(ERM-CE464). For seven of the eight
E
18 ± 4
0.8 ± 0.6
extraction procedures evaluated, the
F
4±2
27 ± 5
CH3Hg values calculated by using
G
4±2
4±1
SIDMS were in good agreement with
H
4±2
1.4 ± 0.5
+
the certified reference value. Only for extraction procedure E, a too high
extraction with glacial acetic acid
occur after spiking.
methylmercury content was found.
(procedure F) and extraction with 4
Because of the unique features
Hence, transformations and losses
mol/L HNO3 (procedure E) was used.
and undisputed benefits of EPA
of Hg and CH3Hg can be directly
Owing to the relatively low ratio of
Method 6800, it is expected that
linked to pretreatment steps.
Hg2+ to CH3Hg+, the high methylation
utilization of SIDMS will increase and
Results obtained by external
rate of 18% in procedure E did not
that this valuable tool for optimizing
calibration: Based upon the
cause a significant error regarding
and validating sample preparation
data shown in Tables 3 and 4,
CH3Hg concentration. In contrast, a
procedures for trace-metal
the concentrations found for
pronounced demethylation rate has
speciation, involving extraction,
methylmercury in the alkaline
a considerable effect if, as in the case
separation and detection, will gain
extraction procedures using either
of procedure F, high CH3Hg+ to Hg2+
much wider acceptance by analytical
the ultrasonic-assisted system
ratios are prevailing. Apparently, the
chemists.
(procedures A and B) or the
elevated Hg concentration of
microwave device (procedure C) were
0.27 mg/kg stems from the
Acknowledgments
in good agreement with the certified
pronounced demethylation rate.
We thank Milestone Inc. for the
values at the 95% confidence level.
The SIDMS protocol is an invaluable
financial support to Laura H. Reyes
These procedures yielded similar
tool for overcoming nonquantitative
and for instrumental support. We
methylation and demethylation rates
recoveries and species
also thank Agilent Technologies,
(~ 6%). Although procedure G was
transformations observed during the
Applied Isotope Technologies, Inc.,
suitable for CH3Hg determination,
evaluation of extraction procedures.
Duquesne University and Metrohm
2+
+
+
2+
+
USA, Inc., for instrumental and
inorganic mercury contamination – 0.45 compared with 0.12 mg/kg
Conclusions
Hg2+ – in the extracting reagent was
Ion chromatography coupled to
observed.
an ICP/MS is a powerful tool to
References
Extraction procedures D, E, F and
determine different organic and
[1] C.W. Levenson and D.M. Axelrad, Nutr. Rev.,
H suffered from too low CH3Hg
inorganic species unambiguously
64 (2006), 139-45.
recoveries (64–86%). Despite
in one single run. In the absence
[2] US EPA Method 6800, elemental
frequent use of acid leaching for
of interconversions, traditional
and speciated isotope-dilution mass
the extraction of mercury species
speciation analysis provides accurate
spectrometry, test methods for evaluating
from tuna fish samples, the lowest
results down to the sub-ppb level.
solid waste, physical/chemical methods SW
concentration of methylmercury
Species interconversions, however,
846, Update IVA. US Government Printing
on the ERM-CE464 and the highest
require correction. SIDMS according
Office (GPO), 2007, Washington, DC.
mercury species transformation
to EPA Method 6800 is capable of
[3] L. Reyes et al., Anal. Bioanal. Chem., 390
occurred when microwave-assisted
tracing any interconversions that
(2008), 2123-2132.
+
separation science — volume 1 issue 6
material support.
feature article — Speciation analysis with IC-ICP/MS
21
A comparison of TLC methods for constructing medicinal plant fingerprints Łukasz Cieśla and Monika Waksmundzka-Hajnos Department of Inorganic Chemistry, Medical University of Lublin, Poland.
Chromatographic fingerprinting is one of the most important quality control methods in the analysis of medicinal plants. It has been widely introduced and accepted by WHO, FDA, EMEA, German Commission E, British Herbal Medicine Association and many others. HPTLC with digital scanning is becoming increasingly popular in the development of chromatographic fingerprints. In case of very complex samples, as well as those of closely related plant species or varieties, the use of multidimensional and multimodal thin-layer chromatography may become a common approach. This paper describes two modes of TLC applied for fingerprint construction: one- and multidimensional modes are described, advantages and disadvantages of the both methods are highlighted, and the techniques compared with HPLC. 22
feature article — TLC fingerprinting
www.sepscience.com
Recent decades have brought a
has gained recent attention,
validated and well described [6].
great interest in herbal medicines
especially when a lack of authentic
In TLC detection can be repeated
and botanicals, which take a more
standard substances for active
several times, with and without
balanced and holistic approach
component identification exist [4].
derivatization. In addition, there
when compared with synthetic
A chromatographic fingerprint is
is no need for complicated clean-
drugs. Dietary supplements,
a chromatogram that represents
up procedures, which are often
nutraceuticals, traditional Chinese
the chemical characteristics of
necessary in HPLC to avoid column
medicines, Ayurvedic drugs are
herbal medicine [5]. Although HPLC
contamination. Sample preparation
only a few of the great diversity of
dominates the chromatographic
does not need to be modified even
phytopharmaceuticals present on
fingerprint literature, HPTLC
if one wants to focus on different
the market.
has several unique features that
substance classes present in the
Botanicals are often considered
can offer advantages over other
extract. Correct chromatographic
to be safe, but at the same time
chromatographic techniques
systems must be chosen in
not very effective; however,
used for fingerprint construction.
order to focus on the desired
nothing could be further from
However, we would not like to
constituents group. However, in
the truth. Many herbal medicines
detract from these other techniques,
case of quantitative measurements,
can cause severe side effects,
but rather underline that HPTLC
during chromatographic fingerprint
when overdosed or not properly
can provide reliable results as far
development, HPLC is a preferable
prepared. For example a popular
as fingerprint development is
technique because of its higher
herbal drug, Stephania tetrandra,
concerned. Identification is believed
separation power [3].
is often adulterated with plants
to be one of the leading applications
All aforementioned advantages
containing hepato- and nephrotoxic
of HPTLC, as the technique offers
become even more important in
aristolochic acids [1] (see also Figure
a very visual representation of
the analysis of very complex natural
1 for S. tetrandra fingerprint and
chromatographic results [3]. The
mixtures, containing structural
reference [2]). A proper amount of
major advantage of this technique is
analogues or substances spanning a
active constituents present in the
its ability to compare many samples
wide polarity range.
herb is responsible for its curing
side by side (see Figure 1). In Figure 1
or health-promoting effects. It is,
all samples on the plate are analysed
One-dimensional HPTLC
therefore, important to confirm
under the same conditions, which
fingerprints
not only the identity, but also the
is impossible to perform using the
In traditional one-dimensional thin-
amount or ratios of the active/
sequential mode of HPLC [3]. This
layer chromatographic fingerprints,
marker compounds. The need for
advantage, in turn, leads to another
the analysed sample is compared
quality assurance tools to ensure
– rapid results, as many samples are
with the botanically authenticated
the identity, purity and quality
analysed at the same time, leadings
raw material or properly chosen
of botanical materials has risen
to solvent savings.
chemical reference standards on
dramatically. Botanical drugs are
Many factors can be changed
the same chromatoplate (Figure
usually complex mixtures, and it is
during the chromatographic process,
1) [7]. All the analysed samples
difficult to analyse them with the
which influence the resolution of
are compared with regard to the
use of techniques developed for
analysed compounds. This can also
number, sequence, colour and
synthetic drugs [3].
be considered as a drawback, as it
intensity of the separated zones.
is more difficult to obtain identical
However in very complex samples
HPTLC for fingerprint construction
conditions during development
coeluting bands may appear.
Among a variety of quality control
of each plate. For reproducible
What is more, plant extracts used
methods for herbal materials,
results, all the steps involved in
for fingerprint construction,
chromatographic fingerprinting
method development should be
very often contain constituents
separation science — volume 1 issue 6
feature article — TLC fingerprinting
23
Figure 1
chromatogram is divided into several sections, where characteristic zones are marked and briefly described [7]. However, such an approach does not provide enough information, for example, with closely related plant species, as their chemical content is very similar [8]. In such instances it is of great importance 1
2
3
4
5
6
7
8
9
10 11 12 13 14
15
to gain as much information, from the chromatogram, as is possible. Chemotaxonomic studies are easier to perform after the application of methods enabling the resolution of all, or almost all, constituents present in the sample. Even the smallest differences in content may contribute to uncovering
16
17 18
19 20 21 22
23 24 25 26
27 28 29
chemotaxonomical relationships between closely related plant species [8].
Figure 1: One-dimensional HPTLC fingerprint of different Chinese plants with respect to aristolochic acids. Chromatographic conditions: SiO2, eluent: toluene/ethyl acetate/water/formic acid (20:10:1:1, v/v/v/v), derivatization agent: Tin(II) chloride reagent, observation under UV 366 nm. 1 = Aristolochic acids mixture, 2 = Aristolochic acid A, 3 = Aristolochic acid B, 4 = Aristolochic acid C, 5-8 = Aristolochia fangji, 9-11 = Stephania tetrandra, 12-15 = Aristolochia manshuriensis, 16 = Aristolochic acids mixture, 17-18 = Clematis armandii (Mutong), 19-20 = Clematis chinensis, 21 = Aristolochia tubiflora, 22 = Akebia trifoliata, 23 = Aristolochia mollissima, 24-26 = Asarum heterotropoides, 27 = Asarum sieboldi, 28-29 = Saussurea costus (Aucklandia lappa) [2].
Multidimensional HPTLC fingerprints As already stated, with very complex
spanning a wide polarity range
phenolic acids, flavonoids, etc.).
samples traditional one-dimensional
[8]. It is very difficult to find one
Sometimes ubiquitous constituents,
fingerprints may appear insufficient,
chromatographic system that would
such as amino acids or sterols can
as it is almost impossible to develop
enable satisfactory resolution of the
also provide plant-specific profiles
appropriate analytical methods to
majority of constituents present in
[3]. When nothing or very little
represent all chemical characteristics
this type of sample. In such instances
is known about the sample, one
of constituents in a chromatogram
some constituents are usually eluted
fingerprint is usually insufficient,
[5]. Several solutions have been
with the mobile phase front, or
and generating multiple fingerprints
proposed to this problem: (i) a
remain at the start, and thus are
is recommended [5]. Traditionally,
combination of analytical methods
lost from the fingerprint. However,
in a one-dimensional fingerprint,
with different separation principles,
the separation of all sample
the sequence of zones is usually
as has been demonstrated with
constituents is not always necessary,
presented relative to a point of
Andrographis paniculata analysis
and is very often not needed for
reference (e.g., mobile phase
[10], (ii) development of “multiple
distinguishing plant species [3]. For
front, markers) [9]. The addition
chromatographic fingerprints”,
a one-dimensional fingerprint it is a
of reference substances into the
used, for example, for distinguishing
good approach to focus only on the
extract, such as dyes, should
between Radix Puerariae Lobatae
constituents that are characteristic
rather be avoided. For proper
and Radix Puerariae Thomsonii [4] or
for the analysed plant species,
chromatogram description the
for developing binary fingerprints
rather than include substances
European Pharmacopoeia proposes
of Danshen Dropping Pill (DSDP) [5],
common to many plants (e.g., some
the use of a table, in which the
(iii) application of multidimensional
24
feature article — TLC fingerprinting
www.sepscience.com
and/or multimodal thin-layer
is a must to obtain reproducible
they cannot be removed from the
chromatography methods; for
and reliable results. It should
plate and may cause difficulties
example, HPTLC fingerprints of
always be checked as to whether
in obtaining reproducible results
furanocoumarins for distinguishing
multiple dimensions lead to analyte
[13]. It is also not advisable to apply
different Heracleum spp [8].
decomposition, because this can
aqueous eluents, because water
In the analysis of botanicals and
be the main reason for obtaining
extends the drying step between
herbal preparations the following
unreliable results [11].
each development and if not quantitatively removed may lead to
modes of multidimensional planar chromatography have been
Repeated developments in one
commonly applied: comprehensive
direction
2D planar chromatography realized
Multiple development techniques
Two-dimensional thin-layer
on mono- and bilayers, coupled-
in one direction have occasionally
chromatography
layer chromatography, combinations
been applied in fingerprint
A greater number of fully resolved
of MD-PC (multidimensional planar
construction [8]. The most popular
spots can be obtained using
chromatography) techniques and
is the unidimensional multiple
two-dimensional thin-layer
hyphenated methods [11].
development (UMD) technique [13],
chromatography [16]. In this
This way of constructing
and so will be discussed here. UMD
technique the analysed sample is
chromatographic fingerprints
has been applied, for example, to
first developed in one direction, and
has several interesting features;
fingerprint development of closely
after drying is subjected to another
for example, all separated
related species of the Heracleum
development, perpendicular to the
constituents can be detected on
genus or in cases of Chelidonium
first one. If systems, orthogonal
one chromatoplate and as such are
majus and Fumaria officinalis alkaloid
in properties, are applied a great
easier to compare. With multiple
fractionation, or the analysis of
spot capacity may be obtained
chromatographic fingerprints
coumarins [14]. This method consists
[11]. This method is very useful for
information contained in two or
of the repeated development
closely related plant species, which
more chromatograms should be
of the same plate, with a mobile
are characterized by comparable
combined before employing the lot-
phase of constant composition, for
chemical profiles [8]. Usually, small
to-lot consistency testing methods;
the same distance [13]. After each
differences between the species,
for example, data fusion-based
development the plate is carefully
varieties and forms are difficult
methods have been employed
dried. In this technique spots are
to notice using one-dimensional
for this purpose [5]. In addition,
better resolved because of a spot-
fingerprints, because of spot co-
multidimensional and multimodal
reconcentration mechanism, as
elution. If orthogonal systems are
chromatography enable the analysis
repeated development leads to
applied, in perpendicular directions,
of highly complex samples, as many
narrowing of spots, similar to the
the spots are spread over the whole
parameters can be used during
effect of a preconcentrating zone
plate, and any differences in the
each development step to affect
[15].
amount and spot location are easily
the outcome of the separation.
This method should not be used
observed.
For good resolution and high spot
with chemically labile constituents;
Planar chromatography allows
capacity values, systems orthogonal
for example, carotenoids as they may
two-dimensional separations using
in properties should be used [12].
undergo chemical changes during
the same stationary phase with
However obtaining reliable multiple
the chromatographic process. The
different eluent systems or by using
chromatographic fingerprints
application of nonvolatile mobile
a stationary phase gradient [11].
with the use of multidimensional/
phase additives, such as acetic acid,
For example, graft TLC with silica
multimodal chromatography is not
DMSO (dimethylsulfoxide) and
and RP-18W plates proved to be the
simple or easy. Proper validation
buffers, should also be avoided as
method of the greatest selectivity
separation science — volume 1 issue 6
ghost peak formation.
feature article — TLC fingerprinting
25
differences for the separation of
of the plate in the second direction
Conclusion
furanocoumarins, when compared
(see Figure 2) [8].
Despite many advantages,
with one-dimensional and
One-dimensional separation of
multiple development thin-layer
comprehensive two-dimensional
structural analogues is a difficult
chromatography techniques are not
thin-layer chromatography on
task, as is observed, for example,
always a good analytical choice, and
one adsorbent (CN-silica) [15, 17].
in coumarin analysis [8]. Coumarin
it should always considered whether
Good results are also obtained
fractions from Apiaceae plant
the application of such a technique is
when different multidimensional
material are poorly resolved after
actually needed. First, these methods
and/or multimodal techniques
one-dimensional development;
are usually time-consuming, and
are combined. For example UMD
that is, the separated peaks
if their application offers no great
may be applied hand in hand with
observed on the densitogram do
improvement their use is simply
two-dimensional developments
not belong to pure compounds,
not warranted. Sometimes, changes
[8]. The application of multiple
as proved by checking the UV
in experimental setup are a better
development improves resolution
spectra at each peak’s centre
idea, as has been proven for the
of partly resolved spots in the
and flanks. Comprehensive two-
separation of fatty oils according
orthogonal direction. The use of
dimensional separation results in
to the European Pharmacopoeia
UMD in the first direction allows the
better separation of the analysed
[3], for which changing the
developing distance in the second
compounds, although some of
application mode resulted in better
dimension to be reduced, leading to
the coumarins still are poorly
separation compared with double
shorter analysis time. It is because
resolved. The application of graft
development. The use of HPTLC
of satisfactory spot resolution from
TLC enables the best separation of
plates instead of TLC ones may
the first dimension development.
these compounds. Coumarins are
also bring satisfactory results. With
In addition, UMD together with
first separated on silica gel according
multiple development techniques
methanol, for transferring spots
to differences in their polarity, and
there is always the threat of artifact
from the first to another adsorbent,
then orthogonally via differences in
formation, which may be the result
results in narrowing of starting
structure and dimension of nonpolar
of chemisorption or decomposition
bands and symmetrical, well
molecule fragments, and structural
of some constituents during the
separated spots after development
analogues are resolved (Figure 2).
chromatography process. To check
Figure 2
All @ 300 nm
700.0 600.0 500.0
700.0
400.0
(AU)
300.0
500.0
200.0
400.0
100.0
300.0 2D
200.0 100.0 0.0
5.0
0.0 100.0 (mm) 80.0 70.0 60.0 10.0 50.0 15.0 20.0 40.0 25.0 30.0 30.0 20.0 35.0 40.0 10.0 (mm) 0.0 50.0
Figure 2: Two-dimensional chromatographic fingerprint of Heracleum sibiricum fruit sample adultered with fruit of unknown Apiaceae species. The sample was first triple developed on silica with the use of eluent: 35% (v/v) AcOEt + n-heptane, after the transfer with MeOH it was redeveloped on RP-18W plate with a mixture: 50% (v/v) MeOH + H2O.Spots of adulterant are marked with arrows.
26
feature article — TLC fingerprinting
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whether the spots, that appear after
[4] S.B. Chen et al., J. Chromatogr. A, 1121
the second or further development,
(2006) 114.
are not artifacts the sample should
[5] X.H. Fan et al., Anal. Chim. Acta, 555 (2006)
be developed in two-dimensional
217.
mode, with application of the same
[6] A. Schibli and E. Reich, J. Planar
eluent in both directions [11]. All
Chromatogr., 18 (2005) 34.
spots should lie on the diagonal,
[7] E. Reich et al., J. Planar Chromatogr., 15
with artifacts located to the side of
(2002) 244.
the line. An example of this is the
[8] Ł. Cieśla et al. J. Chromatogr. A, 1207 (2008)
double development of Angelica
160.
sinensis sample, according to the
[9] C. Fang et al., J. Planar Chromatogr., 19
European Pharmacopoeia method
(2006) 348.
[3]. It must always be proved that
[10] A. Srivastava et al., Phytochem. Anal., 15
additional zones after repeated
(2004) 280.
developments are not the result of
[11] Ł. Cieśla and M. Waksmundzka-Hajnos, J.
artifacts [3]. With two-dimensional
Chromatogr. A, 1216 (2009) 1035.
separations another problem is the
[12] M. Daszykowski et al., Acta Chromatogr.,
limitation of one sample per plate,
20
and some difficulties may occur in
(2008) 283.
the identification of zones because
[13] C.F. Poole and S.K. Poole, J. Chromatogr.
the standards must be separated on
A, 703 (1995) 573.
a separate plate.
[14] M. Waksmundzka-Hajnos and Ł. Cieśla,
Before developing a
Medicinal Plants in Poland and in the World, 17
chromatographic fingerprint, the
(2009) 24.
most appropriate method should
[15] Ł. Cieśla et al., J. Planar Chromatogr., 21
be carefully considered: HPLC,
(2008) 237.
one-dimensional TLC or one of the
[16] C.F. Poole, J. Chromatogr. A, 1000 (2003)
several modes of multidimensional
963.
TLC. The amount of samples to
[17] Ł. Cieśla et al., J. Planar Chromatogr., 21
be analysed, their complexity,
(2008) 447.
chemotaxonomical relationships between the analysed plants, as well as the aim of analysis (e.g., if there is a need for quantitative results) should be taken into account. References [1] A. Blatter and E. Reich, J. Planar Chromatogr., 17 (2004) 355. [2] www.camag.com/laboratory/methods/ identification.html, accessed on 15th April, 2009. [3] E. Reich and A. Schibli, High-Performance Thin-Layer Chromatography for the Analysis of Medicinal Plants. Thieme, New York, 2008.
separation science — volume 1 issue 6
feature article — TLC fingerprinting
27
An Application notes
Artemisinin in Artemisia Annua Leaf Company: Camag Summary: Artemisinin is an antimalarial substance obtained from the dried leaves of Artemisia annua. This application note from Camag outlines an HPTLC method for the quantification of artemisinin performed on silica gel 60 with cyclohexane, ethyl acetate, acetic acid (20:10:1). Derivatization with modified anisaldehyde reagent allows specific densitometric evaluation of fluoresecence at 520 nm with a 540 nm cut-off filter. Click here to view the application
Automating Calculations for Rapid Seed Oil Quality Control and Authenticity Company: Waters Seed oils are important components of foods, cosmetics and personal-care products. They are mainly extracted from 22 oil crops around the world. Production processing, storage, transportation and distribution are all critical to the quality of seed oils, and cross contamination can occur accidentally or intentionally. This technical note describes a streamlined system solution for seed oil quality control and authentication using UPLC with and Empower 2 software custom field calculation function to automatically determine and report if a seed oil sample passes or fails user-set QC criteria. Click here to view the application
28
application notes
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High Speed, Ultra-High Sensitivity, and Robustness Needed for the Quantitation of Pharmaceuticals in Blood Plasma Company: Agilent
High Speed, Ultra-High Sensitivity, and Robustness Needed for the Quantitation of Pharmaceuticals in Blood Plasma
Summary: This application note demonstrates the performance of an Agilent
Application Note Drug discovery and development: Drug metabolism and pharmacokinetics (DMPK)
Authors
1200 Series Rapid Resolution LC (RRLC) system coupled to an Agilent 6460A Triple
0.9
R 2 = 0.9999
0.8
Stephan Buckenmaier
0.7 Relative Responses
Application Scientist Agilent Technologies, Inc. Hewlett-Packard-Straβe 8 76337 Waldbronn Germany
0.6 0.5 0.4 0.3 0.2 0.1
Quadrupole (QQQ) mass spectrometer for the bioanalysis of human plasma.
0 0
100
200
300
400
500
600
Concentration [pg/mL]
Abstract This Application Note demonstrates the performance of an Agilent 1200 Series Rapid Resolution LC (RRLC) system coupled to an Agilent 6460A Triple Quadrupole (QQQ) mass spectrometer for the bioanalysis of human plasma.
High throughput analysis is shown for a test pharmaceutical and an internal
High throughput analysis is shown for a test pharmaceutical and an internal standard with both drugs eluting well within a 1 minute chromatographic window. Ultra-high sensitivity is demonstrated by linearity across a concentration range typically covered in human microdosing pharmacokinetic studies (1–500 pg/mL plasma). Robustness and reproducibility are shown for this concentration range over a 5-day period during which the LC/MS system was exposed to more than 1000 plasma samples.
standard with both drugs eluting well within a 1 minute chromatographic window. Ultra-high sensitivity is demonstrated by linearity across a concentration range
typically covered in human microdosing pharmacokinetic studies (1–500 pg/mL plasma). Robustness and reproducibility are shown for this concentration range over a 5-day period during which the LC/MS system was exposed to more than 1000 plasma samples. Click here to view the application
Hormones Norgestrel and Ethynyl Estradiol on Allure C18 Company: Restek
Hormones Norgestrel and Ethynyl Estradiol on Allure™ C18 Peak List: 1. uracil (marker) 2. ethynyl estradiol 3. norgestrel
1
Sample: Inj.: Conc.: Solvent:
5µL 100µg/mL mobile phase
Column: Cat. #: Dimensions: Particle size: Pore size:
Allure™ C18 9164565 150 x 4.6mm 5µm 60Å
Conditions: Mobile phase: Flow: Temp.: Det.:
Summary: Restek offers and application sheet showing the separation of the hormones norgestrel and ethynyl estradiol
water:methanol (25:75, v/v) 1.0mL/min. 25°C UV @ 220nm 2
on a 150 x 4.6 mm, 5 µm (60 Å pore size) Allure C18 column,
3
using a water-methanol mobile pahse and UV detection at 0
1
2
3
4
5
min.
LC_0123
Restek Corporation 110 Benner Circle Bellefonte, PA 16823 814-353-1300 • 800-356-1688 • Fax: 814-353-1309 • www.restek.com
220 nm. Click here to view the application
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Tu Technology update
Characterizing glycans from glycoproteins Manufacturer: Waters Manufacturer’s description: Waters Corporation has introduced the UltraPerformance LC (UPLC) analytical solution for the characterization of 2-AB labelled glycans from glycoproteins. The UPLC solution combines ACQUITY UPLC BEH Glycan columns with the ACQUITY UPLC system equipped with fluorescence detection. Current FDA regulations require that firms developing and manufacturing therapeutic proteins are able to accurately characterize the
Key
glycans attached to those proteins to ensure the efficacy and safety of a biopharmaceutical product.
Email the company
According to Waters, initial customer feedback has shown the UPLC solution to be up to three times faster than traditional HPLC for the analysis of 2-aminobenzamide (2AB)-labelled
Product information
glycans from human IgG. In addition, to ensure confidence in long-term use in customervalidated methods all ACQUITY UPLC BEH Glycan columns will be
Applications
quality-control tested with relevant labelled glycan standards for consistent batch-to-batch column performance.
Additional information
Expanding the capabilities of UPLC technology Waters ACQUITY UPLC BEH Glycan columns provide superior resolution for a wide range of glycans and represent the fifth applicationtested UPLC column and eleventh UPLC chemistry added to the ACQUITY UPLC column family, claims Waters. ACQUITY UPLC BEH Glycan columns are produced with the Waters Designed for Six Sigma (DFSS) Phase II ACQUITY UPLC column manufacturing process. According to the company, this process produces superior long-term performance by maximizing efficiency while minimizing column-to-column variability. As part of a Waters ACQUITY UPLC total system solution, this new glycan UPLC column is ideally suited to help scientists get more accurate and precise quantitative results than they can get with traditional HPLC columns and systems.
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26–28 August Biopolis Science Park, Singapore
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Day One:
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Advances in Separation Sciences Deriven by the Metabolomics and Proteomics Quest for Biomarkers
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Enviro
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Trace Pollutant Detection in Challenging Environments Solvent-Minimized Sample Preparation for Separation Science An Advanced Proteomic Approach to the Discovery of Microbial Enzymes
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Multidimensional Gel-free Protein Separation Approaches for In-depth Analysis of Complex Proteomes
Gongke Li
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New Approaches to Online Anti-salt Stacking for Direct Capillary Electrophoresis of Biosamples
Molecularly Imprinted Polymers for Trace Analysis of Complicated Samples
Terrorist Explosives by Analysis of Inorganic Residues
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Philip Marriott
GC-MS Analysis of Lipid Oxidation and Cholesterol Metabolism
Headspace Analysis of Plant Materials by Using Comprehensive Two-Dimensional Gas Chromatography: Selected Examples
Thomas Walczyk
Element Separation at the Microscale for High-Precision Isotopic Analysis of Biological Samples
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Multidimensional Gas Chromatographic Analyses of Flavours and Fragrances
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Determination of Biogenic Amines in Food: Conventional and Nonconventional Approaches
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One- and Two- dimensional GC-MS for Hop Metabolics
Current and Future Approaches to Speed Up HPLC Separations
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The applictaion of Quality by Design Principles to Analytical Method Development, Validation and Transfer.
Sanjay Garg
The Role of Analytical Science and Techniques in Early Phase Drug Discovery and Registration for Clinical Studies
Anne Goh
Online Solid Phase Extraction-LC-MS in DMPK Applications
Edward Browne
Biomarker Analysis for Preclinical Pharmaceutical R&D
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Using Narrow Bore Monolithic LC Columns to Enhance Analytical Sensitivity in Equine Doping Control
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HPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines
Yizeng Liang
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ICS-2100 Integrated Reagent-Free IC System Manufacturer: Dionex Manufacturer’s description: Reagent-Free IC systems with Eluent Generation (RFIC-EG systems) have been designed to deliver precise, reproducible results. According to the company, automated eluent generation minimizes the time, labour, costs and errors of manually prepared eluents. The systems are now enhanced by RFIC-ESP: electrolytic sample preparation. RFIC-ESP automates sample preparation so that manual preinjection processing such as matrix elimination and neutralization are removed. The ICS-2100 ensures you have what you need to run your samples, claims Dionex. The eluent organizer tray accommodates 1, 2 or 4 L eluent bottles, and the injection port is ergonomically designed for manual sampling. The eluent valve provides positive shutoff of eluent flow prior to the pump for easy servicing and an easy-access door opens to all chromatography components. Leak detection and management allow fast response to system leaks. The ICS-2100 employs TTL controls for external pump, injection valve, range selection, and signal offset for stand-alone operation. Key features of the ICS-2100 include: • Reagent-Free IC, RFIC-EG (eluent generation), RFIC-ESP, integrated auxiliary sample preparation valve (optional) • LCD front-panel control • Dual-piston pump • Column heater • Electrolytic suppression • Digital conductivity detection • Vacuum degas (option) • USB connectivity, plug-n-play • Optical leak detector • Electronic logbook and trending through virtual channels 32
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LCMS-2020 Manufacturer: Shimadzu Manufacturer’s description: Shimadzu has introduced a new compact LCMS-2020 single quadrupole mass spectrometer, which features the world’s fastest scanning capabilities, according to the company. Utilizing patentpending ultrafast (UF) technology, the LCMS-2020 has fast measurement speed and high sensitivity. This provides more accurate detection of trace impurities in pharmaceuticals, environmental pollutants and other contaminants. The new UFscanning technology achieves mass spectrum measurement speeds of 15,000 u/sec without sacrificing sensitivity or resolution, thus obtaining the best chromatography for the fastest LC conditions, Shimadzu states. The UFswitching technology achieves 15 ms polarity switching, enabling accurate data from the fastest chromatographic peaks without any loss of peak height. Shimadzu redesigned the ion transfer section to provide high sensitivity compared with other quadrupole analyzers for commonly measured substances. Users can now inject less and keep the analyser cleaner for longer. It also has improved high mass operation with sensitivity increased for masses above 1,000. In addition to better performance, the LCMS-2020 allows easier maintenance, permitting users to replace the ionization unit and inlet capillary to the MS from the LC, without breaking the vacuum. LCMS-2020 control and data processing are handled by an updated version of LCMSsolution software. The new software easily displays multiple sets of LC or MS data, allowing overlay and analysis of multiple data results for easy searching and comparison. The new software controls the LCMS-2020 and is fully integrated with Shimadzu’s Prominence series of ultrafast and nano HPLCs.
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Improved sensitivity with 7100 CE system Manufacturer: Agilent Manufacturer’s description: Agilent Technologies has introduced the 7100 Capillary Electrophoresis (CE) system, which it claims providing at least 10 times more sensitivity than any other commercial CE instrument. “We consider electrophoresis to be one of our core technologies and are seeing strong interest in CE in a number of areas, such as new biological drug QA/QC, environmental analysis, food safety and life sciences,” said Nitin Sood, General Manager of Agilent’s Electrophoresis business. “Used in standalone mode, as the separations component of a CE/MS or as a complementary, orthogonal technology to LC, the 7100 CE system brings unprecedented HPLC-like sensitivity to a wide range of analytical challenges.” CE is also attracting attention because the technique uses very small amounts of solvent. The 7100 system also requires 25 per cent less bench space, weighs 30 per cent less than its predecessor and uses less power. The sensitivity improvement is the result of a new detector used in combination with proprietary extended light path capillaries or a high-sensitivity cell. The 7100 offers a wide selection of detectors for flexibility and sensitivity, and the instrument is reverse-compatible to the company’s previous CE platform, so existing methods can continue to be used. 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 34
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of use. The 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 Chemstation software with an easyto-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 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.
Data reprocessing software for GC/MS and LC/MS Manufacturer: Markes International Manufacturer’s description: ClearView software uses sophisticated Dynamic Background Compensation (DBC) algorithms to distinguish between chromatographic peaks and background/baseline anomalies. It reprocesses stored GC/MS and LC/MS data files, eliminating background ions from the total ion chromatogram (TIC) and improving both spectral purity and peak integration, according to Markes. The software is fast, intuitive and works with standard GC/MS and LC/MS data file formats from vendors such as Agilent Technologies, PerkinElmer, Shimadzu, Thermo Scientific and Varian. Files can be processed individually or as a batch for optimum efficiency. The company states that processing takes about three seconds per file and none of the original GC/MS or LC/MS data/information is lost. ClearView is compatible with all standard GC/MS file formats, offering significant tangible benefits and is a must have for anyone involved in trace analysis, reports Markes. Key features include: • Aids automatic identification of trace components • Eliminates noise and background • Improves the repeatability of integration/quantitation • Effective for SIM, SCAN and SIM/SCAN data ClearView is now available as a 12-month or convenient time-unlimited license. ClearView data reprocessing software is available free on a 30-day trial period. Click here to download 30-day free trial.
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