Bioanalytical LC-ICP-MS for pharmacokinetics and drug metabolism studies
Jaap Wieling
Xendo Drug Development, Hanzeplein 1, Groningen, The Netherlands

Metabolic profiling of new compounds includes detection of metabolites, structure characterization and quantitative analysis. In some cases the concentrations of the metabolites may be extremely low and highly specific and sensitive analytical methods are then required.

In the absence of a suitable label, the detection of metabolites can be a source of difficulty, and there is always the possibility that some will be missed, especially if they are significantly different in structure from the parent. Even when the detection and identification of metabolites is successful, quantification is not possible with confidence in the absence of synthetic standards, because of the likelihood of changes in the detector response (e.g., UV extinction coefficient) as compared to the parent.

An alternative approach is LC-ICP-MS, which provides a sensitive and highly specific method for the detection of atoms such as phosphorous, bromine, chlorine, and sulfur. Until now LC-ICP-MS has mainly been applied for metals or metalcontaining drug compounds. In drug research phases with a highly exploratory character LC-ICP-MS offers both additional and complementary data in comparison with existing technologies such as LC-UV, LC-MS/MS and LC with radioflow detection; it offers the unique feature of monitoring elements. Although the technique is not widely applied within pharmacology yet, the efficiency of inductively coupled plasma (ICP) in producing single charged positive ions for most elements makes it an effective ionization source for mass spectrometry (MS). ICP-MS is considered the method of choice for elemental analysis because it combines ppt (pg/mL) detection limits, a linear range over 6 to 7 orders of magnitude, multi-elemental and multi-isotopic measurement capabilities, and limited spectral interferences with a high sample throughput, and almost complete elemental coverage.

In metabolite profiling studies, coupling a powerful chromatographic separation technique such as HPLC with MS using an ICP interface is an attractive approach with excellent sensitivity, high selectivity and applications in many disciplines, (pre-)clinical pharmacology in particular. The main advantages are that the response is dependent on the molecular structure, that the technique is excellent in case of absence of authentic standards (vs. MS and UV) and it is not dependent on radiolabeled substances, thereby avoiding potentially hazardous clinical studies.

Our LC-ICP-MS expertise provides a state-of-the-art technique for the bioanalytical support during development of non-metal and metal-based drugs (Pt, Gd, ..) and other compounds or biomarkers containing specific elements. This presentation provides an overview of the applications that we have run on out LC-ICP-MS platform in the context of pre-clinical and clinical pharmacology.

Quantitative bioanalysis en metabolite identification by LC-MS: Continuous developments and innovations
W.M.A. Niessen
hyphen MassSpec, de Wetstraat 8, 2332 XT Leiden, NL

Bioanalysis is concerned with the analysis of endogenous or exogenous compounds in biological matrices. It plays an essential role in drug development within the pharmaceutical industry. In this presentation, attention is paid to developments in bioanalysis using LC–MS. After a brief discussion on developments in quantitative bioanalysis, most attention is paid to LC–MS technologies for the detection and identification of drug metabolites (and other related drug substances). Various MS-related analytical strategies are discussed and illustrated with examples. The potential of various types of tandem MS instruments in metabolite identification is explored: triple quadrupoles, three-dimensional and linear ion traps, hybrid instruments like quadrupole–time-of-flight, quadrupole–linear-ion-trap, and ion-trap–time-of-flight. In addition, the potential of these instruments is illustrated with various data-acquisition strategies, e.g., full-spectrum product-ion MS–MS, various data-dependent acquisition strategies, data-independent acquisition, and precursor-ion and neutral-loss analysis modes. This critical overview provides a perspective on the current state of the art in metabolite identification by LC–MS.

Design and Validation of Hyphenated Analtytical Technologies in Pharmaceutical Development
Wim Luijten
Department of Structural Analysis, Technologie Servier, France

The need to characterize low level impurities and degradants in drug substances and drug products, combined with an ever increasing demand to speed up pharmaceutical development has led to an extensive use of combined separation and spectrometric methods for structural identification and quantification. These so-called hyphenated techniques have over the past fifteen years changed the way in which impurity analysis is being performed. Instrumental performance and reliability have in an important way contributed to the rapid expansion of LC/MS, (L/C)MS/MS and, more recently, LC-NMR in pharmaceutical development.

Impurities in drug substances can be of a wide variety, ranging from synthetic intermediates to compounds resulting from secondary reactions or degradation. Whereas these compounds are generally related to the chemical structure of the drug substance, impurities originating from cross contamination in, for instance, generic and illicit copies of drug substances are generally more difficult to identify. In drug products, interactions between excipients or impurities in excipients and the drug substance are additional sources of impurities, representing another challenge to the analyst. A common point in these questions is the fact that the analyst is looking for compounds present at typical concentration levels of 0.05 to 0.5 per cent. Obviously, a successful analysis imposes both highly efficient separation methods, combined with sensitive spectroscopic techniques.

Impurities control begins with identification, where the combined efforts of liquid chromatography, NMR and Mass Spectrometry are of the utmost importance. Whereas in LC/MS a variety of methods can be used in on-line experiments, the relatively low intrinsic sensitivity of NMR compared to mass spectrometry limits the use of the various NMR techniques in on-line LC-NMR. Alternatively, experiments under stopped-flow conditions or concentrating elutes on solid phase cartridges are possible to enhance the quality of the NMR spectra. The possibility to obtain both MS and NMR spectral information directly on eluting compounds considerably speeds up the identification process and helps to decide if and which further methods are to be used to unambiguously identify impurities.

In this presentation the various possible methods will be illustrated in some typical examples.

Crystal City III - European Bioanalytical Forum Initiative
Philip Timmerman
Johnson & Johnson, Beerse, Belgium

No abstract available

Abstracts van Posters

Extraction of basic compounds by cation-exchange  SPE followed by normal-phase LC ESI-MS/MS: A METHOD TRANSFER.
Is IT as straightforward as it sounds?
James I. Taylor & Mark Churchill.
Aptuit (Edinburgh) Ltd, Research Avenue South, Heriot-Watt University Research Park, Riccarton, Edinburgh EH14 4AP, UK

One analyte and two metabolites, along with their [¹³C₄] analogues as internal standards, were extracted from human plasma by utilising cation-exchange SPE as part of a multi-site method transfer. Analytical difficulties with the transfer resulted in different wash solutions being evaluated during the SPE after it was discovered that the use of 100 mmol/L HCl caused blocking of the silica-based sorbents. The use of 96-well plate technology was investigated to improve throughput of samples compared to 3 mL barrel cartridges and this was further optimised using two silica-based strong cation exchange sorbents and two polymeric strong cation exchange sorbents to improve compound recovery, selectivity and flow through capabilities. The compounds were separated on a BetaSil 100 column (Thermo, UK) using an isocratic mix of 10 mmol/L ammonium acetate (pH 5) and acetonitrile (33:67% v/v) and quantified using a Sciex API 4000 in positive ion mode. Separation was achieved via a normal phase mechanism, but it was found that the selectivity of the silica phase could be altered by changing the ionic strength of the buffer from 5 to 10 mmol/L. This resulted in a more reversed-phase elution order, due to increased shielding of the silanol groups on the stationary phase by cations in the mobile phase. The presence of ghost peaks and their effect on ion suppression is also discussed.

The result of this optimisation process was a robust, accurate and precise method which met regulatory validation requirements.

UPLC in BioAnalysis: Comparison of 3 HPLC-MS/MS validated methods to UPLC-MS/MS methods
Lieve Dillen¹, Luc Diels¹, Willy Cools¹, Marleen Van Wingerden², Davy Petit², Ronald De Vries¹ and Philip Timmerman¹
1. Johnson & Johnson Pharmaceutical Research and Development, Department of Bioanalysis, Beerse, Belgium
2  Waters, Vilvoorde, Belgium

Technological improvements have made possible chromatographic separations with 1.7 µm particle size columns at elevated pressures. Known as Ultra Performance Liquid Chromatography (UPLC) this technology promises reduced separation times with increased separation efficiency. Therefore, we have evaluated UPLC in a bioanalytical lab for quantitative analysis of drug candidates with the aim to improve throughput without jeopardizing quality. We have transferred 3 validated LC-MS/MS assays with conventional LC separation to UPLC-MS/MS and present data that show that UPLC performs equivalent to HPLC with respect to linearity, accuracy, precision and robustness of the assays. Concerning carryover we have experienced that careful optimization of wash solvents for the injector is needed for optimal results.
An average gain in chromatographic throughput of 4 was realized with a concomitant solvent reduction of a factor 5. Overall the data indicate that UPLC adds value especially for high throughput quantitative analysis of large number of samples and does not compromise the chromatographic separation quality, neither accuracy, precision and robustness.

A sensitive LC-MS/MS method for the quantitative determination of dexamethasone in porcine plasma and porcine muscle, liver and kidney tissue.
F. Venema, M.J. Dröge and E. Oosting
Analytisch Biochemisch Laboratorium (ABL) BV, Assen, The Netherlands

Introduction Dexamethasone is a synthetic glucocorticoid, which has been used for many years in the treatment of metabolic and inflammatory diseases in human and veterinary medicine. Within the European Union, the use of dexamethasone is approved in cattle only for therapeutic indications, and tissues and milk intended for human consumption have to be analysed for their maximal residue limits (MRLs). The following MRLs for dexamethasone in cattle have been established by the Committee for Veterinary Medicinal Products (EMEA): 2 µg/kg in liver, 0.75 µg/kg in kidney and muscle, and 0.3 µg/kg in milk, respectively.  As a result, sensitive analytical methods are required to determine MRLs in edible tissues derived from cattle. The ultimate goal of our work was to develop and validate a sensitive and reliable bioanalytical LC-MS/MS method suitable for the determination of dexamethasone in porcine muscle, liver and kidney tissue and in porcine plasma.

Materials and Methods Porcine tissues were homogenised using a saline solution. Plasma and tissue homogenate samples were supplemented with an internal standard (prednisolone) and subjected to liquid-liquid extraction using acetonitrile. After dilution with water and a washing step with pentane, the samples were extracted using TBME. The evaporated residues were dissolved in injection solvent and injected into the LC-MS/MS system for quantification. The samples were chromatographed on a LUNA C18 LC column (3 µm, 150 x 3.0 mm). The mass-spectrometer consisted of a Sciex API 4000 equipped with a Turbo Ion Spray interface and was operating in the positive ion mode. A full validation of the method was performed according to Volume 8 of The Rules governing Medicinal Products in the European Community (2). The calibration range for dexamethasone covered 0.10 MRL – 10.0 MRL for porcine tissue and 0.100 – 25.0 ng/mL for porcine plasma. The validation included the determination of the parameters: calibration, accuracy and precision, recovery, specificity, dilution, and stability.

Results The assay for dexamethasone was validated in the concentration range of 0.0750–7.50 µg/kg for kidney and muscle tissue, 0.200–20.0 µg/kg for liver tissue, and 0.100–25.0 ng/mL for plasma, respectively. The method showed acceptable accuracies (expressed as bias) and precisions (expressed as CV) for all tissues (Table 1). In porcine plasma, accuracies of -5.8, -6.4, -10.7 and -8.7% and precisions of 11.9, 12.6, 9.1 and 6.5% were obtained at the QC levels LLOQ (0.100 ng/mL), Low (0.500 ng/mL), Medium (5.00 ng/mL) and High (20.0 ng/mL), respectively. A lower limit of quantitation (LLOQ) of 0.10 MRL and 0.100 ng/mL was achieved in porcine tissue and plasma, respectively. In addition, the method was found valid with respect to recovery (>55% in tissue), specificity, the 10-fold dilution of samples with blank matrix, and stability (24 h storage of samples on the autosampler and one additional freeze/thaw cycle) for all matrices.

Table 1. Accuracy and precision of dexamethasone in porcine muscle, liver and kidney tissue.

Discussion and Conclusion At ABL, a sensitive bioanalytical LC-MS/MS method for the determination of dexamethasone in porcine plasma, muscle, liver and kidney tissue has been developed and validated successfully. The method produced accurate and precise results, and very low LLOQs were achieved (up to 0.10 MRL in porcine tissue) in comparison to other available methods for the determination of dexamethasone (usually 0.50 MRL). Due to the low LLOQ, the assay is extremely suitable for the quantitative determination of dexamethasone in porcine plasma, muscle, liver and kidney tissue and, as such, the assays offers a competitive alternative to the methods available for the investigation of the pharmaco- and depletion kinetics of dexamethasone in porcine plasma, muscle, liver and kidney tissue. Moreover, this method can be used as a lead for the validation of dexamethasone in other (cattle) species.

References 1. EMEA 2001. Committee for veterinary Products. Dexamethasone. Summary Report. 2. Volume 8 of The Rules governing Medicinal Products in the European Union. Notice to Applicants and Guideline. Establishment of maximum residue limits (MRLs) for residues of veterinary medicinal products in foodstuffs of animal origin, October 2005.

A Validated method for the analysis of Risperidone in Human Plasma using UPLC/MS/MS.
Iain Gibb¹, Ed Sprake¹, Steve Preece¹, Erin McManus², Diane Diehl²
1. Waters Corporation, MS Technologies Centre, Manchester, UK
2. Waters Corporation, Milford, MA 01757, USA

HPLC/MS/MS is the technique of choice for the quantification of drug substances in biological matrices during drug development and pharmacokinetic studies. The inherent sensitivity and selectivity of this technique allow robust analysis methods with short chromatographic retention times to developed so that fast sample turnaround can be achieved. However, there are potential  challenges with LC/MS/MS.

By reducing analysis times, the probability of the drug substance co-eluting with an interfering compound is increased. Ion suppression due to endogenous   compounds in biological matrixes can lead to deterioration in the limit of quantification (LLOQ) or interferences from co-eluting drug metabolites can give falsely elevated responses for the drug substances.

Improved sample preparation can reduce these effects, but it is often necessary to develop longer chromatographic methods to separate the drugs from interferences. This results in reduced sample throughput.

In this project we have utilized Ultra Performance LC^TM (UPLC^TM) coupled to a tandem quadruple mass spectrometer to develop and validate a bioanalytical method for the determination of Risperidone and it’s major metabolite, 9– Hydroxyrisperidone, in human plasma using Clozapine as an internal standard. UPLC allows the use of shorter run times with maintaining or increasing the  chromatographic resolution reducing the probability of matrix interferences.

A guide for the HPLC Separation of Small Molecules
Vlad Orlovsky¹, Gezinus Grooten², Noud Grimberg²
1. SIELC Technologies, IL 60070, USA
2. Aurora Borealis Control B.V., P.O. Box 2, 7760-AA Schoonebeek, Netherlands

Mixed mode chromatography is a powerful technique for separation of various compounds. These separations are based on a combination of reserved phase, ion-exchange, ion-exclusion or pi-pi interactions. Polar and hydrophobic, acid and basic compounds, zwitterions and neutral molecules can be separated with high selectivity during the run. This guide will help in developing methods for separation of small molecules with different properties.. The flexibility of mixed mode chromatography allows you to find conditions for different detection techniques (UV, MS, ELSD, IR), and provides easy scale-up and high throughput capabilities. This mixed mode technology works well with difficult matrices and a variety of samples diluents.

Ligand fishing with SPR-LC/MS
E. C. A. Stigter, G. J. de Jong, W.P. van Bennekom
Utrecht University, Faculty of Pharmaceutical Sciences, Department of Biomedical Analysis, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands

Biomolecular Interaction Analysis – Mass Spectrometry is an approach that combines Surface Plasmon Resonance (SPR) technology with Mass Spectrometry (MS). SPR sensing is a well-known non-destructive optical technique capable of detecting minute amounts of protein binding to or dissociating from receptor molecules immobilised on a sensor surface. The combination of both techniques provides a means for selective binding, recovery and identification of specific proteins (i.e. based on their molecular mass) from complex biological matrices.
As a rule SPR and MS have been used separately (off line). Originally, MALDI-ToF MS was performed directly on the SPR chip¹, but nowadays the bio molecules isolated with the SPR sensor are released using pre-programmed washing and eluting steps² and used for further study or identification. Direct coupling of SPR and MS can be beneficial in terms of sample throughput, but proves to be difficult due to the bulky nature of most of the current SPR devices, their liquid handling and the use of chaotropic regeneration agents used to recover material from the sensor surface, possibly causing compatibility problems with MS detection. A disadvantage of the SPR-MS approach is that the molecular sequence of the proteins remains unelucidated. In proteomics research the sequence is commonly determined with LC/MS.

Therefore a study was initiated to investigate the coupling of SPR with LC/MS for the efficient transfer of ligands isolated with the SPR sensor for further analysis. The strategy of Natsume et al.³ who used on-chip digestion followed by elution and concentration on a trapping column prior to LC-MS/MS analysis was used as a starting point.
Attention is paid to SPR sensor chip reusability, to the transfer efficiency of the isolated protein as well as to the on-line protein tryptic digestion followed by separation and detection of the formed peptides using LC/MS. Recent data on our investigation will be presented.

(1) Nelson, R. W.; Krone, J. R. Anal. Chem. 1997, 69, 4363-4368.
(2) Sönksen, C. P.; Nordhoff, E.; Jansson, Ö.; Malmqvist, M.; Roepstorff, P. Anal. Chem. 1998, 70, 2731-2736.
(3) Natsume, T.; Nakayama, H.; Jansson, Ö.; Isobe, T.; Takio, K.; Mikoshiba, K. Anal. Chem. 2000, 72, 4193-4198.

Protein depletion and desalting with microchip CE using conductivity and MS detection
L.H.H. Silvertand¹, E. Machtejevas², R. Hendriks³, K.K. Unger², W.P. van Bennekom¹, G.J. de Jong¹
1. Biomedical Analysis, Pharmaceutical Analysis, University of Utrecht,P.O. Box 80082, 3568 TB Utrecht, The Netherlands
2. Institut für Anorganische und Analytische Chemie, Johannes Gutenberg Universität, Duesbergweg 10-14, D55099 Mainz, Germany
3. LSA R&D/ Bioseparation, Merck KGaA, Frankfurterstrasse 250, P.O. Box 64271, 64293 Darmstadt, Germany

One of the problems in the separation of complex biological samples with liquid chromatography and capillary electrophoresis, is the presence of large amounts of salts, proteins, peptides, and other biological compounds. With the microchip CE system described, a part of the sample can be injected into a second separation channel after preconcentration or preseparation. Both columns can be run in different capillary electrophoresis modes with this column coupling microchip device (The Merck IonChipTM), for example isotachophoresis-zone electrophoresis (ITP-CZE) or zone electrophoresis-zone electrophoresis (CZE-CZE). Sample preparation, e.g. desalting or protein depletion, is the first step and is followed by an electrophoretic separation step. The sample used is a mixture of five peptides spiked with Cytochrome C and Human Serum Albumin. A 1 µL sample volume is injected onto the first capillary, which can be used for preconcentration (ITP mode) and/or separation (CZE mode). When the sample reaches a first conductivity detector (CD1), a time-lag can be programmed, which directs part of the sample into the second separation channel, via a T-split implemented on the chip. The rest of the sample is injected into a waste channel. When the depletion or selection of certain compounds is completed, the compounds are separated in the second separation channel and detected with a second conductivity detector. Results of desalting a mixture of proteins with ITP-CZE and depletion of proteins from a mixture of peptides and proteins, using CZE-CZE are presented.
Furthermore we attempted to couple the IonChipTM to an ESI-iontrap mass spectrometer in two different ways. The direct coupling was unsuccessful due to software problems. In the second coupling method, the compounds are first trapped on a C18 monolithic column and also separated by a C18 monolithic separation column and subsequently directed to the MS. First results of transferring a peptide successfully from the IonChipTM to the MS are shown. Depletion and subsequent mobilization of the compounds to the MS has not yet been successful.

Stability studies of recombinant human Growth Hormone in pharmaceutical preparations by capillary zone electrophoresis with bilayer-coated capillaries
Javier Sastre Toraño, Jonatan R. Catai, Gerhardus J. de Jong and Govert W. Somsen
Department of Biomedical Analysis, Utrecht University, P.O. Box 80082, 3508 TB Utrecht.

Pharmaceutical regulatory agencies, such as the European Pharmacopoeia, are starting to recognize capillary zone electrophoresis (CZE) as an effective means for the analysis of biopharmaceuticals (1,2). CZE has a unique, charged-based separation mechanism which can be very useful for the study of protein degradation and glycoforms. Furthermore, CZE uses only minute amounts of sample and provides fast separations and high efficiency. However, interactions of proteins with the internal capillary wall may lead to band broadening, unstable electoosmotic flow (EOF) and poor migration-time reproducibility. This brings serious problems when monitoring the quality of biopharmaceutical preparations and comparing samples. An effective way to avoid these problems can be the coating of the capillary with polybrene (PB) and poly(vinyl sulfonate) (PVS), forming a bilayer of charged polymers. This double coating has shown excellent migration time reproducibility and high plate numbers for proteins (3). Based on this work, we have developed an improved method for studying the stability of the recombinant human growth hormone (hGH) by CZE.

Test samples of hGH were prepared by degradation with 3% hydrogen peroxide or by prolonged exposure to heat (40 °C). Aliquots were taken at appropriate time intervals and analyzed by CZE using PB-PVS coated capillaries and 400 mM TRIS phosphate (pH 8.5) as background electrolyte. Repeatable profiles with narrow and symmetrical peaks were obtained. CZE of hydrogen peroxide treated samples revealed 8 degradation products after 18h. Heated samples showed growing amounts of deamidated and dideamidated hGH in time. After 100h, deamidated hGH started to degrade further, resulting in a total of 10 peaks after 170h. The sum of the peak areas of the degradation products was in good correspondence with the decrease of the peak area of the mother compound hGH. These results show the potential of the system for quantitative degradation analysis in time.
The applicability of the system is illustrated by the analysis of commercially hGH preparations, showing varying degree of degradation. Compared to the Pharmacopoeia method using bare fused silica capillaries (1), the resolution was improved, total analysis times were shorter (8.5 min vs. 24 min), plate numbers were higher (55000 vs. 300000) and migration time reproducibility was better (RSD=1.1% vs. RSD=8%). Moreover, additional degradation products were revealed demonstrating the feasibility of the system for biopharmaceutical quality control.

(1) Monograph: Somatropin for injection, European Pharmacopoeia 5.3 2005, 3619-3621.
(2) Monograph: Erythropoietin concentrated solution, European Pharmacopoeia 5.3 2005, 3494-3498.
(3) Catai, J.R., Tervahauta, H.A., De Jong, G.J., Somsen, G.W., J. Chromatogr. A 2005; 1083, 185-192.

CE-MS of pharmaceutical proteins using bilayer-coated capillaries
Jonatan R.Catai, Javier Sastre Toraño, Gerhardus J. de Jong and Govert W. Somsen
Department of Biomedical Analysis, Utrecht University, P.O. Box 80082, 3508 TB Utrecht

Capillary electrophoresis (CE) is an attractive tool for purity and stability studies of proteins as it provides fast and efficient separations and requires only minute amounts of sample. Changes in protein charge and shape are reflected in the electrophoretic mobility and, moreover, CE can be carried out using quasi-physiological conditions. The combination of CE with mass spectrometry (MS) is even more powerful as it offers both separation and characterization of proteins and their degradation products. However, CE of proteins using bare fused-silica capillaries may suffer from analyte-wall interactions leading to band broadening, unstable electroosmotic flow (EOF) and poor migration time reproducibilities. To overcome these problems, various capillary coating procedures have been proposed but they are often laborious and lack stability. Recently, we have suggested a simple, fast and reproducible coating method based on a bilayer of charged polymers and showed its usefulness for the analysis of proteins and peptides (1-4). In this paper, the feasibility of this coating for the CE-MS analysis of (pharmaceutical) proteins was investigated.

CE was coupled to an ion-trap mass spectrometer via an electrospray ionization (ESI) sheat-flow interface. Capillaries were coated with a bilayer of polybrene (PB) and poly(vinyl sulfonate) (PVS). The coating showed full compatibility with MS detection, and yielded a significant and constant EOF independent of the pH. The influence of several volatile background electrolytes (BGE) (pH 7.0-8.5) on plate number, migration time reproducibility, and MS signal intensity was examined for the tested proteins. Very stable protein separations (migration-time RSDs < 1%) and satisfactory plate numbers (ca. 100,000) were obtained with CE-MS using a BGE of 75 mM ammonium formate (pH 8.5). The applicability of the CE-MS method using bilayer coatings will be demonstrated by the stability monitoring of the biopharmaceutical human growth hormone (hGH) revealing e.g. Deamidation and oxidation products. Furthermore, the results of the CE-MS analysis of several (expired) hGH preparations of commercial sources will be discussed.

(1) Catai, J.R., Somsen, G.W., de Jong, G.J., Electrophoresis 2004, 25, 817-824
(2) Catai, J.R., Tervahauta, H.A., de Jong, G.J., Somsen, G.W., J. Chromatogr. A 2005, 1083, 185-192
(3) Catai, J.R., Sastre Toraño, J., de Jong, G.J., Somsen, G.W., Electrophoresis 2006, 27, 2091-2099
(4) Catai, J.R., Sastre Toraño, J., de Jong, G.J., Somsen, G.W., Analyst, DOI:10.1039/b607178c