Publications by authors named "Laurent Geiser"

29 Publications

  • Page 1 of 1

Toxic doses of caffeine are needed to increase skeletal muscle contractility.

Am J Physiol Cell Physiol 2019 02 19;316(2):C246-C251. Epub 2018 Dec 19.

Institute of Sport Sciences, University of Lausanne , Lausanne , Switzerland.

Discrepant results have been reported regarding an intramuscular mechanism underlying the ergogenic effect of caffeine on neuromuscular function in humans. Here, we reevaluated the effect of caffeine on muscular force production in humans and combined this with measurements of the caffeine dose-response relationship on force and cytosolic free [Ca] ([Ca]) in isolated mouse muscle fibers. Twenty-one healthy and physically active men (29 ± 9 yr, 178 ± 6 cm, 73 ± 10 kg, mean ± SD) took part in the present study. Nine participants were involved in two experimental sessions during which supramaximal single and paired electrical stimulations (at 10 and 100 Hz) were applied to the femoral nerve to record evoked forces. Evoked forces were recorded before and 1 h after ingestion of 1) 6 mg caffeine/kg body mass or 2) placebo. Caffeine plasma concentration was measured in 12 participants. In addition, submaximal tetanic force and [Ca] were measured in single mouse flexor digitorum brevis (FDB) muscle fibers exposed to 100 nM up to 5 mM caffeine. Six milligrams of caffeine per kilogram body mass (plasma concentration ~40 µM) did not increase electrically evoked forces in humans. In superfused FDB single fibers, millimolar caffeine concentrations (i.e., 15- to 35-fold above usual concentrations observed in humans) were required to increase tetanic force and [Ca]. Our results suggest that toxic doses of caffeine are required to increase muscle contractility, questioning the purported intramuscular ergogenic effect of caffeine in humans.
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http://dx.doi.org/10.1152/ajpcell.00269.2018DOI Listing
February 2019

Inhibition screening method of microsomal UGTs using the cocktail approach.

Eur J Pharm Sci 2015 Apr 12;71:35-45. Epub 2015 Feb 12.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address:

A rapid method for the simultaneous determination of the in vitro activity of the 10 major human liver UDP-glucuronosyltransferase (UGT) enzymes was developed based on the cocktail approach. Specific substrates were first selected for each UGT: etoposide for UGT1A1, chenodeoxycholic acid for UGT1A3, trifluoperazine for UGT1A4, serotonin for UGT 1A6, isoferulic acid for UGT1A9, codeine for UGT2B4, azidothymidine for UGT2B7, levomedetomidine for UGT2B10, 4-hydroxy-3-methoxymethamphetamine for UGT2B15 and testosterone for UGT2B17. Optimal incubation conditions, including time-based experiments on cocktail metabolism in pooled HLMs that had been performed, were then investigated. A 45-min incubation period was found to be a favorable compromise for all the substrates in the cocktail. Ultra-high pressure liquid chromatography coupled to an electrospray ionization time-of-flight mass spectrometer was used to separate the 10 substrates and their UGT-specific glucuronides in less than 6 min. The ability of the cocktail to highlight the UGT inhibitory potential of xenobiotics was initially proven by using well-known UGT inhibitors (selective and nonselective) and then by relating some of the screening results obtained by using the cocktail approach with morphine glucuronidation (substrate highly glucuronidated by UGT 2B7). All the results were in agreement with both the literature and with the expected effect on morphine glucuronidation.
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http://dx.doi.org/10.1016/j.ejps.2015.02.001DOI Listing
April 2015

Phenotyping of CYP450 in human liver microsomes using the cocktail approach.

Anal Bioanal Chem 2014 Aug 4;406(20):4875-87. Epub 2014 Jun 4.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211, Geneva 4, Switzerland.

The cocktail approach is an advantageous strategy used to monitor the activities of several cytochromes P450 (CYPs) in a single test to increase the throughput of in vitro phenotyping studies. In this study, a cocktail mixture was developed with eight CYP-specific probe substrates to simultaneously evaluate the activity of the most important CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and the CYP3A subfamily. After cocktail incubation in the presence of human liver microsomes (HLMs), the eight selected substrates and their specific metabolites were analyzed by ultra-high-pressure liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry. Qualitative and quantitative data were simultaneously acquired to produce an overview of the extended phase I biotransformation routes for each probe substrate in the HLMs and to generate phenotypic profiles of various HLMs. A comparison of the cocktail strategy with an individual substrate assay for each CYP produced similar results. Moreover, the cocktail was tested on HLMs with different allelic variants and/or in the presence of selective inhibitors. The results were in agreement with the genetic polymorphisms of the CYPs and the expected effect of the alterations. All of these experiments confirmed the reliability of this cocktail assay for phenotyping of the microsomal CYPs.
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http://dx.doi.org/10.1007/s00216-014-7915-4DOI Listing
August 2014

A cocktail approach for assessing the in vitro activity of human cytochrome P450s: an overview of current methodologies.

J Pharm Biomed Anal 2014 Dec 28;101:221-37. Epub 2014 Mar 28.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address:

An assessment of cytochrome P450 (CYP) enzyme activity is essential for characterizing the phase I metabolism of biological systems or to evaluate the inhibition/induction properties of xenobiotics. CYPs have generally been investigated individually by single probes, and metabolite formation has been monitored by liquid chromatography-mass spectrometry (LC-MS). To increase the throughput, many probes have been applied to assess multiple CYP activities simultaneously within a single experiment. This strategy is called the cocktail approach, and it has already been reviewed for in vivo applications, but never for in vitro ones. This review focuses for the first time on an in vitro cocktail approach, and it references the most notable articles on this topic. The advantages and limitations of applying cocktails for the in vitro activity assessment of major human CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and subfamily CYP3A, are discussed. This article considers the probe reaction selections for each CYP according to regulatory recommendations, probe metabolic properties (i.e., specificity and turnover), probe concentrations and analytical sensitivity, but it also highlights a challenge specific to cocktail design, which is probe-probe interaction. The last part of the review reports some methodologies for incubating these cocktails and discusses some important issues regarding the incubation time, enzyme concentrations and sample preparation.
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http://dx.doi.org/10.1016/j.jpba.2014.03.018DOI Listing
December 2014

Contribution of various types of liquid chromatography-mass spectrometry instruments to band broadening in fast analysis.

J Chromatogr A 2013 Oct 14;1310:45-55. Epub 2013 Aug 14.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.

When performing fast LC with 50mm narrow-bore columns packed with small particles, the LC instrumentation can give rise to non-negligible band broadening. In the present study, the loss in chromatographic efficiency attributed to nine different mass spectrometers of various brands, ionization source geometries and types of analyzers was assessed. In their standard configurations, the extra-column variance of these UHPLC-MS systems was estimated to vary from 20 to >100 μL(2). However, it was demonstrated that these differences arise exclusively from the chromatographic system (i.e., injector, tubing, valves, heater) and from the tubing employed to interface the UHPLC instrument with the MS device. By minimizing the tubing used for each UHPLC system, the extra-column variance was reduced to approximately 17-19 μL(2) at 600 μL/min, for all types of configurations. To achieve optimal chromatographic performance, it is therefore of prime importance to optimize the UHPLC configuration prior to conducting MS. The tubing located between the UHPLC system and the ionization source entrance was found to be particularly critical, as it contributes to band broadening even in the gradient mode. Using an optimized UHPLC-MS configuration, the loss in efficiency with a 50 × 2.1mm I.D. column was negligible for k>7. However, the efficiency loss with 1mm I.D. columns remained non-negligible for all current instrumentation, even for solutes with a value of k>20. Indeed, for a mixture of isobaric substrates and metabolites analyzed in gradient mode, the peak widths decreased by approximately 50% between a standard and optimized UHPLC-MS configuration, considering a 50 × 2.1mm, 1.7 μm column. The peak broadening was changed by 230% on a 50 × 1 mm, 1.7 μm stationary phase, for the same system configurations.
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http://dx.doi.org/10.1016/j.chroma.2013.08.001DOI Listing
October 2013

EasyProt--an easy-to-use graphical platform for proteomics data analysis.

J Proteomics 2013 Feb 28;79:146-60. Epub 2012 Dec 28.

Swiss Center for Applied Human Toxicology (SCAHT), Geneva, Switzerland.

High throughput protein identification and quantification analysis based on mass spectrometry are fundamental steps in most proteomics projects. Here, we present EasyProt (available at http://easyprot.unige.ch), a new platform for mass spectrometry data processing, protein identification, quantification and unexpected post-translational modification characterization. EasyProt provides a fully integrated graphical experience to perform a large part of the proteomic data analysis workflow. Our goal was to develop a software platform that would fulfill the needs of scientists in the field, while emphasizing ease-of-use for non-bioinformatician users. Protein identification is based on OLAV scoring schemes and protein quantification is implemented for both, isobaric labeling and label-free methods. Additional features are available, such as peak list processing, isotopic correction, spectra filtering, charge-state deconvolution and spectra merging. To illustrate the EasyProt platform, we present two identification and quantification workflows based on isobaric tagging and label-free methods.
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http://dx.doi.org/10.1016/j.jprot.2012.12.012DOI Listing
February 2013

Wipe sampling procedure coupled to LC-MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces.

Anal Bioanal Chem 2012 Mar 24;402(8):2499-509. Epub 2011 Jun 24.

Pharmacy, Geneva University Hospitals, 1211 Geneva 14, Switzerland.

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC-MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm(-2)) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.
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http://dx.doi.org/10.1007/s00216-011-5157-2DOI Listing
March 2012

Shotgun proteomics: a relative quantitative approach using Off-Gel electrophoresis and LC-MS/MS.

Methods Mol Biol 2011 ;681:459-72

Swiss Centre for Applied Human Toxicology, Geneva University, Geneva, Switzerland.

Shotgun proteomics originated as a strategy to identify proteins in complex protein mixtures, but it is also possible to obtain information on relative quantitation with some adjustments to the procedure. After protein digestion, the resulting peptide mixture is labelled with isobaric tags. Then, labelled peptides are submitted to two orthogonal techniques: first, peptides are separated according to their isoelectric point (pI) by Off-Gel electrophoresis (OGE), a relatively new isoelectric focusing (IEF) technique; after peptide purification, they are then separated in a second dimension according to their hydrophobic properties by reversed-phase liquid chromatography (RPLC). Finally, following detection by mass spectrometry (MS) and sequencing by tandem mass spectrometry (MS/MS), proteins are matched by means of bioinformatics software, and protein ratios are calculated by comparing isobaric tagged reporter fragments to highlight the different expression of one protein in one sample relative to other samples.
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http://dx.doi.org/10.1007/978-1-60761-913-0_27DOI Listing
February 2011

Shotgun proteomics: a qualitative approach applying isoelectric focusing on immobilized pH gradient and LC-MS/MS.

Methods Mol Biol 2011 ;681:449-58

Swiss Centre for Applied Human Toxicology, Geneva University, Geneva, Switzerland.

Shotgun proteomics is a rapid and near universal strategy to identify proteins in complex protein mixtures. After protein digestion, the resulting peptide mixture is submitted to two orthogonal techniques: peptides are first separated according to their isoelectric point (pI) by isoelectric focusing (IEF) on immobilized pH gradient (IPG); after peptide extraction, they are then separated in the second dimension according to their hydrophobic properties by reverse phase liquid chromatography (RPLC). Finally, they are detected by tandem mass spectrometry (MS/MS) and proteins are matched by means of bioinformatics software.
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http://dx.doi.org/10.1007/978-1-60761-913-0_26DOI Listing
February 2011

Simultaneous quantification of ten cytotoxic drugs by a validated LC-ESI-MS/MS method.

Anal Bioanal Chem 2010 Dec 7;398(7-8):3033-42. Epub 2010 Oct 7.

Pharmacy, Geneva University Hospitals (HUG), 1211 Geneva, Switzerland.

A liquid chromatography separation with electrospray ionisation and tandem mass spectrometry detection method was developed for the simultaneous quantification of ten commonly handled cytotoxic drugs in a hospital pharmacy. These cytotoxic drugs are cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. The chromatographic separation was carried out by RPLC in less than 21 min, applying a gradient elution of water and acetonitrile in the presence of 0.1% formic acid. MS/MS was performed on a triple quadrupole in selected reaction monitoring mode. The analytical method was validated to determine the limit of quantification (LOQ) and quantitative performance: lowest LOQs were between 0.25 and 2 ng mL(-1) for the ten investigated cytotoxic drugs; trueness values (i.e. recovery) were between 85% and 110%, and relative standard deviations for both repeatability and intermediate precision were always inferior to 15%. The multi-compound method was successfully applied for the quality control of pharmaceutical formulations and for analyses of spiked samples on potentially contaminated surfaces.
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http://dx.doi.org/10.1007/s00216-010-4243-1DOI Listing
December 2010

Non-aqueous capillary electrophoresis 2005-2008.

Electrophoresis 2009 Jan;30(1):36-49

Biomedical Proteomics Research Group, Department of Bioinformatics and Structural Biology, University of Geneva, Geneva, Switzerland.

This review article presents recent developments and applications of non-aqueous capillary electrophoresis (NACE): The text covers the period from the previous review (L. Geiser, J. L. Veuthey, Electrophoresis 2007, 28, 45-57) to summer 2008. We focus primarily on the analysis of pharmaceutical drugs by non-aqueous solvents in CZE within different matrices including phytochemical plant extracts and biological fluids. We also extend our discussion to other application fields (e.g. food material and environmental samples) and to chiral separations by NACE. This review focuses on practical aspects of NACE, illustrating which organic solvents and electrolytes are best suited for NACE analyses and their compatibilities with different detection techniques, including UV, LIF and MS. The review emphasizes the interests of using non-aqueous solvents in place of water for the analysis by CZE, and as an alternative to MEKC.
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http://dx.doi.org/10.1002/elps.200800494DOI Listing
January 2009

In-line system containing porous polymer monoliths for protein digestion with immobilized pepsin, peptide preconcentration and nano-liquid chromatography separation coupled to electrospray ionization mass spectroscopy.

J Chromatogr A 2008 Apr 29;1188(2):88-96. Epub 2008 Feb 29.

Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.

The use of two different monoliths located in capillaries for on-line protein digestion, preconcentration of peptides and their separation has been demonstrated. The first monolith was used as support for covalent immobilization of pepsin. This monolith with well-defined porous properties was prepared by in situ copolymerization of 2-vinyl-4,4-dimethylazlactone and ethylene dimethacrylate. The second, poly(lauryl methacrylate-co-ethylene dimethacrylate) monolith with a different porous structure served for the preconcentration of peptides from the digest and their separation in reversed-phase liquid chromatography mode. The top of the separation capillary was used as a preconcentrator, thus enabling the digestion of very dilute solutions of proteins in the bioreactor and increasing the sensitivity of the mass spectrometric detection of the peptides using a time-of-flight mass spectrometer with electrospray ionization. Myoglobin, albumin, and hemoglobin were digested to demonstrate feasibility of the concept of using the two monoliths in-line. Successive protein injections confirmed both the repeatability of the results and the ability to reuse the bioreactor for at least 20 digestions.
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http://dx.doi.org/10.1016/j.chroma.2008.02.075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2435401PMC
April 2008

Clinical comparison of fixation methods for patellar bone quadriceps tendon autografts in anterior cruciate ligament reconstruction: absorbable cross-pins versus absorbable screws.

Am J Sports Med 2007 Dec 11;35(12):2118-25. Epub 2007 Oct 11.

Sportorthopädie Bern, Klinik Sonnenhof, Buchserstrasse 30, CH-3006 Bern, Switzerland.

Background: Recently, the use of the quadriceps tendon transplant with bone block (patellar bone quadriceps tendon autografts) for anterior cruciate ligament reconstruction has increasingly been reported.

Hypothesis: Clinical results after the implantation of a patellar bone quadriceps tendon autograft fixed with cross-pins or screws will show no significant difference between the 2 techniques with regard to stability, function, and subjective satisfaction.

Study Design: Cohort study; Level of evidence, 2.

Methods: Between 1998 and 2004, 193 patients with anterior cruciate ligament ruptures were implanted with a patellar bone quadriceps tendon autograft. For 100 of these patients, fixation was carried out using absorbable cross-pins, and for the remaining 93, fixation was carried out using absorbable screws. The results were evaluated by means of International Knee Documentation Committee, Noyes, and Lysholm scores, as well as KT-1000 arthrometer measurement and subjective satisfaction.

Results: The mean follow-up postoperative control period was 29 months. In the International Knee Documentation Committee overall evaluation, the pin group showed a significantly better result (P =.03). The values of the Noyes score produced no significant differences. The mean value of the Lysholm score was 94 points in the screw group and 89 points in the pin group (P <.001). Overall, 90% of the patients subjectively judged their conditions as good or very good.

Conclusion: With both operating processes examined, 80% to 90% of the cases achieved good to very good results. The use of cross-pins can be recommended for fixing patellar bone quadriceps tendon autografts.
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http://dx.doi.org/10.1177/0363546507307390DOI Listing
December 2007

Optimization of the porous structure and polarity of polymethacrylate-based monolithic capillary columns for the LC-MS separation of enzymatic digests.

J Sep Sci 2007 Nov;30(17):2814-20

Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.

The porous structure as well as the polarity of methacrylate ester-based monolithic stationary phases has been optimized to achieve the separation of various peptides originating from enzymatic digestion. The porous structure, determined by the size of both pores and microglobules, was varied through changes in the composition of porogenic solvents in the polymerization mixture, while the polarity was controlled through the incorporation of butyl, lauryl, or octadecyl methacrylate in the polymer backbone. Both the morphology and the chemistry of the monoliths had a significant effect on the retention and efficiency of the capillary columns. The best resolution of peptidic fragments obtained by digestion of Cytochrome c with trypsin in solution was obtained in a gradient LC-MS mode using a monolithic capillary column of poly(lauryl methacrylate-co-ethylene dimethacrylate) featuring small pores and small microglobules. Raising the temperature from 25 to 60 degrees C enabled separations to be carried out at 40% higher flow rates. Separations carried out at 60 degrees C with a steeper gradient proceeded without loss of performance in half the time required for a comparable separation at room temperature. Our preparation technique affords monolithic columns with excellent column-to-column and run-to-run repeatability of retention times and pressure drops.
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http://dx.doi.org/10.1002/jssc.200700185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759379PMC
November 2007

Rapid determination of pK (a) values of 20 amino acids by CZE with UV and capacitively coupled contactless conductivity detections.

Anal Bioanal Chem 2007 Nov 14;389(6):1869-78. Epub 2007 Sep 14.

Laboratoire de Chimie Analytique Pharmaceutique, Section des sciences pharmaceutiques, Université de Genève, Université de Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland.

A rapid and universal capillary zone electrophoresis (CZE) method was developed to determine the dissociation constants (pK (a)) of the 20 standard proteogenic amino acids. Since some amino acids are poorly detected by UV, capacitively coupled contactless conductivity detection (C(4)D) was used as an additional detection mode. The C(4)D coupling proved to be very successful on a conventional CE-UV instrument, neither inducing supplementary analyses nor instrument modification. In order to reduce the analysis time for pK (a) determination, two strategies were applied: (i) a short-end injection to reduce the effective length, and (ii) a dynamic coating procedure to generate a large electroosmotic flow (EOF), even at pH values as low as 1.5. As a result, the analysis time per amino acid was less than 2 h, using 22 optimized buffers covering a pH range from 1.5 to 12.0 at a constant ionic strength of 50 mM. pK (a) values were calculated using an appropriate mathematical model describing the relationship between effective mobility and pH. The obtained pK (a) values were in accordance with the literature.
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http://dx.doi.org/10.1007/s00216-007-1568-5DOI Listing
November 2007

Controlling the surface chemistry and chromatographic properties of methacrylate-ester-based monolithic capillary columns via photografting.

J Sep Sci 2007 Feb;30(3):407-13

Department of Chemistry, University of California, Berkeley, CA, USA.

Preparation of monolithic capillary columns for separations in the CEC mode using UV-initiated polymerization of the plain monolith followed by functionalization of its pore surface by photografting has been studied. The first step enabled the preparation of generic poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths with optimized porous properties, controlled by the percentages of porogens 1-decanol and cyclohexanol in the polymerization mixture, irradiation time, and UV light intensity. Ionizable monomers [2-(methacryloyloxy)ethyl]trimethylammonium chloride or 2-acryloamido-2-methyl-1-propanesulfonic acid were then photografted onto the monolithic matrix, allowing us to control the direction of the EOF in CEC. Different strategies were applied to control the grafting density and, thereby, the magnitude of the EOF. To control the hydrophobic properties, two approaches were tested: (i) cografting of a mixture of the ionizable and hydrophobic monomers and (ii) sequential grafting of the ionizable and hydrophobic monomers. Cografting resulted in similar retention but higher EOF. With sequential grafting, more than 50% increase in retention factors was obtained and a slight decrease in EOF was observed due to shielding of the ionizable moieties.
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http://dx.doi.org/10.1002/jssc.200600316DOI Listing
February 2007

Stability and repeatability of capillary columns based on porous monoliths of poly(butyl methacrylate-co-ethylene dimethacrylate).

J Chromatogr A 2007 Jan 19;1140(1-2):140-6. Epub 2006 Dec 19.

College of Chemistry, University of California, Berkeley, CA 94720-1460, USA.

Monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) capillary columns have been prepared via either thermally or photochemically initiated polymerization of the corresponding monomers and the repeatability of their preparation has been explored. Three separate batches of 5 columns each were prepared using thermal and photochemical initiation for a total of 30 columns. All 30 capillary columns were tested in liquid chromatography-electrospray ionisation mass spectrometry mode for the separation of a model mixture of three proteins--ribonuclease A, cytochrome c and myoglobin. Excellent repeatability of retention times was observed for the proteins as evidenced by relative standard deviation (RSD) values of less than 1.5%. Somewhat broader variations with RSD values of up to 10% were observed for the pressure drop in the columns. The stability of retention times was also monitored using a single monolithic column and no significant shifts in either retention times or back pressure was observed in a series of almost 2200 consecutive protein separations.
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http://dx.doi.org/10.1016/j.chroma.2006.11.079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680358PMC
January 2007

Nonaqueous capillary electrophoresis in pharmaceutical analysis.

Electrophoresis 2007 Jan;28(1-2):45-57

Laboratory of Analytical Pharmaceutical Chemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne,Geneva, Switzerland.

This review presents different solvents and electrolytes commonly used as BGEs in NACE for the analysis of pharmaceutical compounds. Most NACE applications carried out since 1998 for the analysis of compounds of pharmaceutical interest are presented in four tables: (i) analysis of drugs and related substances, (ii) analysis of chiral substances, (iii) analysis of phytochemical extracts and (iv) analysis of drugs in biological fluids. These selected examples are used to illustrate the interest in NACE versus conventional aqueous CE.
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http://dx.doi.org/10.1002/elps.200600265DOI Listing
January 2007

Determination of pKa values by capillary zone electrophoresis with a dynamic coating procedure.

J Sep Sci 2005 Nov;28(17):2374-80

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, EPGL, University of Geneva, Switzerland.

CZE allows to measure the acidic dissociation constant (pKa) of many drug substances. However, determining the EOF intensity may be time-consuming, especially at a low pH. In order to overcome this drawback, a dynamic coating procedure of the capillary was carried out to increase microEOF, and thus to reduce the analysis time. In addition, this coating procedure enhanced migration time stability. The effective mobilities of 15 compounds were measured at different pH, producing pK'a values dependent on BGE ionic strength. The latter values were corrected with the activity coefficient to obtain a "true" pKa value. The 15 investigated compounds were (i) five acids: namely, salicylic acid, benzoic acid, ketoprofen, phenobarbital, and phenol, (ii) four bases: lidocaine, propafenone, propranolol, and quinine, (iii), five ampholytes: sulfanilamide, sulfabenzamide, sulfadimethoxine, sulfadoxine, and sulfisoxazole, and (iv) one zwitterion: cetirizine. The range of determined pKa values was between 1.2 and 11.2, and close to the pKa values available from the literature.
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http://dx.doi.org/10.1002/jssc.200500213DOI Listing
November 2005

Capillary zone electrophoresis for the estimation of transdermal iontophoretic mobility.

J Pharm Sci 2005 Dec;94(12):2667-75

School of Pharmaceutical Sciences, University of Geneva, 30 Quai Ernest-Ansermet, 1211 Geneva, Switzerland.

The objective of the study was to investigate the relationship between transdermal iontophoretic flux--specifically, the electromigratory component--and electrophoretic mobility as determined by capillary zone electrophoresis (CZE). First, the steady-state iontophoretic transport rates of a series of dipeptides across porcine skin were determined in vitro. Co-iontophoresis of acetaminophen was used to quantify the respective contributions of electroosmosis (EO) and electromigration (EM). Second, the electrophoretic mobilities of the dipeptides and three other cationic drugs (lidocaine, propranolol, and quinine) were determined, under equivalent experimental conditions, using CZE. Analysis of the transport data using the results of the CZE experiments revealed a linear dependence (r2 > 0.9) between EM flux and electrophoretic mobility. The CZE measurements also provided insight into the charge state of "zwitterionic" dipeptides, H-Glu-epsilon-Lys-OH and H-Tyr-Gln-OH, revealing that these molecules had partial net negative charges under the formulation conditions, accounting for the absence of anodal iontophoretic delivery. The results suggest that CZE might (i) enable identification of ionization states of complex molecules, (ii) serve as a preliminary screen to identify electrically mobile compounds suitable for iontophoretic delivery, and (iii) prove useful for predicting the EM contribution to transdermal iontophoretic flux.
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http://dx.doi.org/10.1002/jps.20483DOI Listing
December 2005

Rapid stereoselective separations of amphetamine derivatives with highly sulfated gamma-cyclodextrin.

Electrophoresis 2005 Oct;26(20):3910-20

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, EPGL, University of Geneva, Geneva, Switzerland.

The highly sulfated gamma-CD (HS-gamma-CD) is a chiral selector widely used in CE for the enantioseparation of pharmaceutical compounds. This paper investigated different approaches to reduce the stereoselective analysis time of amphetamine (AT) derivatives according to the chiral selector concentration in the BGE. With high HS-gamma-CD concentration, tested analytes were separated in 3.5 min as anionic complexes with short-end injection technique in reversed polarity mode. However, this procedure presented some limitations in terms of efficiency and resolution, excessive Joule heating and poor compatibility with MS detection. With low HS-gamma-CD concentration, compounds were separated as cations. Conventional approaches to reduce CE analysis time demonstrated critical resolution between some analytes. Therefore, the use of the partial-filling technique compatible with MS detection was carried out. Under optimized conditions, the analysis time for the chiral separation of seven AT like compounds was reduced to 6 min. Moreover, sensitivity of CE-MS was sufficient for the determination of ATs in plasma following a simple liquid-liquid extraction.
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http://dx.doi.org/10.1002/elps.200500177DOI Listing
October 2005

Strategies for rapid chiral analysis by capillary electrophoresis.

J Pharm Biomed Anal 2006 Feb 8;40(2):235-41. Epub 2005 Sep 8.

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, University of Geneva, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland.

The aim of this study was to investigate four strategies to decrease chiral CE analysis time: (1) short-end injection technique, (2) high electric field through a capillary length reduction, (3) external pressure application and (4) capillary dynamically coated to generate an important electroosmotic flow. These approaches were applied for a simultaneous enantiomeric separation of amphetamine and four related compounds using a neutral derivatised cyclodextrin (hydroxypropyl-beta-cyclodextrin) as chiral selector. Analysis time and CE performances, in terms of peak efficiency and resolution, were examined. Among the investigated strategies, the dynamic coating procedure appeared to be the most suitable approach to decrease analysis time (inferior to 7 min) and improve sensitivity. Furthermore, it exhibited very good migration time repeatability (0.1%). This benefit is of utmost interest in chiral analysis for an unambiguous peak identification, especially for a complex mixture such as reported in this study.
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http://dx.doi.org/10.1016/j.jpba.2005.07.023DOI Listing
February 2006

Decreasing analysis time in capillary electrophoresis: validation and comparison of quantitative performances in several approaches.

Electrophoresis 2005 Jun;26(12):2293-302

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, EPGL, University of Geneva, Geneva, Switzerland.

Capillary electrophoresis (CE) can be used for the rapid determination of pharmaceuticals, particularly in routine quality control analysis. This paper focuses on several approaches aimed at decreasing the analysis time with commercially available instrumentation by (i) application of a high electric field through a reduced capillary, (ii) use of a dynamically coated capillary to increase the electroosmotic flow, (iii) short-end injection (SEI) technique, and (iv) application of multiple sample injections. Moreover, SEIs were combined with the three other approaches. A pharmaceutical formulation containing lidocaine as an active component was selected, and the methods were validated according to the ICH guidelines. The seven approaches investigated fulfilled different statistical requirements and demonstrated their linearity and trueness, with good recoveries and confidence limits always inferior to 1.5%. Furthermore, relative standard deviation (RSD) values for repeatability and intermediate precision were inferior to 1.1 and 1.8%, respectively. These results confirmed that each approach is of utmost interest to increase the analyte throughput in CE.
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http://dx.doi.org/10.1002/elps.200410242DOI Listing
June 2005

Determination of electroosmotic flow in nonaqueous capillary electrophoresis.

J Chromatogr A 2005 Mar;1068(1):75-81

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmacy, University of Geneva, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.

Mobility of the electroosmotic flow (mu(EOF)) in fused-silica capillaries strongly depends on the nature of the background electrolyte. In this study, 27 solvent systems were investigated, namely water, methanol, ethanol, 2-propanol, 1-butanol, acetonitrile (MeCN), formamide, N-methylformamide (NMF), N,N-dimethylformamide and dimethyl sulfoxyde, as well as 8 hydroorganic and 9 organic mixtures. For each system, six mu(EOF) were determined at a different ionic strength in basic conditions, and an absolute electroosmotic flow mobility (mu(EOF,0)) was extrapolated according to the Debye-Huckel Onsager model. The obtained mu(EOF,0) values were correlated with the solvent's relative permittivity (epsilon) and viscosity (eta). A good correlation (r2=0.867) between mu(EOF,0) and the solvent's epsilon/eta ratio was demonstrated, except for two solvents (MeCN and NMF). Furthermore, the donor number (DN) of a solvent took into account the possible zeta potential modification in the electric double layer near the capillary wall. Consequently, the relationship between mu(EOF,0) and epsilon/(eta x DN) was superior, with a r2 of 0.943 for 10 pure solvents.
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http://dx.doi.org/10.1016/j.chroma.2005.02.001DOI Listing
March 2005

Validation of capillary electrophoresis--mass spectrometry methods for the analysis of a pharmaceutical formulation.

Electrophoresis 2003 Sep;24(17):3049-56

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmacy, University of Geneva, Geneva, Switzerland.

The coupling of capillary electrophoresis (CE) to mass spectrometry (MS) with an electrospray ionization (ESI) technique is generally performed for qualitative applications, and only few quantitative results have been reported. This paper investigates the validation of a CE-ESI-MS method for the analysis of a pharmaceutical formulation containing lidocaine. Some important ESI criteria are discussed including sheath-liquid composition, nebulizing gas pressure and position of the CE capillary outlet. After optimization of these parameters, an intermediate precision of about 5% was achieved. The latter, as well as efficiency and resolution, were compared to those achieved with UV detection. Besides, a multiple injection procedure was developed to reduce analysis time per sample and was successfully applied to both UV and MS detectors. The validation results achieved by multiple injections were identical to those obtained with classical injection, but afforded a gain of time by a factor of 2.5.
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http://dx.doi.org/10.1002/elps.200305564DOI Listing
September 2003

Infinite enantiomeric resolution of basic compounds using highly sulfated cyclodextrin as chiral selector in capillary electrophoresis.

Electrophoresis 2003 Aug;24(15):2633-41

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmacy, University of Geneva, 20 Boulevard d'Yvoy, CH-1211 Geneva, Switzerland.

Four chiral basic analytes, namely methadone, fluoxetine, venlafaxine, and tramadol, were selected as model compounds for investigating their stereoselective separation with highly sulfated gamma-cyclodextrin (HS gamma-CD) by capillary electrophoresis (CE)-UV and CE-mass spectrometry (MS). At high concentration of chiral selector, the preferentially bonded enantiomer migrated faster in the anodic mode to the detector and high resolutions were obtained for all analytes. In the cathodic mode, at lower highly sulphated cyclodextrin (HS-CD) concentration, basic compounds could be detected, with the weakly bonded enantiomer migrating first (enantiomeric migration order inversion). It was also then possible, at intermediate HS-CD concentration, that only one enantiomer migrated to the detector as cation while the other enantiomer complexed with the CD was negatively charged and presented an opposite mobility. The latter never reached the detector achieving a perfect enantiomeric selectivity. Infinite chiral resolutions were thus achieved by CE-UV as well as by CE-electrospray ionisation (ESI)-MS where concentrations of HS-CD were adapted according to the negative contribution of the nebulization gas pressure of the interface.
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http://dx.doi.org/10.1002/elps.200305481DOI Listing
August 2003

Influence of electrolyte nature on the separation selectivity of amphetamines in nonaqueous capillary electrophoresis: protonation degree versus ion pairing effects.

Electrophoresis 2003 May;24(10):1577-86

Laboratoire d'Electrochimie et Chimie Analytique, UMR CNRS 7575, Ecole Nationale Supérieure de Chimie de Paris, Paris, France.

The simultaneous analysis of Ecstasy and its derivatives in an acetonitrile-methanol (80:20 v/v) mixture was previously shown to be strongly dependent on the nature of the electrolyte (acetate versus formate). To elucidate the phenomena involved, systematic experiments were conducted in this solvent medium. Conductivity measurements allowed to evaluate the ion-pairing rate in the background electrolyte (BGE) and thereby distinguish between electrolyte concentration and ionic strength. The influence of electrolyte concentration on analyte effective mobilities micro(eff)) was studied by capillary electrophoresis (CE). As micro(eff) extrapolated to infinite dilution proved to be independent of the nature of the electrolyte, selectivity changes could not be attributed to a modification in the protonation degree of amphetamines. Experimental mobility data were then confronted to existing theoretical mobility models to discriminate between ion pairing or simple ionic strength effect. Ion-pair formation in a BGE containing acetate was highlighted with an ion-pairing model and ion-pair formation constants between each amphetamine and acetate ion were calculated.
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http://dx.doi.org/10.1002/elps.200305372DOI Listing
May 2003

Simultaneous analysis of metabisulfite and sulfate by CE with indirect UV detection. Application to and validation for a pharmaceutical formulation.

J Pharm Biomed Anal 2003 Apr;31(6):1059-64

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmacy, University of Geneva, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland.

Metabisulfite is used as an antioxidant agent in a number of pharmaceutical formulations. In order to quantify simultaneously both metabisulfite and its oxidation product (sulfate), a capillary zone electrophoretic (CZE) method with indirect UV detection was developed. Best results were achieved with a background electrolyte (BGE) constituted of 15 mM pyromellitic acid, 15 mM tris-(hydroxymethyl)-aminomethane and 0.2 mM tetradecyltrimethylammonium bromide at pH 8.3 and an applied electrical field of 123 V/cm in a 32.5 cm fused silica capillary. Indirect UV detection was performed at a wavelength of 225 nm. In order to validate this method, an internal standard (IS), namely ammonium formate, was used. Moreover, due to the high chloride concentration in the pharmaceutical formulation, conductivity was adjusted by adding sodium chloride into standard solutions to prevent matrix effect. Linearity and accuracy were successfully tested in a concentration range of 33.3-250 microg/ml for sodium metabisulfite and of 50-375 microg/ml for sodium sulfate. Method precision was determined on six samples each day. Thereby, relative standard deviations (R.S.D.) of 6% and 12-13% were obtained for intra-day and inter-day precision, respectively. Considering the instability of metabisulfite and its use as an antioxidant agent and not as an active principle, the method was accepted and used for routine analyses.
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http://dx.doi.org/10.1016/s0731-7085(02)00651-9DOI Listing
April 2003

Potential of formamide and N-methylformamide in nonaqueous capillary electrophoresis coupled to electrospray ionization mass spectrometry. Application to the analysis of beta-blockers.

J Chromatogr A 2002 Dec;979(1-2):389-98

Laboratory of Pharmaceutical Analytical Chemistry, University of Geneva, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.

A nonaqueous capillary electrophoresis (NACE) method, coupled with either UV or electrospray mass spectrometry (ESI-MS), is described for the simultaneous analysis of seven beta-blockers. The same electrolyte, namely 25 mM ammonium formate and 1 M formic acid, was used with different investigated organic solvents. In addition to frequently used organic solvents such as methanol (MeOH) and acetonitrile (MeCN), formamide and its derivatives were investigated. Formamide (FA) and N-methylformamide (NMF) present several interesting physico-chemical properties, one of them being a high dielectric constant (e). Since FA and NMF possess a high UV cutoff, beta-blockers with an absorbance above 250 nm were selected as model compounds in order to compare NACE-UV and NACE-MS performances. FA and NMF showed different selectivity compared to water, MeOH or MeCN, and also demonstrated a higher efficiency in terms of the number of theoretical plates (especially NMF). To overcome their unfavorable optical properties, hyphenation with MS detection appears as a promising technique, thanks to its benefits in terms of selectivity, sensitivity and universality. The practical compatibility of FA and NMF with ESI-MS detection in combination with a sheath liquid configuration was demonstrated. In comparison to UV detection, sensitivity was increased, while a high efficiency was maintained. In addition, the low and stable generated currents observed were evidences for the successful hyphenation with ESI-MS. Hence, FA and NMF seemed to be promising alternatives in NACE-ESI-MS, either used as pure organic solvent or as a mixture with MeOH or MeCN.
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http://dx.doi.org/10.1016/s0021-9673(02)01254-2DOI Listing
December 2002
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