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

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 [Ca2+] ([Ca2+]i) 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 [Ca2+]i 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 [Ca2+]i. Our results suggest that toxic doses of caffeine are required to increase muscle contractility, questioning the purported intramuscular ergogenic effect of caffeine in humans.

Inhibition screening method of microsomal UGTs using the cocktail approach.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

Non-aqueous capillary electrophoresis 2005-2008.

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.

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.

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.

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

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.

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

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.

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

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.

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

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.

Nonaqueous capillary electrophoresis in pharmaceutical analysis.

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.

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

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.

Strategies for rapid chiral analysis by capillary electrophoresis.

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.

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

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.