Sandrine Souverain

Column-switching procedures for the fast analysis of drugs in biologic samples.

Abstract: Liquid chromatography coupled with mass spectrometry in single and dual mode (LC-MS and LC-MS/MS) is the method of choice for the quantification of drugs and their metabolites in biologic fluids. Following the new challenges encountered in the process of drug development, liquid chromatography-mass spectrometry has been found to achieve high-throughput analysis. With this impressive tool, the sample preparation step before analysis is simplified, and the analytic process speeded up. Several generic approaches have recently been developed for the sample extraction coupled on line with a LC-MS system. In this paper, different extraction supports allowing the direct injection of biologic fluids were investigated, namely, restricted-access media, large-size particle, and monolithic phases. In the column-switching configuration, these supports, coupled with microbore analytic columns, were found suitable for the fast analysis (total analysis time of less than 10 minutes) of different drugs and their metabolites in biologic matrices at the nanogram per milliliter level.

Development and validation of a heart-cutting liquid chromatography–mass spectrometry method for the determination of process-related substances in cetirizine tablets

A heart-cutting liquid chromatography–mass spectrometry (LC–MS) method was developed for the simultaneous analysis of seven process related substances (PRS) at ng ml −1 level in tablets containing cetirizine (CTZ) as active ingredient. After dissolution and filtration, 10 l of sample was injected onto a cyano-column coupled to an electrospray ionization–mass spectrometer (ESI–MS) equipped with a single quadrupole. A switching valve placed between the analytical column and the MS detector allowed for the transfer of only the PRS fractions into the MS. The separation of PRS and CTZ was achieved in <14 min with a binary gradient using a 50 mM ammonium acetate solution (pH 7) and acetonitrile. The ESI–MS detection was performed using single ion monitoring (SIM). The method was validated. Weighted linear regression was found appropriate in a concentration range of 2.5–250 ng ml −1 and the limit of quantification (LOQ) was estimated at 10 ng ml −1 for each analyte. Method selectivity and robustness was demonstrated. Method performance was evaluated in terms of accuracy and precision using quality control (QC) samples over the investigated range. Finally, the heart-cutting LC–MS method was successfully applied to commercially available tablets.

Use of large particles support for fast analysis of methadone and its primary metabolite in human plasma by liquid chromatography-mass spectrometry.

A bioanalytical method was developed for the quantitation of methadone (MTD) and its primary metabolite, (EDDP) in plasma. The extraction step was performed within a capillary column packed with large particles (35x0.3 mm I.D.; d(p) 30 micrometer) at high flow-rate conditions (450 microliter/min). The separation was performed on a microbore analytical column (55x2 mm I.D.; d(p) 3 micrometer) coupled to a mass spectrometer (MS). This procedure was based on a column-switching unit. Analytes of interest were retained on the precolumn by hydrophobic interactions and backflushed from the precolumn to the analytical column. The detection was carried out with a MS single quadrupole equipped with an electrospray interface. The total analysis time was 6 min. The limits of quantification were evaluated at 10 and 25 ng/ml for MTD and EDDP, respectively. At this level, good accuracies were obtained for both analytes with repeatability values less than 18%.

Automated LC-MS Method for the Fast Stereoselective Determination of Methadone in Plasma

Abstract Methadone (MTD) is a chiral drug widely used for the treatment of opioid dependence for which a rapid analytical method for the determination of each enantiomer would be advantageous. In order to improve method sensitivity and to automate the entire analytical process, a column-switching configuration has been developed. An online extraction system coupled to a cellulose-based chiral stationary phase (CSP), namely Chiralcel OJ-R, was used and detection was performed by mass spectrometry. Fifty μl of plasma were injected into the liquid chromatography-mass spectrometry (LC-MS) system after addition of acetonitrile (ACN) containing methadone deuterated D9 (MTD-D9) (internal standard) and centrifugation. For the rapid extraction step, a large particle size support was selected. A baseline separation of MTD enantiomers was obtained in less than 12 min. Trueness and precision were evaluated with control samples at 500 ng/ml of (R,S)-methadone. Trueness values were 106.6% and 103.0% for (R)-MTD and (S)-MTD, respectively, with a coefficient of variation inferior to 2.5% for both compounds. Finally, a good concordance was found using this method for analysis of plasma samples from patients in maintenance treatment as compared to a previously described HPLC-UV method after liquid-liquid extraction.

Rapid liquid chromatographic-mass spectrometric analysis of withanolides in crude plant extracts by use of a monolithic column

SummaryA rapid analytical method has been developed for the mutual resolution of three steroidal compounds, withaferin A, iochromolide, and withacnistin. Liquid chromatography was performed on a Chromolith analytical column (4.6 mm i.d.×50 mm), made from a cylindrical silica rod, operated at a flow rate of 4 mL min−1 with a simple linear gradient prepared from 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile. Under optimum conditions simultaneous separation of the compounds was achieved in less than 7 min, one eighth the time required for conventional LC separation. The overall analysis time was reduced without sacrificing chromatographic performance—essential for the resolution of positional isomers such as iochromolide and withacnistin. The column was coupled to a single-quadrupole mass spectrometer and the method was characterized by good performance in terms of repeatability, selectivity, linearity, and sensitivity. Detection limits in the single-ion-monitoring mode were 0.15 μg mL−1 or below. Finally, the developed method was successfully applied to the determination of withanolides in extracts fromlochroma gesnerioides obtained by three different processes—traditional Soxhlet extraction and two faster methods, microwave-assisted extraction and pressurized solvent extraction.

Use of monolithic supports for fast analysis of drugs and metabolites in plasma by direct injection

SummaryCommercial monolithic columns were evcluated as extraction supports for the analysis of various drugs and their metabolites from plasma. For this purpose, a precolumn packed with a monolithic phase was used as extraction and enrichment support and integrated in a column-switching system for the direct and simultaneous analysis of fluoxetine and norfluoxetine, methadone and EDDP, flunitrazepam and norflunitrazepom in humon plasma. In this configuration, plasma samples were directly injected onto a Chromolith Flash precolumn (25×4.6 mm I.D.) coupled to a microbore analytical column packed with a conventional reversed-phase malerial. Detection was by an electrospray-ionization mass spectrometer (ESl-MS). Total analysis time, including extraction and separation of the six analytes, was <10 min. For all the investigated compounds, the limits of quantitation (LOQ) were estimated in the ng.mL1 range for an injection volume of 50 μL. The method was also applied to a real clinical case to demonstrate its applicability.