Jean-Marc Brisson

Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography-tandem mass spectrometry.

Direct plasma injection technology coupled with a LC-MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within +/- 15%. The advantages and challenges of using direct single column LC-MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.

Simultaneous fast HPLC-MS/MS analysis of drug candidates and hydroxyl metabolites in plasma

A rapid bioanalytical method was evaluated for the simultaneous determination of drug discovery compounds and their potential metabolites in plasma samples within 1 min run time by fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). The fast HPLC-MS/MS system is achieved by using mini-column HPLC coupled to tandem mass spectrometer which is advantageous over regular HPLC-MS/MS systems, such as a shorter chromatographic region of ionization suppression, less solvent consumption and higher throughput. Matrix ionization suppression effect of the test compounds in plasma samples when using fast HPLC-MS/MS method was examined by a post-column infusion technique. In the described example, the proposed approach has been successfully employed to determine the plasma concentration of the test compound and its hydroxyl metabolite (M+16) in monkey in the low ng/ml region. The monkey pharmacokinetic results obtained by the proposed fast HPLC-MS/MS method were in good agreement within 20% error with those obtained by the regular HPLC-MS/MS method based on the same sample preparation procedure.

Direct simultaneous analysis of plasma samples for a drug discovery compound and its hydroxyl metabolite using mixed-function column liquid chromatography-tandem mass spectrometry

A polymer-coated mixed-function (PCMF) column was evaluated for direct plasma injection for the simultaneous determination of a drug candidate and its hydroxyl metabolite by high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS-MS) in support of pharmacokinetic studies. Each diluted monkey plasma sample containing internal standard was directly injected on to the PCMF column for sample clean-up, enrichment and chromatographic separation. The proteins and macromolecules were first eluted from the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column were then eluted with a strong mobile phase using a gradient separation technique at a constant flow rate of 1.0 ml min(-1). When not diverted, the column effluent was connected either to the atmospheric pressure chemical ionization (APCI) source or the electrospray ionization (ESI) source as part of the mass spectrometer system used for quantification. The calibration curve was linear over the range 5-2500 ng ml(-1) for both analytes. The retention times for the analytes and the internal standard were both consistent and no column deterioration was observed for at least 500 injections. The recovery through the column and reproducibility of the dosed compound and its hydroxyl metabolite in monkey plasma samples were > 90% (RSD < 6%). The total analysis time was < 8 min per sample. The analytical results obtained by the proposed direct plasma injection method were in good agreement with those obtained by the conventional LC-MS-MS method.

Permeability evaluation of peptidic HCV protease inhibitors in Caco-2 cells-correlation with in vivo absorption predicted in humans

The permeability of six peptidic hepatitis C virus (HCV) protease inhibitors, with molecular weights ranging from 500 to 780, was examined in the Caco-2 cell system. All six compounds permeated the cells transcellularly; paracellular permeability, evaluated in the Caco-2 cell system by reducing the calcium concentration in the media to increase the pore size of the tight junctions, most likely contributes only minimally to the oral absorption of the compounds. All six compounds were shown to be efflux substrates displaying concentration-dependent saturation of efflux. The efflux could be blocked by cyclosporine A, a specific P-glycoprotein (P-gp) inhibitor, suggesting that P-gp may be the responsible transporter. Oral absorption in rats was calculated using in vivo oral bioavailability and hepatic extraction ratios. Human oral absorption was projected to be similar to that of rats, as reported previously by comparing rat and human absorption values for 23 marketed drugs. Upon comparison of human oral absorption predicted by Caco-2 permeability and by rat pharmacokinetics, we show a better correlation with Caco-2 permeability obtained at higher compound concentrations, where efflux is saturated, than at lower concentrations. The higher concentrations are likely reflecting the lumen concentrations after in vivo oral dosing. The results presented in this study demonstrate that, when tested at relevant compound concentrations, Caco-2 permeability is useful for predicting the oral absorption of peptidic compounds.