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

Abstract

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.