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 plas ma 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. Eac h diluted monkey plasma sample containing internal standard was directly in jected on to the PCMF column for sample clean-up, enrichment and chromatogr aphic separation. The proteins and macromolecules were first eluted from th e column while the drug molecules were retained on the bonded hydrophobic p hase. The analytes retained on the column were then eluted with a strong mo bile 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 eithe r to the atmospheric pressure chemical ionization (APCI) source or the elec trospray ionization (ESI) source as part of the mass spectrometer system us ed for quantification. The calibration curve was linear over the range 5-25 00 ng ml(-1) for both analytes. The retention times for the analytes and th e internal standard were both consistent and no column deterioration was ob served for at least 500 injections. The recovery through the column and rep roducibility of the dosed compound and its hydroxyl metabolite in monkey pl asma samples were > 90% (RSD < 6%). The total analysis time was < 8 min per sample. The analytical results obtained by the proposed direct plasma inje ction method were in good agreement with those obtained by the conventional LC-MS-MS method.