COLUMN-SWITCHING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY COMBINED WITHIONSPRAY TANDEM MASS-SPECTROMETRY FOR THE SIMULTANEOUS DETERMINATION OF THE PLATELET INHIBITOR RO-44-3888 AND ITS PRO-DRUG AND PRECURSOR METABOLITE IN PLASMA

A liquid chromatographic/mass spectrometric (LC/MS) assay was develope d for the simultaneous determination of a pro-drug (Ro 48-3657), its a ctive metabolite (platelet inhibitor, no 44-3888) and precursor metabo lite (no 48-3656) in human, dog and rat plasma, utilizing on-line colu mn-switching solid-phase extraction (SPE) for clean-up and high-perfor mance liquid chromatography (HPLC) for separation of the analytes, wit h on-line detection by ionspray (pneumatically assisted electrospray) tandem mass spectrometry in the selected reaction monitoring (SRM) mod e. The assay was validated for the quantification of all three analyte s. The method involves protein precipitation with perchloric acid, enr ichment of the analytes on a standard bore trapping column (i.d. 4.6 m m) and separation on a narrow-bore analytical column (i.d. 2 mm). Exce pt for the plasma precipitation step, the assay was fully automated, a llowing unattended operation. The lower limits of quantification were 0.20 ng ml(-1) (no 48-3657, no 44-3888) and 0.50 ng ml(-1) (no 48-3656 ) using a 0.5 ml plasma aliquot. The mean inter-assay precision and ac curacy derived from quality control samples were 5.3% and 101%, respec tively, utilizing the calibration range 0.2-200 ng ml(-1). Using the u nique features of column-switching HPLC combined with MS/MS, it was po ssible to develop the method in a short period of time. The method has been successfully applied to map complete concentration-time courses for the kinetic evaluation of the drug and its metabolites in man, dog and rat. This LC/MS assay is sensitive, specific, accurate, precise a nd robust.