ORAL PLATELET-AGGREGATION INHIBITOR RO-48-3657 - DETERMINATION OF THEACTIVE METABOLITE AND ITS PRODRUG IN PLASMA AND URINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING AUTOMATED COLUMN-SWITCHING

A sensitive and highly automated high-performance liquid chromatograph y (HPLC) column-switching method has been developed for the simultaneo us determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perch loric acid (0.5 M), while urine samples could be processed directly af ter dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) wer e backflushed onto the analytical column, separated by gradient elutio n and detected by means of UV detection at 240 nm. The limit of quanti fication for both compounds was 1 ng/ml (500 mu l of plasma) and 25 ng /ml (50 mu l of urine) for plasma and urine, respectively. The practic ability of the new method was demonstrated by the analysis of about 60 00 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.