Picogram determination of a novel dopamine D-4 receptor antagonist in human plasma and urine by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive and specific assay for the determination of 3-{[4-(4-chlorophenyl)piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine (I, L-745,870), a potential antipsychotic agent, has been developed, utilizing high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. The analyte and the internal standard (II, 3-{[4-(4-trifluoromethyl)piperazin-1-yl]methyl})-1H-pyrrolo[2,3-b] pyridine) were isolated from a basified biological matrix using liquid-liquid extraction with methyl-tert.-butyl ether. The organic extract was evaporated to dryness, the residue was reconstituted in a mobile phase and injected into the HPLC system. The chromatographic conditions used for the analysis were a Keystone Scientific C-18 BDS 150x4.6 mm, 5 mu m column with a mobile phase consisting of a 40:60 (v/v) mixture of acetonitrile and water containing 10 mM ammonium acetate and 0.1% formic acid pumped at a flow-rate of 1.2 ml/min, yielding retention times of 3.4 and 5.0 min for I and II, respectively. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring using the parent-->product ion combinations of m/z 327-->131 and 361-->131 were utilized to quantitate I and II, respectively. The assays were validated in the concentration range of 0.1 to 100 and 0.5 to 500 ng/ml for plasma and urine, respectively. The precision of the assays, expressed as coefficients of variation were less than 10% over the entire concentration range, with adequate assay accuracy, sensitivity and specificity to determine the pharmacokinetics in human subjects following a single 1-mg dose.