Development and validation of a selective and robust LC–MS/MS method for high-throughput quantifying rizatriptan in small plasma samples: Application to a clinical pharmacokinetic study

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC–MS/MS) was developed for the determination of a potent 5-HT1B/1D receptor agonist, rizatriptan in human plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 100 μL plasma samples by liquid–liquid extraction (LLE) and chromatographed on a Lichrospher C18 column (4.6 mm × 50 mm, 5 μm) with a mobile phase consisting of acetonitrile–10 mM aqueous ammonium acetate–acetic acid (50:50:0.5, v/v/v) pumped at 1.0 mL/min. The method had a chromatographic total run time of 2 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 270 → 201 (rizatriptan) and 313.4 → 138 (granisetron) used for quantitation. The assay was validated over the concentration range of 0.05–50 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 98%. The intra-day accuracy of the assay was within 12% of nominal and intra-day precision was better than 13% C.V. Following a 10 mg dose of the compound administered to human subjects, mean concentrations of rizatriptan ranged from 0.2 to 70.6 ng/mL in plasma samples collected up to 24 h after dosing. Inter-day accuracy and precision results for quality control samples run over a 5-day period alongside clinical samples showed mean accuracies of within 12% of nominal and precision better than 9.5% C.V.