Study on quantification of verapamil in dog plasma by liquid chromatography-mass spectrum

Abstract

This study aims to develop and validate an Liquid chromatography - Tandem mass spectrometry (LC-MS/MS) method for the quantification of verapamil in canine plasma, compliant with FDA and Vietnamese Pharmacopoeia requirements, and to apply this method for the in vivo bioavailability evaluation of a gastro-retentive extended-release verapamil tablet. Plasma samples were processed using protein precipitation combined with solid-phase extraction employing acetonitrile, magnesium sulfate, and sodium chloride. Verapamil and the internal standard diltiazem were analysed using LC-MS/MS with electrospray ionisation in positive mode (ESI+) and multiple reaction monitoring (MRM) acquisition. The method was validated for specificity, linearity, accuracy, precision, lower limit of quantification (LLOQ), extraction recovery, stability, and carry-over. Results show that the method achieved an LLOQ of 5.0 ng/ml, a linear range of 5.0-495.0 ng/ml; a coefficient of determination of R²=0.9999, accuracy between 87.1 and 106.6%, and extraction recovery ranging from 70.5-79.7%. The analyte was stable after three freezethaw cycles, during sample preparation, and under long-term storage at -40°C. Carry-over was within acceptable limits. The method was successfully applied to quantify verapamil in canine plasma (n=6) following oral administration of a 120 mg floating tablet; C _max=232.9±18.1 ng/ml; T_max=5.3±1.2 h; AUC_0-24=2907.5±290.3 h.ng/ml; AUC_0-∞=3310.6±365.0 h.ng/ml. The developed LC-MS/MS method exhibited high sensitivity, accuracy, and reliability, making it suitable for bioavailability studies of verapamil formulations.