A NOVEL VALIDATED BIO-ANALYTICAL METHOD DEVELOPMENT FOR AZELNIDIPINE (DIHYDROPYRIDINE) IN INDIAN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY QUADRUPLE TANDEM MASS SPECTROMETRY (LC-ESI-MS/MS, API-4000) WITH AN APPLICATION TO IN VIVO PHARMACOKINETIC AND BIOEQUIVA

Abstract

Azelnidipine is a dihydropyridine used as a calcium channel blocker. The main aim of this study was to develop a validated bio-analytical method (as per US-FDA and EMA guidelines) for in vivo pharmacokinetic and bioequivalence study of azelnidipine in human plasma by LC-MS/MS, API-4000. In this method, the drug was ionized in negative mode and gave adequate response because this drug was highly sensitive and had high electron affinity due to the presence of the electron-withdrawing the nitro group in the structure of azelnidipine. The deprotonated precursor ions [M-H]- at mz-1 581.2 and consistent fragment ion selected was mz-1 491.0. For internal standard, the deprotonated precursor ions [M-H]- at mz-1 269.0 (highest peak) was observed in Q1 MS and characteristic product ions or fragment ions found in Q3 MS were at mz-1 169.8. For plasma extraction, the liquid-liquid extraction technique was used. The calibration concentrated points of azelnidipine were 0.15 to 10.00 ng mL-1 including LLOQ 0.15 ng mL-1, LQC 0.46 ng mL-1, MQC 3.75 ng mL-1 and HQC 7.50 ng mL-1. The LOD value was 0.07 ng mL-1. The result of matrix effect of internal standard (tolbutamide) ranges between 93.51% - 98.68% and 91.94% - 95.07% for azelnidipine, recovery result after extraction of plasma of azelnidipine was 90.73% to 100.46% and for IS it was 95.95% to 98.82%. After administration of film-coated azelnidipine 8mg of test drug at 2.92±0.77h. Cmax obtained was 5.98±1.93ng mL-1 whereas for reference drug it was 6.18±1.96ng mL-1 Cmax at 3.03±0.98h. This method was validated as per regulatory guidelines and is highly selective, specific, highly sensitive and reproducible with low ionic suppression and high recovery, High throughput screening method was successfully applied to in vivo pharmacokinetic and bioequivalence study of azelnidipine.