Liquid Chromatography-Electro Spray Ionization Tandem Mass Spectrometry for Simultaneous Determination of Amlodipine, Benazepril and its Active Metabolite Benazeprilat in Human Plasma

Abstract

A selective, sensitive, rapid, and reproducible liquid chromatographyelectro spray ionization tandem mass method has been developed and subsequently validated for the simultaneous determination of Amlodipine (AML), Benazepril (BENZ), and its active metabolite Benazeprilat (BENZT) in spiked human plasma, using Moexipril (MOX) as an internal standard (IS). Various modes were tried and the Multiple Reaction Monitoring (MRM) mode was found the most suitable one. The three analytes and Moexipril (IS) were extracted from human plasma by simple protein precipitation using acetonitrile as the precipitating solvent. The stationary phase used was a C18 Sunfire column while water and acetonitrile at 0.1 % formic acid (30:70, v/v) was used as a mobile phase. The flow rate used was 0.8 mL/min. FDA guidelines were followed for the method validation. The linearity range was found to be 10-200 ng/mL for Amlodipine, 0.5-100 ng/mL for Benazepril and 5-200 ng/mL for Benazeprilat and the correlation coefficient was more than 0.9980 for each analyte. The equation of the calibration curve was: y=0.0068x - 0.0072 (r2=0.9981, n=3) for Amlodipine, y=0.6098x - 0.135 (r2 =0.9988, n=3) for Benazepril and y=0.0173x - 0. 0312 (r2=0.9987, n=3) for Benazeprilat. Results for accuracy and precision showed satisfactory results. Also the method was compared with reported HPLC method and no significant difference was found.