A rapid, simple, sensitive and specific LC-MS/MS method has been developed and validated for the simultaneous estimation of atorvastatin (ATO), amlodipine (AML), ramipril (RAM) and benazepril (BEN) using nevirapine as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Analytes and IS were extracted from plasma by simple liquid-liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on C(18) column by pumping 0.1% formic acid-acetonitrile (15:85, v/v) at a flow rate of 1 mL/min. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 0.26-210 ng/mL for ATO; 0.05-20.5 ng/mL for AML; 0.25-208 ng/mL for RAM and 0.74-607 ng/mL for BEN with mean correlation coefficient of ≥0.99 for each analyte. The intra-day and inter-day precision and accuracy results were well with in the acceptable limits. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers.
Copyright © 2010 John Wiley & Sons, Ltd.