Development, optimization and validation of a highly sensitive UPLC–ESI-MS/MS method for simultaneous quantification of amlodipine, benazeprile and benazeprilat in human plasma: Application to a bioequivalence study

• Simultaneous determination of amlodipine, benazepril and benazeprilat in plasma was done.
• The method was applied for a bioequivalence study.
• A validated, highly sensitive UPLC–ESI-MS/MS method was developed.

A rapid, simple, sensitive and specific LC–MS/MS method has been developed and validated for the simultaneous estimation of amlodipine (AML), benazepril (BEN) and benazeprilat (BNT) using eplerenone and torsemide as internal standards (IS). The Xevo TQD LC–MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Sample preparation involves both extraction and precipitation techniques. The reconstituted samples were chromatographed on Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column by pumping 0.1% formic acid and acetonitrile in a gradient mode at a flow rate of 0.45 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 0.1–5 ng/ml for AML; 5–1200 ng/ml for both BEN and BNT. The intra-day and inter-day precision and accuracy results were within the acceptable limits. A run time of 2.5 min for each sample made it possible to analyze more than 300 human plasma samples per day. The developed assay method was successfully applied to a bioequivalence study in human volunteers.

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