UPLC-MS/MS assay for the simultaneous quantification of carvedilol and its active metabolite 4'-hydroxyphenyl carvedilol in human plasma to support a bioequivalence study in healthy volunteers.

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the simultaneous determination of carvedilol and its pharmacologically active metabolite 4'-hydroxyphenyl carvedilol in human plasma using their deuterated internal standards (IS). Samples were prepared by solid-phase extraction using 100 μL human plasma. Chromatographic separation of analytes was achieved on UPLC C18 (50 × 2.1 mm, 1.7 µm) column using acetonitrile-4.0 mM ammonium formate, pH 3.0 adjusted with 0.1% formic acid (78:22, v/v) as the mobile phase. The multiple reaction monitoring transitions for both the analytes and IS were monitored in the positive electrospray ionization mode. The method was validated over a concentration range of 0.05-50 ng/mL for carvedilol and 0.01-10 ng/mL for 4'-hydroxyphenyl carvedilol. Intra- and inter-batch precision (% CV) and accuracy for the analytes varied from 0.74 to 3.88 and 96.4 to 103.3% respectively. Matrix effect was assessed by post-column analyte infusion and by calculation of precision values (coefficient of variation) in the measurement of the slope of calibration curves from eight plasma batches. The assay recovery was within 94-99% for both the analytes and IS. The method was successfully applied to support a bioequivalence study of 12.5 mg carvedilol tablets in 34 healthy subjects.

RCT Entities:   Population Interventions Outcomes