A UPLC-MS/MS method for quantification of perindopril and perindoprilat and applied in a bioequivalence study for their pharmacokinetic parameter measurement

To investigate the pharmacokinetic parameters of perindopril and perindoprilat in healthy volunteers, a simple and sensitive UPLC-MS/MS method with isotope-labeled internal standards of perindopril-d4 and perindoprilat-d4 was established and further applied in a bioequivalence study.A simple and sensitive UPLC-MS/MS method with isotope-labeled internal standards of perindopril-d4 and perindoprilat-d4 was validated and applied in a single-center, randomized, cross-over, and two-period bioequivalence study. 20 healthy Chinese subjects (16 males and 4 females) were enrolled and had their plasma concentrations of perindopril and perindoprilat quantified and calculated for the pharmacokinetic parameters. After acetonitrile precipitation, the analytes and internal standards were gradient eluted with methanol-acetonitrile-ammonium acetate on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm) column. Detection was carried out in a multireaction monitoring mode using positive ionization electrospray mass spectrometry.The total chromatographic run time was 4 minutes with retention time for perindopril and perindopril-d4 of ~ 1.86 minutes, whereas perindoprilat and perindoprilat-d4 was ~ 1.79 minutes. The calibration curves of perindopril and perindoprilat were linear over 0.4 - 80 ng/mL and 0.2 - 40 ng/mL, respectively. The method was fully validated to meet the requirement for bioassay in accuracy (89.6 - 112.4%), precision (coefficient of variation (CV) ≤ 13.8%), recovery (79.65 - 97.83%), matrix effect (CV ≤ 5.9%), and stability (CV ≤ 10.0%). The 90% confidence intervals (CIs) for the geometric mean ratios of Cmax, AUC0-tlast, and AUC0-∞ of perindopril and perindoprilat all fell within the bioequivalence acceptance criteria (80 - 125%). There were no significant differences between the two formulations in terms of tmax and T1/2 of perindopril and perindoprilat. There was no adverse event in this clinical study. Interestingly, it was found that the pharmacokinetics of perindoprilat in 1 subject were significantly different from that of the others which may be associated with genetic diversity.This method was successfully applied to the bioequivalence test of two perindopril tert-butylamine tablets. The two one-sided t-tests showed that these two products were bioequivalent.