A UPLC-MS/MS method for simultaneous quantification of sildenafil and N-desmethyl sildenafil applied in pharmacokinetic and bioequivalence studies in a Chinese population.

OBJECTIVE: To evaluate the pharmacokinetic parameters and bioequivalence of two sildenafil tablets (20 mg) in healthy Chinese subjects. MATERIALS AND METHODS: A random, crossover, self-control design was used. 20 healthy subjects including males and females were randomized into two groups. A single oral dose of the trial or reference preparation was given to the two groups of subjects after an overnight fast of 10 hours. Blood samples were taken at scheduled time points. Plasma concentrations of sildenafil and N-desmethyl sildenafil were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). ANOVA was used to check the difference of the mean values of the pharmacokinetic parameters between the two preparations. Bioequivalence was determined by two one-sided t-tests and 90% confidence intervals. RESULTS: The quantitative range of sildenafil and N-desmethyl sildenafil was 2.000 - 200.0 ng/mL and 0.800 - 80.00 ng/mL, respectively. Plasma samples were stable, and there was no mutual interference between the analyte and internal standard. With the 90% confidence limit, the trial preparations AUC0→t and Cmax fall within 80.00 - 125.00% of the reference preparation's AUC0→t and Cmax. The tmax of sildenafil and N-desmethyl sildenafil was ~ 0.9 hours and 1 hour, and the T1/2 was 2.4 hours and 3.7 hours, respectively. The relative bioavailability of sildenafil and N-desmethyl sildenafil was 99.28 ± 3.30% and 99.20 ± 3.39%. No significant difference was found in every factor between the trial preparation and the reference preparation. CONCLUSION: The UPLC-MS/MS method was successfully established to evaluate the pharmacokinetic parameters of sildenafil in healthy Chinese subjects. The trial preparation was bioequivalent to the reference preparation.

A UPLC-MS/MS method for quantification of perindopril and perindoprilat and applied in a bioequivalence study for their pharmacokinetic parameter measurement
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OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.