Abstract
Purpose: To develop new sensitive, accurate, rapid and reproducible high performance liquid chromatography (HPLC) method to determine metronidazole levels in human plasma and to apply the method in a bioequivalence study.
Methods: Metronidazole was extracted from human plasma through one step of protein precipitation by methanol using carbamazepine as internal standard (IS). After centrifugation of the plasma sample, the supernatant layer was separated and injected into HPLC system using Eclipse XDB-phenyl column. The mobile phase consisted of phosphate buffer (pH 4.5): acetonitrile (95: 5, v/v). The UV detector was set at 320 nm. The bioavailability of the test metronidazole product (Brand A) was compared to a commercial metronidazole brand as reference product in 24 healthy volunteers who received a single dose equivalent to 500 mg of the test and reference products in a randomized balanced two-way cross-over design separated by two-week wash-out period.
Results: Mean standard calibration curves of metronidazole over the concentration range of 0.05 - 30 mu g/ml were linear. No significant differences were found based on analysis of variance of the pharmacokinetics parameters required for the assessment of bioequivalence of test and reference formulations. The mean value and 90 % CI of test/reference ratios for the derived parameters were: C-max, 9.64 vs. 8.38 (0.93 - 1.10), AUC(0-24), 124.6 vs. 122.3 mu g.h/mL (0.973 - 1.051) and AUC(0-infinity), 140.9 vs. 128.4 h/mL (1.15 - 1.23).
Conclusion: The test metronidazole product was bioequivalent to the reference. The method is suitable for bioequivalence and pharmacokinetic studies in humans with a low limit of quantification of 0.05 mu g/ml.