rapid and sensitive HPLC method for the analysis of metronidazole in human plasma: application to single dose pharmacokinetic and bioequivalence studies

ABSTRACT

A sensitive, accurate and rapid reverse phase HPLC method was developed to quantitate plasma levels of metronidazole in order to conduct a comparative bioavailabllity studies. The drug and internal standard were added to plasma samples, vortexed and then zinc sulfate solution was added in order to precipitate the plasma proteins. Samples were centrifuged at 3000 rpm for 10 min. The supernatant layer was separated and analyzed on a phenyl [300 X 4.6mm] column, with 5% acetonitrile in 0.1 M KH [2] PO[4] buffer [pH = 4.5] at 324 nm. The standard curve covering 0.15-30 micro g/ml concentration range, was linear [r [2] = 0.9999], relative errors were within 2.48 to 9.15% and the CV% ranged from 2.999 to 10.796. The method is suitable for bioavailability, pharmacokinetic, and bioequivalent studies in human. The in-vivo study was carried out in 12 healthy volunteers according to a single dose, two-sequence, cross over randomized design. The bioavailabllity was compared using the total area under the plasma level versus time curve [AUC [0-48], AUC [0-infinity], peak plasma concentration [C[max]] and time to [C[max] [T[max]]. No statistically significant difference was found between the AUC [0-infinity], C[max] and T[max] values of the test and reference, Flagyl [R] [p > 0.05]. The 90% CI for the ratio of the AUC [0-infinity], [0.94-1.07] and C[max] [0.88-1.03] and the logarithmically transformed AUC[0-infinity] [0.99-1.01] and C[max] [0.94-1.01] values of the generic product over those of Flagyl [R] was calculated to be within the acceptable limit of 0.80-1.20 and 0.80-1.25, respectively. It was, therefore, concluded that the generic metronidazole was bioequivalent with the innovator formulation