Development and Validation of a HPLC-MS-MS Method for Quantification of Metronidazole in Human Plasma

Abstract

The objective of this study was to describe an analytical validation of a simple, rapid, sensitive, and selective liquid chromatography (LC)-tandem mass spectrometry (MS-MS) method for quantification of metronidazole in human plasma samples from bioavailability studies. The plasma samples were pre-treated by liquid-liquid extraction (LLE) with ethyl acetate. Metronidazole and the internal standard (IS) zidovudine were analyzed by combined reversed-phase LC-MS-MS with positive ion electrospray ionization. Multiple reactions monitoring (MRM) of the transitions were used as follows: m/z 171.97 > 127.97 for metronidazole and m/z 268.08 > 126.96 for IS. The main chromatography conditions used were a Varian C(18) Microssorb model (150 mm x 4.6 mm i.d., 5 microm particle size) column, and the mobile phase was composed of acetonitrile and 10 mM ammonium acetate (80:20, v/v) and 0.1% formic acid. Flow rate used was 1.0 mL/min with splitter. The method was linear over a concentration range of 0.05-8.00 microg/mL. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The developed method was validated and successfully applied to a pilot bioequivalence study of metronidazole in healthy volunteers.