Abstract

A VALIDATED REVERSED PHASE HPLC ASSAY FOR THE DETERMINATION OF METRONIDAZOLE IN HUMAN PLASMA

Nada H. Binhashim, Syed N. Alvi, and Muhammad M. Hammami*

ABSTRACT

A simple and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of metronidazole in human plasma was developed and validated. Using tinidazole as an internal standard (IS), separation was achieved on Symmetry shield RP18 column. The mobile phase, sodium acetate 0.05 M (pH=4, adjusted with phosphoric acid), and acetonitrile (85:15,v: v) delivered at flow rate 1.0 ml/min. 0.25ml plasma samples were deproteinized with methanol containing 2% perchloric acid and centrifuged. 100µl supernatant clear solution was injected to HPLC system. The eluent was monitored spectrophotometricly at 320 nm. No interference in blank plasma or of commonly used drugs was observed. The relationship between the concentration of metronidazole in plasma and peak area ratio of metronidazole to the IS was linear over the range of 0.05-15.0 μg/ml. Intra-day and inter-day coefficient of variation (CV) and bias were ≤ 6.2% and ≤ 9.0%, and ≤7.3% and ≤ 11.0%, respectively. Mean extraction recovery of metronidazole and the IS from plasma samples was ≥ 88% using the method, metronidazole was found stable under various conditions generally encountered in the clinical laboratory (≥93% and ≥90% in processed and unprocessed samples, respectively). Further, the method was successfully employed to measure metronidazole levels in plasma samples from healthy volunteers.

Keywords: Metronidazole, Tinidazole, Human plasma, HPLC.