ASSAY OF METRONIDAZOLE BY HPLC COMPARED WITH MICROBIAL METHOD

  • 1 January 1984
    • journal article
    • research article
    • Vol. 19, 31-43
Abstract
A high-performance liquid chromatography (HPLC) method for the assay of metronidazole and its 2-hydroxymethyl metabolite in sera was comapred with a microbiological method, an agar well diffusion technique with Clostridium perfringens as indicator strain. The HPLC technique involved separation of metronidazole from its 2 active major metabolites (the 2-hydroxymethyl and the 1-carboxymethyl derivatives) on a .mu.-Bondapak C18 column and UV detection at 313 nm. The mobile phase was 35% acetonitrile in 0.02 M acetate buffer at pH 4 with a low rate of 2.0 ml/min. Tinidazole was used as an internal standard; metronidazole and its 2-hydroxymethyl metabolite were quantitated by peak height ratios. The 1-carboxymethyl derivative was well separated from the other peaks. The HPLC procedure was superior with respect to sensitivity (detection limits: 0-1 .mu.g/ml serum for both compounds), speed and precision. It discriminated between metronidazole and its 2 major active metabolites and quantitated the total amounts present. The microbial technique co-determined all antibacterially active compounds and monitored the free, not protein-bound, moieties. Data on the activity of the 2-hydroxymethyl metabolite against C. perfringens relative to the metronidazole and results on the protein binding of metronidazole were used to correlate data from the 2 methods on individual serum samples collected during the early, intermediate and late periods after a single i.v. dose of metronidazole to volunteers.