Assay of metronidazole by HPLC compared with microbial method

A high-performance liquid chromatography (HPLC) method for the assay of metronidazole and its 2-hydroxymethyl metabolite in sera was compared with a microbiological method, an agar well diffusion technique with Clostridium perfringens as indicator strain. The HPLC technique involves separation of metronidazole from its two 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 pH 4 with a low rate 2.0 ml/min. Tinidazole was used as an internal standard and metronidazole and its 2-hydroxymethyl metabolite quantitated by peak height ratios. The 1-carboxymethyl derivative was well separated from the other peaks. The HPLC procedure proved to be superior with respect to sensitivity (detection limits: 0-1 microgram/ml serum for both compounds), speed and precision. It discriminates between metronidazole and its two major active metabolites and quantitates the total amounts present. The microbial technique codetermines all antibacterial active compounds and monitors the free, not protein bound moieties. Published data on the activity of the 2-hydroxymethyl metabolite against Cl. perfringens relative to metronidazole and published results on the protein binding of metronidazole were used to correlate data from the two methods on individual serum samples collected during the early, intermediate and late periods after a single intravenous dose of metronidazole to volunteers.