High-performance liquid chromatographic determination of ampicillin and its metabolites in rat plasma, bile and urine by post-column degradation with sodium hypochlorite.

A high-performance liquid chromatographic method has been developed for the determination of ampicillin (1) and its metabolites [(5R,6R)-ampicilloic acid (2), the (5S,6R)-epimer (3) and the corresponding (2R)-piperazine-2',5'-dione (4)] in rat plasma, bile and urine. The method involves the separation of 1-4 from the background components of the biological fluids on a reversed-phase C18 column, using sodium heptanesulphonate as an ion-pairing agent and methanol in the mobile phase, followed by post-column degradation with 1.5 M sodium hydroxide-0.02% sodium hypochlorite solution at ambient temperature, and detection of the degradation product(s) of each compound at 270 nm. The detection limits were about 25 ng for each compound at a signal-to-noise ratio of 3. At concentrations of 2-5 micrograms/ml of each compound, the within- and between-run precisions (relative standard deviation) were 0.77-7.15% and 1.76-5.96%, respectively. The plasma, biliary and urinary levels of 1 and its metabolites were determined by the proposed method after intravenous administration of 1 to rats.