Liquid chromatographic determination of amoxicillin and its metabolites in human urine by postcolumn degradation with sodium hypochlorite.

A high-performance liquid chromatographic method has been developed for the determination of amoxicillin and its metabolites [(5R,6R)-amoxicilloic acid, the (5S,6R) epimer, and the (2R)-piperazine-2',5'-dione] in human urine. They were separated from the background components of urine on a reversed-phase C18 column using sodium heptylsulphonate as an ion-pairing agent and methanol as an organic mobile phase modifier. The eluent was led to the postcolumn degradation with 1.5 M sodium hydroxide plus 0.02% sodium hypochlorite solution at ambient temperature. The degradation product(s) of each compound was detected at 270 nm. The proposed method permits detection of I, II, III, and IV down to 1 microgram/ml in neat urine samples. At a concentration of 5 micrograms/ml of each compound, within- and between-run precisions (relative standard deviation) were 1.12-5.79 and 0.80-2.70%, respectively. The urinary levels of I and its metabolites were determined by the proposed method after administration of I to humans.