Liquid chromatographic determination of ampicillin and its metabolites in human urine by postcolumn alkaline degradation.

A high-performance liquid chromatographic method has been developed for the determination of ampicillin (I) and its metabolites [5R,6R)-penicilloate (II), the (5S,6R)-epimer (III), and piperazine-2,5-dione (IV)) in human urine. The assay was based on the measurement of the absorbance at 300 nm following the postcolumn alkaline degradation with 0.75 M sodium hydroxide, 2 X 10(-3) M mercuric chloride, and 1 X 10(-2) M ethylenediaminetetraacetic acid disodium salt in solution. The limits of accurate determination were 0.5 microgram mL-1 for I, 2.0 microgram mL-1 for II and III, and 1.0 microgram mL-1 for IV in neat urine samples with a 10 microL injection. At concentrations of compounds I-IV of 5 micrograms mL-1, within- and between-run precisions were 1.10-4.03% and 0.93-2.34%, respectively. The urinary levels of I and its metabolites were quantified by the proposed method.