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

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Abstract
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 × 10−3 M mercuric chloride, and 1 × 10−2 M ethylenediaminetetraacetic acid disodium salt in solution. The limits of accurate determination were 0.5 μg mL−1 for I, 2.0 μg mL−1 for II and III, and 1.0 μg mL−1 for IV in neat urine samples with a 10 μL injection. At concentrations of compounds I-IV of 5 μg 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.