Rapid high-performance liquid chromatographic assay for the simultaneous determination of probenecid and its glucuronide in urine. Irreversible binding of probenecid to serum albumin.

A reversed-phase high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of probenecid and its glucuronide in urine has been developed. The genuine glucuronide conjugate was isolated from urine by the use of solid-phase extraction on Amberlite XAD-2 and finally purified by the use of preparative HPLC on a Sepharon Hema 1000 RP-18 column. The purity of the product obtained was 88.9%. The isolated glucuronide was used as a standard sample. Of a p.o. dose of 500 mg to two volunteers, 26 and 29% were excreted as the ester glucuronide, while 1.0 and 2.7% were excreted unmetabolized. The stability of the ester glucuronide was investigated in aqueous buffers, buffered urine and human serum albumin solutions. The glucuronide was unstable in neutral and mildly alkaline solutions, and special precautions have to be taken during sampling and sample treatment in order to preserve the genuine glucuronide. The presence of human serum albumin in the solution stabilized the glucuronide against isomerization/rearrangements but catalysed the hydrolysis of the glucuronide. When incubating human serum albumin with the ester glucuronide, probenecid was shown to be covalently bound to the protein probably via a transacylation reaction.