Direct measurement of probenecid and its glucuronide conjugate by means of high pressure liquid chromatography in plasma and urine of humans.

Probenecid with its phase-I metabolites, and phase-II glucuronide conjugate can be analysed by a gradient high pressure liquid chromatographic method. Probenecid glucuronide in plasma with pH 7.4 is not stable and declines to 10% of the original value within 6 h (t1/2 approximately 1 h). Probenecid glucuronide is stable in urine with pH 5.0, moderately unstable at pH 6.0 (t1/2 approximately 10 h), and unstable at pH 8.0 (t1/2 approximately 0.5 h). Probenecid glucuronide is stable in water and 0.01 mol/l phosphoric acid in the autosampler of the high pressure liquid chromatograph. The decrease in concentration in water is 5.5% during 9 h and 0% in diluted acid. Probenecid glucuronide and the phase-I metabolites were not detectable in plasma. The main compound in fresh urine is the phase-II conjugate probenecid glucuronide (62% of a 500 mg dose); the phase-I metabolites are present and only a trace of probenecid is present. The percentage of the dose of the phase-I metabolites varies between 5 and 10, while hardly any probenecid is excreted unchanged (0.33%).