AN HPTLC METHOD FOR THE ASSAY OF UDP-GLUCURONOSYLTRANSFERASE USING P-NITROPHENOL AS SUBSTRATE

An HPTLC method is described for the assay of UDP- glucuronosyltransfe rase (UGT) in rat liver microsomes, using p- nitrophenol as substrate. The glucuronides formed in the UGT catalyzed reaction were quantified by use of an authentic reference standard of p-nitrophenyl-beta-D-glu curonide on RP-18 plates. The method was validated by studying the sel ectivity, the repeatability of sample application, the stability of th e analyte, the limit of quantification, and the reproducibility and ac curacy of the method. Determination of the enzyme kinetic parameters K -m (mean 109.7 mu M; n = 6) and V-max (mean 48.2 nmol min(-1) mg(-1) p rotein, n = 6) for p-nitrophenol revealed good reproducibility (the RS D values [%] were 6.1 and 6.3, respectively) and the suitability of th e method for kinetic studies. The HPTLC method was also demonstrated t o be suitable for the separation of some catechol glucuronides from th eir parent compounds.