An Hptlc Method For The Assay Of Udp-Glucuronosyltransferase Using P-Nitrophenol As Substrate

An HPTLC method is described for the assay of UDP- glucuronosyltransferase (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-glucuronide on RP-18 plates. The method was validated by studying the selectivity, the repeatability of sample application, the stability of the analyte, the limit of quantification, and the reproducibility and accuracy 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) protein, n = 6) for p-nitrophenol revealed good reproducibility (the RSD values [%] were 6.1 and 6.3, respectively) and the suitability of the method for kinetic studies. The HPTLC method was also demonstrated to be suitable for the separation of some catechol glucuronides from their parent compounds.