ONLINE DECONJUGATION OF GLUCURONIDES USING AN IMMOBILIZED ENZYME REACTOR BASED UPON BETA-GLUCURONIDASE

An immobilized enzyme reactor based upon beta-glucuronidase (BG-IMER) has been developed for the on-line deconjugation of substrates. The ac tivity of the BG-IMER and its applicability to on-line deconjugation w as investigated. The BG-IMER was coupled to a reversed-phase column (C -8 or C-18) and the latter column was used to separate substrates and products eluted from the beta-glucuronidase reactor. The activity of t he BG-IMER was followed by measurement of percent deconjugation and th e parameters investigated were: substrate concentration, pH (4 to 6), temperature (r.t., 37 degrees C), enzyme-substrate contact time using flow-rates of 0.1 to 1.0 ml/min (15-1.5 min). The glucuronides used in the evaluation of the BG-IMER were: 4-methylumbelliferyl-beta-D-glucu ronide, p-acetaminophen-beta-D-glucuronide, 3'-azido-3'-deoxy-thymidin e-beta-D-glucuronide, phenyl-beta-D-glucuronide, chloramphenicol-beta- D-glucuronide, estradiol-17-beta-D-glucuronide and morphine-beta-D-glu curonide. The development of on-line HPLC deconjugation of glucuronide substrates using the BG-IMER will facilitate the identification of me tabolites and quantification of aglycones in metabolic and pharmacokin etic studies. (C) 1998 Published by Elsevier Science B.V. All rights r eserved.