On-line 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 activity of the BG-IMER and its applicability to on-line deconjugation was investigated. The BG-IMER was coupled to a reversed-phase column (C8 or C18) and the latter column was used to separate substrates and products eluted from the beta-glucuronidase reactor. The activity of the BG-IMER was followed by measurement of percent deconjugation and the 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 min/min (15-1.5 min). The glucuronides used in the evaluation of the BG-IMER were: 4-methylumbelliferyl-beta-D-glucuronide, p-acetaminophen-beta-D-glucuronide, 3'-azido-3'-deoxythymidine-beta-D-glucuronide, phenyl-beta-D-glucuronide, chloramphenicol-beta-D-glucuronide, estradiol-17-beta-D-glucuronide and morphine-beta-D-glucuronide. The development of on-line HPLC deconjugation of glucuronide substrates using the BG-IMER will facilitate the identification of metabolites and quantification of aglycones in metabolic and pharmacokinetic studies.