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.
Di Marco
M P
MP
Faculté de Pharmacie, Université de Montréal, PQ, Canada.
Felix
G
G
Descorps
V
V
Ducharme
M P
MP
Wainer
I W
IW
D002701:Chloramphenicol / Q000031:analogs & derivatives / Q000652:urine; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005965:Glucuronates / Q000378:metabolism*; D005966:Glucuronidase / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015203:Reproducibility of Results; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D005965:Glucuronates; C027810:chloramphenicol glucuronide; D002701:Chloramphenicol; D005966:Glucuronidase