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.

On-line deconjugation of chloramphenicol-beta-D-glucuronide on an immobilized beta-glucuronidase column. Application to the direct analysis of urine samples.

An immobilized HPLC column has been developed for the on-line deconjugation of beta-glucuronides. The enzymatic activity of this column has been previously demonstrated [1]. This study reports on the application of the immobilized beta-glucuronidase column to the analysis of glucuronide metabolites in the urine. The system utilized in this work was composed of an internal-surface reversed-phase (ISRP) column (50 x 4.6 mm) containing a hydrophobic inner phase and a hydrophilic outer phase, a beta-glucuronidase immobilized enzyme reactor (BG-IMER) column (50 x 4.6 mm) and a C8 reversed-phase column (150 x 4.6 mm). The columns were connected with three six-port switching valves. A coupled-column procedure was developed for urine samples containing chloramphenicol-beta-D-glucuronides (0.07-1.1 mM/injection). Urine samples were injected into the ISRP column where the glucuronides were separated from the biological matrix, with matrix contaminants eluting off-line to waste. Eluent from the ISRP column containing the glucuronides was then transferred on-line to the beta-glucuronidase column for deconjugation and passed directly on-line to the C8 column. In this portion of the chromatographic procedure, the mobile phase consisted of 0.01 M ammonium acetate at pH 6.7. The analyte concentrated on the top of the reversed-phase column was then eluted using a gradient mobile phase system of acetonitrile and 0.01 M ammonium acetate (pH 5.0) and detected at UV wavelength of 280 nm.