On-line deconjugation of chloramphenicol-β-D-glucuronide on an immobilized β-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 C-s 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. fluent 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 C-s 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. (C) 1998 Elsevier Science B.V. All rights reserved.