On-line deconjugation of chloramphenicol-β- d-glucuronide on an immobilized β-glucuronidase column : Application to the direct analysis of urine samples

Abstract

An immobilized HPLC column has been developed for the on-line deconjugation of β-glucuronides. The enzymatic activity of this column has been previously demonstrated [1]. This study reports on the application of the immobilized β-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 (50×4.6 mm) containing a hydrophobic inner phase and a hydrophilic outer phase, a β-glucuronidase immobilized enzyme reactor (BG-IMER) column (50×4.6 mm) and a C 8 reversed-phase column (150×4.6 mm). The columns were connected with three six-port switching valves. A coupled-column procedure was developed for urine samples containing chloramphenicol-β- d-glucuronides (0.07–1.1 m M/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 β-glucuronidase column for deconjugation and passed directly on-line to the C 8 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.