Use of a post-column immobilized beta-glucuronidase enzyme reactor for the determination of diastereomeric glucuronides of fenoldopam in plasma and urine by high-performance liquid chromatography with electrochemical detection

Abstract

A post-column enzyme reactor, containing beta-glucuronidase immobilized on controlled-pore glass beads, was developed for use in the high-performance liquid chromatographic (HPLC) analysis of glucuronide metabolites using electrochemical detection. The reactor performance was evaluated with glucuronide conjugates of the new antihypertensive agent, fenoldopam [6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-di ol]. These conjugates, which are electrochemically inactive at 0.6 V vs. Ag/AgCl, were separated by HPLC and passed directly into the post-column beta-glucuronidase reactor, which converted the glucuronides to their electrochemically active aglycone, fenoldopam. The enzyme reactor converted greater than 80% of the entering glucuronide to fenoldopam and produced a linear response for fenoldopam glucuronide in the range 0.4-200 ng injected on-column. The reactor performance was optimal when the mobile phase (methanol-acetate buffer) contained 0-25% methanol, but the efficiency gradually declined thereafter until, at 50% methanol, the reactor was inactive. The working pH range for the mobile phase was 5.5-8.0, with a performance optimum at pH 6.0. The reactor displayed marked stability during usage (greater than 4 months) and during storage (greater than 6 months). The reactor did not hydrolyze the 8-O-sulfate conjugate of fenoldopam but did convert the 1(R) and 1(S) diastereomers of fenoldopam-7-O-beta-glucuronide and 1(S)-fenoldopam-8-O-beta-glucuronide to fenoldopam. An assay was developed for 1(R)-fenoldopam-7-O-beta-glucuronide in plasma and urine by using the deschloro, des-4'-hydroxy analogue of fenoldopam glucuronide as the internal standard. The assay was linear in the range 4-1600 ng/ml. The within-day and between-day coefficients of variation for the method were less than 7% at three plasma fenoldopam glucuronide concentrations.