High-performance liquid chromatographic analysis of glucuronic acid conjugates after derivatization with...

In order to enhance the detection sensitivity of various glucuronic acid conjugates (phenol, menthol borneol, estrone and testosterone) in high-performance liquid chromatography (HPLC), the compounds were esterified with 4-bromomethyl-7-methoxycoumarin in the presence of potassium carbonate and 18-crown-6 in acetone. The resulting esters were chromatographed on either a normal-phase (NP) column (LiChrospher DIOL) with hexane-ethanol mixtures as eluents or a reversed-phase column (LiChrospher CH-18) with methanol-water mixtures. They were detected by UV spectrophotometry at 328 nm. The structure of the derivatives was confirmed by mass spectrometry by direct introduction and chemical ionization. Prior to this step, their isolation on a semi-preparative scale was performed by NP-HPLC. The extraction of the studied glucuronides from microsomal solutions was tested by ion-suppression and ion-pair liquid-liquid partition and liquid-solid chromatography (on octadecylsilica cartridges). Extraction and chromatographic data are discussed with regard to the determination of glucuronyltransferase activity towards the aglycones cited above.