Separation and detection of bile acid 24-glucuronides in human urine by liquid chromatography combined with electrospray ionization mass spectrometry

A method for the separation and determination of bile acid 24-glucuronides using liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) has been developed. In the ESI mode, 24-glucuronides were characterized by an intense deprotonated molecule [M-H](-) with. fragment ion [M-H-176](-) formed by elimination of the glucuronic acid moiety. The ratio of these negative ions was significantly influenced by the orifice-1 and the ring lens voltages. Bile acid 24-glucuronides in human urine were extracted with an OASIS HLB cartridge and then subjected to the LC/ESI-MS analysis employing an O-18-labeled internal standard. The simultaneous separation was attained on a Develosil ODS-HG-5 column using 20 mM ammonium acetate (pH 6.0)-acetonitrile (3:1-->3:2, v/v, 35 min) in a linear gradient elution mode, where the 24-glucuronides were monitored with a characteristic negative ion [M-H](-); the limit of detection was 10 fmol. This newly developed method was applied to the quantitation of bile acid 24-glucuronides in human urine with satisfactory selectivity and reliability.