A method for the separation and detection of bile acid 3-glucuronides by liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) has been developed. On the ESI mode, glucuronides were characterized by an intense pseudo-molecular ion [M-h]- with a doubly charged ion [M-2H]2-, and the ratio of these negative ions were markedly influenced by an acidic component of salt added to a mobile phase, according to a pKa value of an acidic moiety at C-24. Bile acid 3-glucuronides in human urine were extracted with a SepPak C18 cartridge, followed by purification on lipophylic ion exchange gel, piperidinohydroxypropyl Sephadex LH-20. Subsequent resolution into unconjugated, glycine and taurine conjugated bile acid 3-glucuronides was attained by LC on a Develosil ODS-HG-5 column using 20 mM ammonium acetate (pH 7.0)-methanol (10:12, v/v) as a mobile phase, where 3-glucuronides were monitored with a characteristic negative ion [M-H]-. The newly developed method was applied to the quantitatiation of bile acid 3-glucuronides in human urine with satisfactory sensitivity and reliability.
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