Ikegawa S, Murao N, Motoyama T, Yanagihara T, Niwa T, Goto J
Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Biomed Chromatogr. 1996 Nov-Dec;10(6):313-7. doi: 10.1002/(SICI)1099-0801(199611)10:6<313::AID-BMC603>3.0.CO;2-F.
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.
已开发出一种通过液相色谱（LC）/电喷雾电离（ESI）-质谱（MS）分离和检测胆汁酸3-葡萄糖醛酸苷的方法。在ESI模式下，葡萄糖醛酸苷的特征在于具有强的准分子离子[M-h]-和双电荷离子[M-2H]2-，根据C-24处酸性部分的pKa值，这些负离子的比例受到添加到流动相中的盐的酸性成分的显著影响。人尿中的胆汁酸3-葡萄糖醛酸苷用SepPak C18柱提取，然后在亲脂性离子交换凝胶哌啶羟丙基葡聚糖凝胶LH-20上进行纯化。随后，通过在Develosil ODS-HG-5柱上进行LC，使用20 mM醋酸铵（pH 7.0）-甲醇（10:12，v/v）作为流动相，将其分离为未结合的、甘氨酸和牛磺酸结合的胆汁酸3-葡萄糖醛酸苷，其中3-葡萄糖醛酸苷用特征负离子[M-H]-进行监测。新开发的方法应用于人尿中胆汁酸3-葡萄糖醛酸苷的定量分析，具有令人满意的灵敏度和可靠性。
