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本研究应用超高效液相色谱串联四极杆质谱(UPLC-MS/MS)在12分钟内有效地检测了10种激素和激素葡糖醛酸代谢产物。激素睾酮(T)、二氢睾酮(DHT)、雄甾酮(ADT)、本胆烷醇酮(ETIO)、雌二醇(E_2)以及它们的葡糖醛酸化产物能够在Eclipse Plus C_(18)色谱柱(2.1 mm×50 mm,RRHD 1.8μm)上很好地分离。流动相为甲醇和含1 m M甲酸铵水溶液(60:40,v/v),流速为0.25 m L/min。液相流出液使用电喷雾离子源(ESI)在正负模式下进行检测。使用中性丢失(丢失分子量为正模式下的176、194、211和229 Da)和母离子扫描(母离子分子量m/z为正模式下的141、159、177和负模式下的75、85、133)的方法鉴定激素的葡萄醛酸化产物。质谱多反应监测模式(MRM)在正模式下检测睾酮、二氢睾酮、雄甾酮、本胆烷醇酮和雌二醇的检测离子分别为m/z 289.3→97.1、291.3→105、291.3→199.2、273.2→145.4和255.2→159.1;在负模式下睾酮的葡糖醛酸产物(T-G)的检测离子为m/z 463.3→85,二氢睾酮、雄甾酮、本胆烷醇酮的葡糖醛酸产物(DHT-G、ADT-G、ETIO-G)的检测离子为m/z 465.3→75,雌二醇的葡糖醛酸产物(E_2-G)的检测离子为m/z 447.3→271。此外,本分析方法还用于检测人肝微粒体孵育体系中激素的葡糖醛酸化产物,这可能为深入研究激素在人体内外的代谢过程奠定基础。
In this study, UPLC-MS / MS was used to detect 10 hormones and glucuronide metabolites effectively in 12 minutes. Hormone testosterone, dihydrotestosterone (DHT), androstone (ADT), the present cholecalciferolone (ETIO), estradiol (E_2) and their glucuronidation products were able to be used in Eclipse Plus C 18 ) Column (2.1 mm × 50 mm, RRHD 1.8 μm). The mobile phase consisted of methanol and 1 M aqueous ammonium formate (60:40, v / v) with a flow rate of 0.25 m L / min. The liquid phase effluent was detected using electrospray ionization (ESI) in positive and negative mode. Using neutral loss (176,194,211 and 229 Da for loss of molecular weight positive mode) and precursor ion scan (parent ion molecular weight m / z is 141, 159, 177 for positive mode and 75, 85 for negative mode , 133) for the identification of glucuronidation products of hormones. The detection mass of testosterone, dihydrotestosterone, androsterone, the present cholagrenolone and estradiol in the positive mode of the mass spectrometry multiple reaction monitoring mode (MRM) were m / z 289.3 → 97.1, 291.3 → 105, 291.3 → 199.2, 273.2 → 145.4 and 255.2 → 159.1; in the negative mode the test ion of testosterone glucuronate product (TG) is m / z 463.3 → 85, dihydrotestosterone, androsterone, The detected ion of the uronic acid products (DHT-G, ADT-G, ETIO-G) is m / z 465.3 → 75, and the detection ion of the glucuronic acid product of estradiol (E 2 -G) is m / z 447.3 → 271. In addition, this method is also used to detect the glucuronidation products of the hormone in the human liver microsomal incubation system, which may lay a foundation for the further study of hormone metabolism in and outside the human body.