许丽,袁波,金艺,甄慧娟,赵怀清,徐海燕
XU Li,YUAN Bo,JIN Yi,ZHEN Hui-juan,ZHAO Huai-qing,XU Hai-yan(School of Pharmacy

• 1:沈阳药科大学药学院

摘要(Abstract)：

目的建立同时测定硫酸雌酮与染料木素的细胞孵化液中硫酸雌酮、染料木素及它们主要代谢物含量的LC-MS/MS法,并用该法研究硫酸雌酮与染料木素在人乳腺细胞中的代谢相互作用。方法细胞孵化样品经处理后,采用Diamonsil C18柱分离,流动相为乙腈和5 mmol.L-1醋酸铵(pH7.8),梯度洗脱,以大豆苷元为内标。采用电喷雾电离(ESI)源,以多反应监测(MRM)方式进行检测,用于定量分析雌酮(estrone,E1)、雌二醇(estradiol,E2),硫酸雌酮(estrone-3-sulfate,E1S)、硫酸雌二醇(estradiol-3-sulfate,E2S)、β-D-葡萄糖醛酸雌酮(estrone-3-glucuronide,E1G)、β-D-葡萄糖醛酸雌二醇(estradiol-3-glucuronide,E2G)、染料木素(genistein,GEN)、染料木素-4'-硫酸结合物(genistein-4'-sulfate,G-4'-S)、染料木素-7-硫酸结合物(genistein-7-sulfate,G-7-S)、染料木素-4'-葡萄糖醛酸结合物(genistein-4'-glucuronide,G-4'-G)和染料木素-7-葡萄糖醛酸结合物(genistein-7-glucu-ronide,G-7-G)。结果该方法中E1、E1S、E2S、G-4'-S、G-7-G的线性为2.45～5 000.00 nmol.L-1,E2的线性为9.80～5 000.00 nmol.L-1,E1G的线性为0.25～500.00 nmol.L-1,E2G的线性为0.98～500.00 nmol.L-1,GEN的线性为19.50～10 000.00 nmol.L-1,G-7-S的线性为2.45～1250.00 nmol.L-1;日内和日间精密度均不大于6.7%,相对误差在±12.5%以内。在人乳腺细胞模型中,GEN促进E1S的代谢,而E1S抑制GEN代谢。结论该法适用于细胞孵化液中硫酸雌酮、染料木素及它们主要代谢物的同时测定,并成功应用于人乳腺细胞中硫酸雌酮与染料木素代谢相互作用的研究。
Objective To develop an LC-MS/MS method for simultaneous assay of estrone,genistein and their main metabolites in cell incubation solutions and study the metabolic interacting of estrone and genistein in breast cells.Methods The analytes(E1,E2,E1S,E2S,E1G,E2G,GEN,G-4′-S,G-7-S,G-7-G)and the internal standard(daidzein)were separated on a Diamonsil C18 column with an elution gradient consisting of 5 mmol · L-1 ammonium acetate(pH 7.8)and acetonitrile.A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the negative ion mode.Results The method showed a good linearity in a concentration range of 2.45-5 000.00 nmol · L-1 for E1,E1S,E2S,G-4′-S and G-7-G,9.80-5 000.00 nmol · L-1 for E2,2.45-1 250.00 nmol · L-1 for G-7-S,0.25-500.00 nmol · L-1 for E1G,0.98-500.00 nmol · L-1 for E2G,19.50-10 000.00 nmol · L-1 for GEN.The intra-and inter-day precisions were within 6.7% in terms of relative standard deviation(RSD/%)and the accuracy was within±12.5% in terms of relative error(RE/%).When genistein and E1S were simultaneously incubated in breast cells,the metabolism of genistein was inhibited,while the metabolism of E1S was promoted.Conclusions The developed method is simple,rapid,sensitive,specific and suitable for the simultaneous determination of estrone,genistein and their main metabolites in cell incubation samples.

关键词(KeyWords)： 雌激素;染料木素;LC-MS/MS;药物相互作用;人乳腺细胞
estrogen;genistein;LC-MS/MS;metabolic interaction;human breast cell

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(30801420)

作者(Author): 许丽,袁波,金艺,甄慧娟,赵怀清,徐海燕
XU Li,YUAN Bo,JIN Yi,ZHEN Hui-juan,ZHAO Huai-qing,XU Hai-yan(School of Pharmacy

DOI: 10.14066/j.cnki.cn21-1349/r.2012.08.010

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