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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 establish a LC-MS / MS method for the simultaneous determination of estrone sulfate, genistein and their major metabolites in estrone sulfate and genistein-containing cell incubation solution. The method was used to study the effect of estrone sulfate and genistein on human Metabolic interactions in breast cells. Methods After incubation, the samples were separated on a Diamonsil C18 column. The mobile phase consisted of acetonitrile and 5 mmol·L-1 ammonium acetate (pH 7.8) with gradient elution and daidzein as internal standard. The electrospray ionization (ESI) source was used for the detection of estrone (E1), estradiol (E2) and estrone-3-sulfate , E1S), estradiol-3-sulfate (E2S), estrone-3-glucuronide (E1G), estradiol- 3-glucuronide, E2G), genistein (GEN), genistein-4’-sulfate (G- 4’-S), genistein- Genistein-7-sulfate (G-7-S), genistein-4’-glucuronide (G-4’-G) and genistein- The genistein-7-glucu-ronide (G-7-G). Results The linearities of E1, E1S, E2S, G-4’-S and G-7-G were 2.45-5000.00 nmol.L-1 and the linearities of E2 were 9.80-5000.00 nmol.L- The linearity of E2G was 0.98-500.00 nmol.L-1, the linearity of GEN was 19.50-10 000.00 nmol.L-1, the linearity of G-7-S was 2.45-1250.00 nmol.L-1. The intra- and inter-day precision was no more than 6.7% with the relative error within ± 12.5%. In the human mammary gland cell model, GEN promotes the metabolism of E1S while E1S inhibits the metabolism of GEN. Conclusion The method is suitable for the simultaneous determination of estrone sulfate, genistein and their major metabolites in cell incubation solution and has been successfully applied to the metabolic interaction between estrone sulphate and genistein in human mammary gland cells.