摘要
目的:建立快速、灵敏、简便的超高效液相色谱—串联质谱(UPLC-MS/MS)法同时测定乳腺癌细胞中雌二醇及其代谢产物2-OH雌二醇和4-OH雌二醇的含量的方法。方法:以炔雌醇为内标,细胞样品经甲基叔丁基醚(MTBE)萃取后、氮气吹干,用吡啶-3-磺酰氯衍生化、再次用MTBE萃取、氮气吹干、用80%甲醇复溶,通过ACQUITY UPLC CSHTM C18柱梯度洗脱,采用电喷雾离子源(ESI),阳离子扫描模式,以多反应监测方式(MRM)进行含量测定。结果:雌二醇在0.4~20 ng/m L,2-OH雌二醇、4-OH雌二醇在0.2~10 ng/m L浓度内具有良好的线性关系,雌二醇、2-OH雌二醇、4-OH雌二醇的定量下限分别为0.4 ng/m L、0.2 ng/m L、0.2 ng/m L;日内和日间精密度分别为5.63%~17.37%和1.19%~18.95%、准确度为85.29%~113.9%、稳定性和基质效应等均符合生物样品分析的要求。结论:本方法简便、准确、专属性强,适用于雌二醇及其代谢物在乳腺癌细胞中的药代动力学研究。MCF-7细胞主要将雌二醇代谢为2-OH雌二醇。
Objective： A fast,sensitive and simple method using ultra performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS） was established for simultaneous determination of estradiol and its metabolite 2-hydroxyestradiol and 4-hydroxyestradiol in breast cancer cell. Methods： Ethinylestradiol was used as the internal standard,the cell sample was extracted with methyl tert-butyl ether（MTBE）,dried in nitrogen,derivatized by pyridine-3-sulfonyl chloride,extracted with MTBE again,dried with nitrogen and reconstituted with 80% methanol. Chromatographic separation was performed on a Waters ACQUITY UPLC CSHTM-C18（2. 1 × 100 mm,1. 7 μm） column. The analytes were identified by Electrospray ionization in positive ion modes and multiple reaction monitoring（MRM）. Results： The calibration curves were linear in the concentration of 0. 4 ~ 20 ng/m L for estradiol,in the concentration of 0. 2 ~ 10 ng/m L for 2-hydroxyestradiol,4-hydroxyestradiol with correlation coefficients（r） more than 0. 998. The limits of quantification of estradiol,2-hydroxyestradiol and 4-hydroxyestradiol were 1. 2 ng/m L,0. 6 ng/m L,0. 6 ng/m L,respectively. The intra-day and inter-day precision were 5. 63% ~ 17. 37% and 1. 19% ~ 18. 95%,the accuracy was 85. 29% ~ 113.9%,and the stability and matrix effect were all in accordance with the requirements of biological sample analysis. Conclusion： Thissimple,accurate and specific assay is suitable for the pharmacokinetic study of estradiol and its metabolites in breast cancer cells. MCF-7 cells can mainly metabolize estradiol to 2-hydroxy estradiol.
出处
《江西中医药大学学报》
2018年第2期77-81,共5页
Journal of Jiangxi University of Chinese Medicine
基金
国家"重大新药创制"科技重大专项(2009ZX09103-399)
安徽省高校科研创新团队项目(2016hz23)
安徽省科技厅公益联动项目(1501Id04004)
