超高效液相色谱-串联质谱法测定斑马鱼性腺中13种类固醇激素

Analysis of Steroid Hormones in Ovaries of Zebrafish(Danio rerio) by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

建立了超高效液相色谱-串联质谱测定雌性斑马鱼性腺中13种类固醇激素的分析方法。雌鱼性腺分别经预冷的乙腈、乙酸乙酯浸提后,氨基（NH2）、亲水-亲脂平衡介质（HLB）固相萃取柱富集净化,ACQUITY UPLCBEH C18色谱柱进行梯度分离。采用多反应监测模式扫描,基质匹配外标法定量。研究了不同提取溶剂对13种类固醇激素的提取效果,考察了固相萃取柱的净化效果,并对斑马鱼性腺进行类固醇激素加标回收率实验,结果表明,13种分析物的平均加标回收率为74.8%-127.0%,相对标准偏差为3.9%-15.8%,方法检出限为0.006-0.300μg/kg。该方法简便、准确、灵敏,可应用于斑马鱼性腺类固醇激素生理水平的检测。

An ultra performance liquid chromatography-tandem mass spectrometric（UPLC-MS /MS） method was developed for the simultaneous determination of steroid hormones in ovaries of zebrafish.Homogenized ovary samples were purified with NH2 and HLB solid phase extraction column after extracted with acetonitrile and ethyl acetate at 4 ℃.The samples were transported and eluted on an analytical column ACQUITY UPLCBEH C18（2.1 mm × 100 mm,1.7 μm）.All the analytes were detected in selected reaction monitoring（SRM） mode and quantified by the external matrixmatched standard method.The extraction efficiencies of different extraction solvents and cleanup effects of solid phase extraction columns for each analyte were further investigated.The average recoveries of 13 steroid hormones in spiked zebrafish ovary samples ranged from 74.8% to 127.0% with relative standard deviations（RSD） of 3.9%-15.8%.The results showed that the limits of detection（LODs,S / N = 3） were in the range of 0.006-0.300 μg / kg.The method was sensitive and reliable,and could meet the requirements for the quantification of steroid hormones in zebrafish at physiological level.