同位素稀释超高效液相色谱-串联质谱法检测牛奶中类固醇激素

Determination of steroid hormones in milk by isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry

目的建立一种牛奶中睾酮、孕酮及氢化可的松类固醇激素同位素稀释超高效液相色谱-串联质谱法(isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry,ID-UPLC-MS/MS)的分析方法。方法样品加氯化钠盐析、沉淀蛋白,经0.5%甲酸乙腈溶液提取,正己烷反萃取后,氮吹干燥,用10%甲醇溶液复溶,过HLB固相萃取柱净化,甲醇溶剂洗脱、氮吹干燥,复溶后上机检测。采用0.1%甲酸(A)和0.1%甲酸-甲醇(B)作为流动相进行梯度洗脱,采用电喷雾离子化(ESI+)质谱多级反应监测模式(multiple reactions monitoring,MRM)对分析物的定量离子和定性离子进行监测。结果本方法在8 min内完成3种目标化合物的分离分析。3种目标化合物在0.1~100μg/kg的浓度范围内有良好的线性关系,相关系数r2均大于0.9985。睾酮、孕酮和氢化可的松的方法检出限分别为0.043、0.027和0.085μg/kg,方法定量限分别为0.122、0.083和0.227μg/kg。在1、10和50μg/kg 3个添加水平下,3种目标化合物同位素稀释法回收率为97.29%~102.71%,日内相对标准偏差为0.30%~2.96%,日间相对标准偏差为0.84%~4.08%。结论该方法快速、准确、稳定性高,适用于同时检测牛奶中3种类固醇激素含量。

Objective To establish a method for the determination of testosterone, progesterone and hydrocortisone in milk by isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry（ID-UPLC-MS/MS）. Methods Milk samples were added with sodium chloride to salt out and precipitate proteins. After extraction with acetonitrile（contains 0.5% formic acid）, n-hexane was back-extracted, and then dried with nitrogen. The residue was dissolved with 10% methanol and purified by HLB solid phase extraction cartridge and eluted by methanol. The gradient elution was carried out using 0.1% formic acid aqueous solution（A） and 0.1% formic acid in methanol solution（B）. UPLC-MS/MS（ESI＋） with multiple reactions monitoring（MRM） was used for qualitative and quantitative analysis of the analytes. Results The separation and analysis of 3 kinds of target compounds were completed within 8 min. In the range of 0.1-100 μg/kg, 3 kinds of target compounds had good linear relationships and the correlation coefficients（r2） were more than 0.9985. The limits of detection（LOD） of testosterone, progesterone and hydrocortisone were 0.043, 0.027 and 0.085 μg/kg, and limits of quantification（LOQ） were 0.122, 0.083 and 0.227 μg/kg, respectively. At 3 spiked levels of 1, 10 and 50 μg/kg, the recoveries ranged from 97.29% to 102.71%. The intra-day and inter-day RSDs were in the range of 0.30%-2.96% and 0.84%-4.08%, respectively. Conclusion This method is fast, accurate and stable, which is suitable for the simultaneous determination of 3 kinds of steroid hormones in milk.