[关键词]
[摘要]
建立了黄油中雌酮、α/β-雌二醇、雌三醇和孕酮5种类固醇激素的气相色谱/燃烧炉/同位素比质谱(GC/C/IRMS)溯源方法。样品经乙酸乙酯-环己烷(1:1,V/V)提取,经凝胶渗透色谱(GPC)净化和半制备液相色谱(Pre-HPLC)纯化,纯化液经HP-5MS(30 m×0.25 mm,0.25 μm)柱分离,GC/C/IRMS溯源分析和气相色谱-质谱(GC/MS)定性和定量分析。黄油中外源性激素δ13C值<-30‰,内源性激素δ13C值>-27‰,其中黄油中外源性孕酮δ13C值=-30.59‰±0.12‰,内源性孕酮δ13C值范围在-26.83‰±0.25‰与-23.80‰±0.33‰之间,单因素方差分析(p值=0.009<0.05)显示内源性孕酮和外源性孕酮的δ13C值存在显著差异性。经模拟实验显示,实际样品中引入外源性激素的δ13C值与外源性激素δ13C值具有同源性。方法灵敏度为15~100 ng,方法批内精密度为0.11‰~0.17‰,批间精密度为0.16‰,Pre-HPLC的馏分接收是同位素分馏现象发生和影响测定准确性的主要阶段。结果表明,本方法准确性和特异性好,GC/C/IRMS是鉴别激素来源的有效工具。
[Key word]
[Abstract]
A gas chromatography-combustion-isotope ratio mass spectrometry method was developed for investigating the traceability of five steroid hormones (estrone, α/β-estradiol, estriol and progesterone) in butter. The samples were first extracted by ethylacetate/cyclohexane (1:1, V/V). The sample was extracted with ethyl acetate-cyclohexane (1:1, V/V), purified by gel permeation chromatography (GPC) and semi-preparative liquid chromatography (Pre-HPLC), separated through a HP-5MS column (30 m × 0.25 mm, 0.25 μm), before traceability analysis by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) and qualitative and quantitative analyses by GC/MS. The δ13C values of the exogenous hormones in butter were <-30‰ while the δ13C values of endogenous hormones were >-27‰, among which the δ13C value of exogenous progesterone in butter was -30.59‰±0.12‰ and the δ13C value of endogenous progesterone ranged from -26.83‰±0.25‰ to -23.80‰±0.33‰. One-way analysis of variance (p-value=0.009<0.05) indicated a significant difference in the δ13C values between the endogenous and exogenous progesterone. The simulation experiments showed that the δ13C value of the sample spiked with an exogenous hormone was homologous with the δ13C value of the exogenous hormone. The sensitivity of the method for the five hormones was in the range of 15~100 ng. The within-batch precision of the method ranged from 0.11‰ to 0.17‰ and the between-batch precision was 0.16‰. The step of Pre-HPLC fraction collection was critical for isotopic fractionation and analysis accuracy. The obtained results showed that the established method is accurate and specific, and GC/C/IRMS is an effective tool for investigating the origin of steroid hormones.
[中图分类号]
[基金项目]
上海市科委科研项目(16142201500;17DZ2293700;18DZ2201200;17DZ2201100);长三角科技合作项目(17395810102);中央引导地方科技发展专项(YDZX20173100004528)