牛肉中孕酮的气相色谱/燃烧炉/同位素比质谱检测

Determination of Progesterone in Beef Sample by Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry

建立了牛肉中孕酮的气相色谱/燃烧炉/同位素比质谱(GC/C/IRMS)检测方法。牛肉样品用乙腈振荡和超声辅助提取,经Na Cl脱水,有机相离心和旋转蒸发后以Zn Cl2脱脂,然后用LC-C18、LC-Si、LC-NH2固相萃取柱净化,过滤液经半制备液相色谱(Pre-HPLC)的C18柱纯化,最后分析物以GC/C/IRMS系统分析。牛肉中加标外源性孕酮δ13C值为-30.64±0.24‰(n=6),牛肉内源性孕酮δ13C值为-25.70±0.13‰(n=6),单因素方差分析(ANOVA,p值=2.23×10-14<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,且牛肉中加标外源性孕酮δ13C值与孕酮标准溶液的δ13C值无差异性。同时,经模拟实验可知,实际样品中内外源性孕酮混合物的δ13C值与外源性孕酮的δ13C值具有同源性。结果表明,本方法特异性和准确性好,GC/C/IRMS是鉴别激素来源的有效工具,该方法填补了国内鉴别激素来源技术空白。

The method of gas chromatography/combustion/isotope ratio mass spectrometry was developed for the determination of progesterone in beef samples in this study. The beef samples were extracted by acetonitrile oscillation and ultrasonic-assisted extraction, and were dehydrated by Na Cl to induce separation of water and organic layers. After centrifugation and evaporation, the extracts were further treated with Zn Cl2 to remove lipids and purified by solid phase extraction using LC-C18, LC-Si and LC-NH2 cartridges. After Pre-HPLC purification using a C18 column, the samples were analyzed for progesterone by GC/C/IRMS system. The δ13 C value of endogenous progesterone in beef was about-25.70±0.13‰（n=6）, and the δ13 C value of exogenous progesterone spiked in the sample was about-30.64±0.24‰（n=6）. One-way analysis of variance（ANOVA, p=2.23×10-140.05） indicated that there was a highly significant difference between the endogenous and exogenous δ13 C value of progesterone, while there was no significant difference between the exogenous and standard solution δ13 C value of progesterone. Meanwhile, the simulation experiment showed that the δ13 C value of mixture（endogenous and exogenous） of progesterone in actual sample was homologous with the exogenous δ13 C value of progesterone. The results showed that the method was specificity and reliable. GC/C/IRMS can be a powerful tool to investigate the origin of natural hormones. The method will fill the domestic technical gap for identifying the origin of hormones.