气相色谱-质谱联用法测定小鼠血清非胆固醇甾醇

Determination of non-cholesterol sterols in mouse serum by gas chromatograph-mass spectrometer

目的本研究旨在建立气相色谱-质谱联用法(gas chromatograph-mass spectrometer,GC-MS)测定小鼠血清中非胆固醇甾醇的方法。方法采集32周龄雄性C57BL/6J小鼠血清,以5α-胆甾烷为内标,经过氢氧化钾乙醇溶液皂化、正己烷萃取、硅烷化试剂衍生等步骤,进行小鼠血清样本进样前处理。运用气相色谱-质谱联用仪对小鼠血清非胆固醇甾醇进行定性及定量分析。配制不同浓度混标溶液进行GC-MS法分析制备标准曲线。制备高、中、低浓度混标溶液与等量小鼠血清混合处理后测定非胆固醇甾醇含量进行加标回收率、精密度及重复性试验。采用酶比色法测定小鼠血清总胆固醇(total cholesterol,TC)含量。结果 GC-MS法测得小鼠血清角鲨烯(squalene)、去氢胆甾醇(desmosterol)、菜油甾醇(campesterol)、豆甾醇(stigmasterin)和β-谷甾醇(β-sitosterol)的含量依次为(4.52±0.94、1.71±1.57、22.17±9.86、0.69±0.05和8.82±3.92)μg/mL;标准化后角鲨烯/TC、去氢胆甾醇/TC、菜油甾醇/TC、豆甾醇/TC和β-谷甾醇/TC,分别为(5.92±2.06、1.89±2.26、26.52±9.54、0.90±0.18和9.08±5.51)μg/mg,平均加标回收率依次为95.65%、96.75%、99.14%、103.52%和100.57%,日内精密度及重复性试验相对标准偏差(relative standard deviation,RSD)均在5.00%以下。结论 GC-MS法具有检测速度快、结果准确、精密性高和重复性好的优点,可用于实验小鼠血清非胆固醇甾醇的检测,为深入研究实验动物胆固醇合成和吸收的机制提供研究手段。

Objective To establish a method of gas chromatograph-mass spectrometer（GC-MS） to determine the non-cholesterol sterols in mouse serum.Methods Serum samples collected from male mice aged 32 weeks were preprocessed by saponification with potassium hydroxide ethanol solution,extraction by hexane,and derivation by silylation reagents with 5α-cholestane as an internal standard.The non-cholesterol sterols content in mouse serum were determined by GC-MS.Different concentrations of mixed standard solutions by GC-MS analysis were made for the standard curve.Three different concentrations of mixed standard solutions were put into the same amount of mouse serum,and were used to determine the content of non-cholesterol sterols by GC-MS for the recoveries,precision and repetition test.The serum total cholesterol（TC） was determined by enzyme colorimetric method.Results The contents of squalene,desmosterol,campesterol,stigmasterin and β-sitosterol by GC-MS were as follows（4.52±0.94,1.71±1.57,22.17±9.86,0.69±0.05 and 8.82±3.92） μg/mL,respectively.The results were standardized as ratio to TC（total cholesterol）.Squalene/TC,desmosterol/TC,campesterol/TC,stigmasterin/TC and β-sitosterol/TC were as follows（5.92±2.06,1.89±2.26,26.52±9.54,0.90±0.18 and 9.08±5.51） μg/mg.The mean recoveries of the non-cholesterol sterols were between 95.65%and 103.52%.The intra-day RSDs of precision and repetition test were both below 5.00%.Conclusion GC-MS can be used for quickly measuring serum non-cholesterol sterols of mouse with high sensitivity,specificity and reproducibility.This method will provide a reliable basis for further study of the mechanisms of cholesterol synthesis and absorption in experimental animals.