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目的建立同时测定人血浆中植物固醇和胆固醇合成前体物质的气相色谱-质谱(GC-MS)定量分析方法,为研究患者胆固醇代谢提供检测手段。方法色谱柱为HP-5MS石英毛细管柱(30 m×0.25μm×0.25 mm),初始柱温200℃,保持2 min,程序升温15℃.min-1至280℃,保持30 min,不分流进样。血浆经碱性醇溶液皂化后用正己烷萃取,再用衍生化试剂(BSTFA+1%TMCS)衍生后进样,采用全扫描(Scan)定性,选择离子监测(SIM)内标法定量。结果胆固醇、2,4-脱氢胆固醇、7-烯胆固醇、菜油固醇、β-谷固醇线性关系良好,日内日间精密度均分别小于7.9%与9.8%,加样回收率分别在89.5%～106.3%之间。应用本方法分别测定20名高血脂患者中非胆固醇类固醇的含量,其中2,4-脱氢胆固醇的含量为(0.39±0.08)mg.L-1,7-烯胆固醇的含量为(3.41±0.83)mg.L-1,菜油固醇的含量为(3.12±0.53)mg.L-1,β谷固醇的含量为(4.22±0.67)mg.L-1。结论 GC-MS法可同时测定人血浆中4种非胆固醇类固醇含量,并具有良好的准确度与精密性,适用于患者胆固醇代谢个体差异的分析。
OBJECTIVE To establish a gas chromatography-mass spectrometry (GC-MS) quantitative method for the simultaneous determination of phytosterols and cholesterol precursors in human plasma and to provide a means of detecting cholesterol metabolism in patients. Methods The column was HP-5MS quartz capillary column (30 m × 0.25 μm × 0.25 mm). The initial column temperature was 200 ℃ and kept for 2 min. The program temperature was raised from 15 ℃ to min-1 to 280 ℃ for 30 min. kind. The plasma was extracted with n-hexane after saponification with alkaline alcohol solution, and then derivatized with derivatization reagent (BSTFA + 1% TMCS). The samples were quantified by full scan (Scan) and selected by internal standard ion trap (SIM). Results Cholesterol, 2,4-dehydrocholesterol, 7-ene cholesterol, oleyl-sterol and β-sitosterol had good linearity. The intra- and inter-day precision were less than 7.9% and 9.8%, respectively. The recovery rates were 89.5 % ~ 106.3% between. The content of non-cholesterol steroids in 20 hyperlipidemic patients was determined by this method, and the content of 2,4-dehydrocholesterol was (0.39 ± 0.08) mg. The content of L-1,7-ene cholesterol was (3.41 ± 0.83 ) mg.L-1, the content of campesterol was (3.12 ± 0.53) mg.L-1, and the content of β-sitosterol was (4.22 ± 0.67) mg.L-1. Conclusion GC-MS method can simultaneously determine the content of four non-cholesterol steroids in human plasma with good accuracy and precision, and is suitable for the analysis of individual differences in cholesterol metabolism.