高效液相色谱-四极杆/静电场轨道阱高分辨质谱法测定血清中胆固醇及其6种代谢标志物

利用高效液相色谱-四极杆/静电场轨道阱高分辨质谱（HPLC-Q/Orbitrap HRMS）测定血清中的胆固醇及其6种代谢标志物（2，4-脱氢胆甾醇、7-烯胆甾醇、菜油固醇、豆固醇、β-谷固醇和角鲨烯）。以乙腈作为提取溶剂，超声提取，采用Acquity UPLC BEH C₁₈色谱柱（100 mm×2.1 mm，1.7 μm），以95%（v/v）的甲醇-乙腈（2：8，v/v）-5%（v/v）水做流动相，等度洗脱15 min，流速0.4 mL/min。在大气压化学电离源（APCI）正离子扫描模式下，质谱采用全扫描/数据依赖二级扫描（Full MS/dd MS²）监测模式，同时获得定性和定量结果，一级分辨率为70000 FWHM，二级分辨率为17500 FWHM。7种目标物质在其线性范围内的线性相关系数（r²）均不小于0.992，检出限为0.8~62.1 μg/L，3个加标水平下的回收率为82.1%~97.5%，相对标准偏差（RSD）为1.6%~7.4%。方法准确、简单、快捷，可以作为血清中胆固醇及其6种代谢标志物的检测方法。

Determination of cholesterol and its six metabolism markers in serum by high performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry

A method for the determination of cholesterol and its six metabolism markers (desmosterol, lathosterol, campesterol, stigmasterol, β-sitosterol, squalene) in serum by high performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (HPLC-Q/Orbitrap HRMS) was developed. The sample was ultrasonically extracted with acetonitrile. The separation was performed by using 95%(v/v) methanol-acetonitrile (2:8, v/v)-5%(v/v) water as the mobile phase. Isocratic elution was performed at a flow rate of 0.4 mL/min on an Acquity UPLC BEH C₁₈ column (100 mm×2.1 mm, 1.7 μm) in 15 min. The atmospheric pressure chemical ionization (APCI)/positive mode was used in the MS detection. Cholesterol and its six metabolism markers were qualitatively and quantitatively determined by using the full MS scan/date dependent MS² (Full MS/dd MS²) mode. The resolution of the precursor mass was 70000 FWHM, while the resolution of the product mass was 17500 FWHM. The correlation coefficients (r²) of all the seven target compounds were no less than 0.992 in the linear range. The limits of detection were between 0.8 μg/L and 62.1 μg/L, the recoveries were in the range of 82.1%-97.5% with the relative standard deviations in range of 1.6%-7.4%. The method is accurate, simple, and rapid, and can be used for the determination of cholesterol and its six metabolism markers in serum.