脂质组学血清样品处理方法及其快速高分辨液相色谱-质谱分析

Development of Method for Serum Preparation and Lipidomics Analysis Based on Rapid Resolution Liquid Chromatography-Mass Spectrometry

通过系统考察血清样本前处理方法、优化液相色谱条件及质谱检测参数,建立了脂质组学血清样本前处理及其快速高分辨液相色谱-质谱(RRLC-MS)分析方法。血清样本中加入二氯甲烷-甲醇(CH2Cl2-CH3OH,2∶1,V/V)混合溶剂进行脂质提取,采用ACQUITY UPLC CSH C18色谱柱(100 mm×2.1mm,1.7μm),以10 mmol/L甲酸铵溶液(含0.1%甲酸)为流动相A,乙腈-异丙醇(1∶1,V/V)为流动相B,梯度洗脱,采用正负离子检测模式电喷雾电离(±)ESI、正离子检测模式大气压化学电离(+)APCI方式进行质谱检测。对血清中脂肪酸、甘油脂、鞘磷脂、甘油磷脂和胆固醇(酯)5类14种代表性脂质类成分进行了方法学考察。结果表明,方法灵敏度高、精密度及稳定性良好。对穿插在检测序列中的质控样本检测结果进行主成分分析(PCA),证明了本方法获得数据的可靠性良好,适用于脂质组学研究。

A rapid resolution liquid chromatography-mass spectrometric（RRLC-MS） method was developed and validated for the determination of endogenous lipid metabolites in human serum.The samples were pretreated by lipid extraction with methylene chloride-methanol（2 ∶ 1,V/V） mixture solvent and introduced to RRLC-MS under intensive optimization of chromatography conditions and mass spectrometry parameters.The chromatographic separation was performed on an ACQUITY UPLC CSH C18 column（100 mm×2.1 mm,1.7 μm） with mobile phase containing 10 mmol/L ammonium formate（0.1% formic acid）（A） and acetonitrile-isopropyl alcohol（1 ∶ 1,V/V） mixture solution（B） in a linear gradient elution.The MS detection was carried out by the integrated ionization approach using electrospray ionization（ESI） in both positive and negative ion modes and atmosphere pressure chemical ionization（APCI） in positive ion mode.Typical lipid molecules such as free fatty acids（FFAs）,phospholipids（PLs）,sphingophosphalipids（SPs）,glycerolipids（GLs）,cholesterol（Chol.） and cholesterol esters（CEs） had been detected with high sensitivity,good precision and reliable stability.Furthermore,the principal component analysis（PCA） results of the quality control（QC） samples among the real sample queue indicated the data reliability of the method in lipidomics applications.