液相色谱串联质谱法测定全血磷脂酰乙醇

Determination of blood phosphatidylethanol by liquid chromatography tandem mass spectrometry

目的建立一种基于液相色谱串联质谱技术（LC－MS/MS）测定全血磷脂酰乙醇（PEth）的方法，用以客观、量化反映饮酒者酒精摄入水平。方法全血样品中加入磷脂酰丙醇（16∶0/16∶0）作为内标，用异丙醇溶液沉淀蛋白，震荡、离心、提取上清、氮气吹干，复溶，液相色谱分离后进入串联质谱分析。考察方法的线性、检测限、定量限、回收率和精密度。收集40名有一年以上稳定日常饮酒习惯的志愿者全血样本，测定PEth，分析其与自述酒精摄入量的相关性；分析轻度、中度和重度饮酒者全血PEth水平的差异。结果本方法线性相关系数大于0.999 2，检测限和定量限分别低于0.74 μg/L和2.48 μg/L。测定不同浓度PEth样本的批内和总不精密度分别为0.77%～3.18%和2.30%～6.95%。平均加样回收率为96.88%～102.99%。相关性分析显示，总PEth水平与志愿者自述饮酒量呈正相关（r＝0.769, P〈0.001）。Kruskal－Wallis分析显示不同程度饮酒者的总PEth水平差异有统计学意义（χ2＝18.850, P〈0.001）。结论建立LC－MS/MS测定全血PEth的方法，该方法简便、精密、可靠，可以有效区分轻、中、重度饮酒者，有望为评价酒精摄入状况以及饮酒与疾病关系提供方法学基础。

ObjectiveTo establish a method for measuring blood phosphatidylethanol by liquid chromatography coupled with tandem mass spectrometry （LC-MS/MS）, which can be applied for objective and quantitative of alcohol intake.MethodsWhole blood samples were treated with isopropanol to precipitate protein, and phosphopropanol （16∶0/16∶0） was used as the standard.After centrifugation, the supernatants were transferred and evaporated under a stream of nitrogen until dryness.Then the residuals were analyzed by LC-MS/MS.Various methodological parameters, including linearity, limit of detection （LOD）, limit of quantitation （LOQ）, recovery, and precision, were investigated.Finally, blood samples from 40 Chinese individuals with more than one year of regular drinking habits were analyzed, and distributions of phosphatidylethanol were evaluated.ResultsThe correlation coefficients were higher than 0.9992.The LOD and LOQ were lower than 0.74 and 2.48 ng/ml, respectively.The inter- and total assay coefficient of variations were 0.77%-3.18% and 2.30%-6.95%, respectively, with recoveries ranged from 96.88% to 102.99%.The relationship between phosphatidylethanol level and self-reported alcohol consumption was significantly and positively correlated （r=0.769, P〈0.001）. Furthermore, Kruskal-Wallis analysis showed a significant difference in total phosphatidylethanol levels among individuals with different levels of alcohol intake （χ2=18.850, P〈0.001）.ConclusionsAn LC-MS/MS method for whole blood phosphatidylethanol detection has been developed. This method is simple, sensitive and accurate and can effectively reflect light, moderate and heavy alcohol intake. The method will be applied to the assessment of alcohol consumption and its association with the risks of drinking related diseases.