基于智能化分时区间采集技术的液相色谱-串联质谱法测定全血中磷脂酰乙醇及人群水平调查

磷脂酰乙醇是具有潜在临床应用价值的酒精摄入生物学标志物，准确测定其含量可以为酒精摄入监测提供客观、量化的重要参考依据。本研究建立了一种基于智能化分时区间采集技术的液相色谱-串联质谱分析方法，能够同时实现对人全血样品中18种磷脂酰乙醇的准确测定。采用甲醇-甲基叔丁基醚-水作为萃取体系，选用XBridge C18色谱柱，以2.5 mmol/L乙酸铵异丙醇溶液和2.5 mmol/L乙酸铵水溶液-乙腈（50∶50， v/v）为流动相进行梯度洗脱，采用电喷雾离子源、智能化分时间段-负离子多反应选择离子监测模式。经验证，该方法线性关系良好，相关系数≥0.9998，线性范围在10~2500 ng/mL之间，检出限和定量限分别为0.7~2.8 ng/mL、2.2~9.4 ng/mL，加标回收率在91.0%~102.2%之间，日内精密度和日间精密度在0.4%~7.4%之间。该方法简便、快速、精密，智能化分时区间采集方法为每个离子通道分配合适的扫描时间段，增加了每个目标物离子通道的有效采集时长，提高了各目标分析物的信号响应，从而实现了对人全血样本中18种磷脂酰乙醇的有效分析测定。使用本方法测定了359名有规律饮酒习惯的志愿者全血样本，样本中的总磷脂酰乙醇含量范围为51.13 ng/mL~2.89 μg/mL，平均为363.16 ng/mL。磷脂酰乙醇16∶0/18∶1、16∶0/18∶2是两个丰度最大的同系物，平均含量分别为74.21和48.75 ng/mL，各约占总量的20.43%和13.42%。Spearman相关性分析结果显示，磷脂酰乙醇之间相关性良好，并与临床现有酒精生物学标志物γ-谷氨酰转肽酶呈正相关；同时磷脂酰乙醇与肝肾功能相关临床生化指标显著相关。所建方法可以准确、精密检测人血磷脂酰乙醇含量，能够客观、可靠、量化反映人体酒精摄入状况，并可为临床酒精摄入监测提供有潜在应用价值的分析手段。

Determination of phosphatidylethanol in whole-blood by liquid chromatography-tandem mass spectrometry based on intelligent scheduled time-zone acquisition technology and the application to population level survey

Alcohol intake is an important risk factor for cardiovascular disease， liver disease， and diabetes. The accurate and objective evaluation of alcohol intake is important for disease prevention and intervention， as well as alcohol intake monitoring. Phosphatidylethanol （PEth） is a potential clinical biomarker of alcohol consumption. Monitoring PEth levels can provide an objective and quantitative basis for alcohol intake studies. Unlike other current alcohol biomarkers， PEth can only be produced in the presence of alcohol. Therefore， PEth is highly specific for alcohol intake and not affected by confounding factors， such as age， gender， hypertension， kidney disease， liver disease， and other comorbidities. Because of its long half-life and high specificity for alcohol intake， PEth may be used as a tool for monitoring drinking behavior in the clinical， transportation， and other fields. Given rapid developments in mass spectrometry technology over the past decade， liquid chromatography-tandem mass spectrometry （LC-MS/MS） has become the preferred method for PEth detection. However， most current LC-MS/MS methods focus on the determination of one or several PEth homologs， and the number of PEth homologs that can be determined simultaneously is relatively limited. Moreover， the detection capacity of the available methods remains insufficient， and their analytical sensitivity for some PEth homologs must be further improved. In this study， a novel LC-MS/MS method based on an intelligent scheduled time-zone negative multiple reaction monitoring acquisition technology （Scheduled-MRM） was developed. The technology monitors two ion channels in each PEth to ensure reliable results and can quantify 18 PEth homologs in human whole blood simultaneously. Methanol-methyl tert-butyl ether-water was used as the extraction system. An XBridge C18 column （100 mm×2.1 mm， 3.5 μm） was selected for gradient elution with 2.5 mmol/L ammonium acetate isopropanol solution and 2.5 mmol/L ammonium acetate aqueous solution-acetonitrile （50∶50， v/v） as the mobile phases. Negative electronic spray ionization in scheduled-MRM mode was applied for MS/MS detection. The method was validated to have a linear range of 10-2500 ng/mL with correlation coefficients greater than 0.9999. The limits of detection and quantification were 0.7-2.8 and 2.2-9.4 ng/mL， respectively， and the spiked recoveries ranged from 91.0% to 102.2%. The method was confirmed to be simple， rapid， and precise， and subsequently validated for the measurement of 18 PEth homologs in human blood. Scheduled-MRM can assign a suitable scan time to each ion channel and effectively reduce the number of concurrent ion pairs monitored per unit time. This technology overcomes the problem of insufficient dwell time caused by an excessive number of ion channels， thereby avoiding the redundant monitoring of non-retention times. Scheduled-MRM significantly improved the detection sensitivity， data acquisition quality， and signal response of the proposed method. Whole blood samples from 359 volunteers with regular drinking habits were analyzed using this method. The total PEth concentrations ranged from 51.13 ng/mL to 2.89 μg/mL， with a mean of 363.16 ng/mL. PEth 16∶0/18∶1 and 16∶0/18∶2 were the two most abundant homologs， with mean concentrations of 74.21 and 48.75 ng/mL， accounting for approximately 20.43% and 13.42%， respectively， of the total PEth. Spearman correlation analyses showed that the PEth homologs correlated well with each other， γ-glutamyltransferase， a clinically available biological marker of alcohol， and other clinical biochemical parameters related to liver and kidney function. Overall， the method was demonstrated to be sensitive， precise， and accurate； thus， it may be an effective tool for monitoring alcohol intake in the clinical and other fields.