Phosphatidylethanol (PEth) is a direct biomarker for alcohol consumption consisting of a fraction of different ethanol‐modified, homologue phospholipids. The aim of this study was to validate an ultra‐high‐performance liquid chromatography–tandem mass spectrometry method to quantitate six different homologues of PEth (16:0/18:1, 16:0/18:2, 16:0/20:4, 18:0/18:1, 18:0/18:2, and 18:1/18:1) from dried blood spots (DBSs). DBSs were prepared volumetrically (20 μL of whole blood) and extracted with 1 mL of methanol (0.02 ng/μL internal standards). PEth homologues were separated on a BEH C18 column (2.1 × 150 mm, 1.7 μm) using methanol and ammonium acetate buffer (25 mM) in a 7 min isocratic run. Multiple reaction monitoring mode was used for the detection of PEth and the internal standards. Calibrators (10–1000 ng/mL) and quality controls (40, 400, and 700 ng/mL) were prepared from spiked whole blood; external control samples were obtained from proficiency testing schemes. After a comprehensive validation of the method, quantitative patterns of the different homologues were investigated in PEth positive samples (n = 57) from patients in a transplant setting. Satisfactory chromatographic separation, sensitive detection, and reliable quantification of the PEth homologues in DBSs can be achieved using the liquid chromatography–tandem mass spectrometry (LC/MS/MS) procedure. Validation results, including accuracy, linearity, recovery, matrix effects, and in‐process stability, complied with international standards, and the analytical performance of the procedure was not affected by the hematocrit of the blood samples. Different quantitative patterns of the investigated PEth homologues were observed in authentic samples from liver transplant patients. This method will enable the study of the kinetics of six PEth homologues simultaneously and investigate the meaning of the homologues' distribution in individuals.

中文翻译：

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使用液相色谱-串联质谱法分析来自干血斑的六种不同的磷脂酰乙醇同系物。

磷脂酰乙醇 (PEth) 是酒精消耗的直接生物标志物，由一小部分不同的乙醇修饰的同源磷脂组成。本研究的目的是验证超高效液相色谱 - 串联质谱法定量 PEth 的六种不同同系物 (16:0/18:1, 16:0/18:2, 16:0/20 :4、18:0/18:1、18:0/18:2 和 18:1/18:1) 来自干血斑 (DBS)。DBS 是按体积（20 μL 全血）制备的，并用 1 mL 甲醇（0.02 ng/μL 内标）萃取。在 7 分钟等度运行中，使用甲醇和醋酸铵缓冲液 (25 mM) 在 BEH C18 柱（2.1 × 150 mm，1.7 μm）上分离 PEth 同系物。PEth和内标的检测采用多反应监测模式。校准品 (10–1000 ng/mL) 和质量控制 (40, 400, 和 700 ng/mL) 由加标全血制备；外部对照样本来自能力验证计划。在对该方法进行全面验证后，在移植环境中的患者的 PEth 阳性样本 (n = 57) 中研究了不同同源物的定量模式。使用液相色谱-串联质谱 (LC/MS/MS) 程序可以实现 DBS 中 PEth 同系物的令人满意的色谱分离、灵敏的检测和可靠的定量。验证结果，包括准确度、线性、回收率、基质效应和过程稳定性，符合国际标准，并且该程序的分析性能不受血样血细胞比容的影响。在来自肝移植患者的真实样本中观察到所研究的 PEth 同系物的不同定量模式。该方法将能够同时研究六个 PEth 同系物的动力学，并研究同系物在个体中分布的意义。