Department of Legal Medicine, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
Department of Medicine (Med Klinik I), University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
Drug Test Anal. 2021 Jan;13(1):140-147. doi: 10.1002/dta.2910. Epub 2020 Sep 1.
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.
磷脂酰乙醇（PEth）是一种直接的酒精消耗生物标志物，由不同乙醇修饰的同系物磷脂的一部分组成。本研究的目的是验证一种超高效液相色谱-串联质谱法来定量分析六种不同同系物的 PEth（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）的含量，这些物质来自于干血斑（DBS）。DBS 通过体积法（20 μL 全血）制备，并使用 1 mL 甲醇（0.02ng/μL 内标）提取。PEth 同系物在 BEH C18 柱（2.1×150mm，1.7μm）上分离，采用甲醇和 25mM 乙酸铵缓冲液在 7 分钟等度运行。采用多反应监测模式对 PEth 和内标物进行检测。校准器（10-1000ng/mL）和质控品（40、400 和 700ng/mL）均由加标全血制备；外部对照样品取自能力验证计划。在对该方法进行全面验证后，研究了在移植环境中患者的阳性 PEth 样本（n=57）中不同同系物的定量模式。使用液相色谱-串联质谱（LC/MS/MS）程序可以在 DBS 中实现 PEth 同系物的良好色谱分离、灵敏检测和可靠定量。验证结果，包括准确性、线性、回收率、基质效应和过程稳定性，均符合国际标准，且该程序的分析性能不受血液样本的血细胞比容影响。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种 PEth 同系物的动力学，并研究同系物分布在个体中的意义。
