Andreassen Trine Naalsund, Havnen Hilde, Spigset Olav, Falch Berit Margrethe Hasle, Skråstad Ragnhild Bergene
Department of Clinical Pharmacology, St. Olav University Hospital, 7006 Trondheim, Norway.
Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology, Trondheim, Norway.
J Anal Toxicol. 2018 Jan 1;42(1):33-41. doi: 10.1093/jat/bkx075.
Phosphatidylethanol (PEth) is an alcohol biomarker formed in the presence of ethanol in the body. Both due to its specificity and because it has a detection window of up to several weeks after alcohol intake, its application potential is broader than for other ethanol biomarkers. The aim of this study was to develop and validate a robust method for PEth in whole blood with fast and efficient sample extraction and a short analytical runtime, suitable for high throughput routine purposes. A validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC®-MSMS) method for quantification of PEth 16:0/18:1 in the range 0.05-4.00 μM (R2 ≥ 0.999) is presented. PEth 16:0/18:1 and the internal standard (IS) PEth-d5 (0.55 μM), were extracted from whole blood (150 μL) by simple protein precipitation with 2-propanol (450 μL). Chromatography was achieved using a BEH-phenyl (2.1 × 30 mm, 1.7 μm) column and a gradient elution combining ammonium formate (5 mM, pH 10.1) and acetonitrile at a flow rate of 0.5 mL/min. Runtime was 2.3 min. The mass spectrometer was monitored in negative mode with multiple reaction monitoring (MRM). The m/z 701.7 > 255.2 and 701.7 > 281.3 transitions were monitored for PEth 16:0/18:1 and the m/z 706.7 > 255.3 for PEth-d5. Limit of quantification was 0.03 μM (coefficient of variation, CV = 6.7%, accuracy = 99.3%). Within-assay and between-assay imprecision were 0.4-3.3% (CV ≤ 7.1%). Recoveries were 95-102% (CV ≤ 4.9%). Matrix effects after IS correction ranged from 107% to 112%. PEth 16:0/18:1 in patient samples were stable for several days at 30°C. Repeated freezing (-80°C) and thawing did not affect the concentration. After thawing and analysis patient samples were stable at 4-8°C for at least 4 weeks. Results from a proficiency test program, showing |Z| values ≤1.2, confirm the validity of the method. Analysis of the first 3,169 samples sent to our laboratory for routine use has demonstrated its properties as a robust method suitable for high throughput purposes.
磷脂酰乙醇（PEth）是一种在体内有乙醇存在时形成的酒精生物标志物。因其特异性以及在酒精摄入后长达数周的检测窗口期，其应用潜力比其他乙醇生物标志物更广泛。本研究的目的是开发并验证一种用于全血中PEth的稳健方法，该方法具有快速高效的样品提取和较短的分析运行时间，适用于高通量常规检测。本文介绍了一种经过验证的超高效液相色谱串联质谱（UPLC®-MSMS）方法，用于定量0.05 - 4.00 μM范围内的PEth 16:0/18:1（R2≥0.999）。通过用2 - 丙醇（450 μL）进行简单的蛋白沉淀从全血（150 μL）中提取PEth 16:0/18:1和内标（IS）PEth - d5（0.55 μM）。使用BEH - 苯基（2.1×30 mm，1.7 μm）色谱柱和梯度洗脱，流动相为甲酸铵（5 mM，pH 10.1）和乙腈，流速为0.5 mL/min。运行时间为2.3分钟。质谱仪采用负离子模式下的多反应监测（MRM）。监测PEth 16:0/18:1的m/z 701.7 > 255.2和701.7 > 281.3跃迁以及PEth - d5的m/z 706.7 > 255.3跃迁。定量限为0.03 μM（变异系数，CV = 6.7%，准确度 = 99.3%）。批内和批间不精密度为0.4 - 3.3%（CV≤7.1%）。回收率为95 - 102%（CV≤4.9%）。内标校正后的基质效应范围为107%至112%。患者样品中的PEth 16:0/18:1在30°C下可稳定保存数天。反复冷冻（-80°C）和解冻不影响其浓度。解冻并分析后，患者样品在4 - 8°C下至少稳定4周。能力验证计划的结果显示|Z|值≤1.2，证实了该方法的有效性。对发送到我们实验室进行常规检测的前3169个样品的分析证明了其作为适用于高通量检测的稳健方法的特性。
