High Throughput UPLC®-MSMS Method for the Analysis of Phosphatidylethanol (PEth) 16:0/18:1, a Specific Biomarker for Alcohol Consumption, in Whole Blood.

ABSTRACT

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 160/181 in the range 0.05-4.00 µM (R2 ≥ 0.999) is presented. PEth 160/181 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 160/181 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 160/181 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.