Beyond the baseline: quantification of two phosphatidylethanol homologues in whole blood by LC–MS-MS and retrospective data analysis from a National Reference Laboratory

Alcohol is the most abused substance in Western society, resulting in major economic losses and negative health consequences. Therefore, there is a need for a selective and robust detection method for alcohol consumption in various clinical and forensic settings. This study aimed to validate a mass spectrometry method for quantifying phosphatidylethanol (PEth) and perform retrospective data analysis from the patient population of a national reference laboratory. Quantification of PEth in whole blood was accomplished using an LC-MS-MS assay. Isotopically labeled internal standard for the two PEth homologues was added to the whole-blood specimen, followed by protein precipitation with a mixture of acetonitrile and isopropyl alcohol. After centrifugation, an aliquot of the supernatant was buffered with ammonium acetate before LC-MS-MS analysis on an Agilent 6470 triple quadrupole mass spectrometer coupled to an Agilent 1260 Infinity II LC system. This LC-MS-MS assay was validated for clinical use in accordance with Clinical & Laboratory Standards Institute guidelines. The analytical measurement range, 10-2000 ng/mL, was linear with R2 of 0.999. The within-run and total imprecision was < 5% CV for the low (20 ng/mL), medium (200 ng/mL), and high QC (1000 ng/mL). Results from accuracy and method comparison experiments met the bias criteria of ±15%. Retrospective data analysis showed ∼27% of patients had PEth concentrations <20 ng/mL. Males and females had similar positivity rates for PEth and the positivity rate of women of reproductive age (15-44 years old) was 35% in comparison to 25% in women 45-89 years old. This study's LC-MS-MS method showed acceptable analytical performance in quantifying PEth as a sensitive and specific biomarker for evaluating alcohol consumption. Results from this study may provide an opportunity to educate women of reproductive age on drinking during pregnancy and the long-term effects of alcohol use.