An improved and economical LLE-LC/MS method for quantifying phosphatidylethanol: Identification and separation of isotopic cross-talk in clinical alcohol testing

  • Clin Biochem. 2025 Oct:139:110994. doi: 10.1016/j.clinbiochem.2025.110994.
Naga Veera Yerra  1 ,  Christopher Pini  2 ,  Carlos Cordon-Cardo  3 ,  Damodara Rao Mendu  4
Affiliations
  • 1. Clinical Chemistry Service, Department of Pathology, Molecular and Cell-Based Medicine, The Mount Sinai Hospital, New York 10029 NY, USA. Electronic address: [email protected].
  • 2. Clinical Chemistry Service, Department of Pathology, Molecular and Cell-Based Medicine, The Mount Sinai Hospital, New York 10029 NY, USA. Electronic address: [email protected].
  • 3. Clinical Chemistry Service, Department of Pathology, Molecular and Cell-Based Medicine, The Mount Sinai Hospital, New York 10029 NY, USA. Electronic address: [email protected].
  • 4. Clinical Chemistry Service, Department of Pathology, Molecular and Cell-Based Medicine, The Mount Sinai Hospital, New York 10029 NY, USA. Electronic address: [email protected].
PMID: 40850364 DOI: 10.1016/j.clinbiochem.2025.110994
Abstract

Objective: Phosphatidylethanol (PEth) is a group of Phospholipids formed in the presence of ethanol on the red blood cell membrane. Due to their stability in blood for 3-4 weeks, they have become reliable direct biomarkers for long-term monitoring of alcohol use. This study aimed to develop and validate a robust, high-throughput liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for the routine clinical quantification of the two most common PEth homologues, PEth 16:0/18:1 (POPEth) and PEth 16:0/18:2 (PLPEth), while addressing common analytical challenges.

Methods: An established quantification method employing liquid-liquid extraction was used with optimized LC-MS/MS parameters. The method was validated for correlation studies, precision, analytical measurement range, analytical sensitivity, analytical specificity, carryover, dilution linearity, stability, matrix effect and extraction recovery, with specific attention to eliminating isotopic cross-talk and chromatographic interferences. A method comparison was performed using specimens analyzed by an external reference laboratory.

Results: The method demonstrated excellent linearity for both POPEth and PLPEth across the analytical measurement range (10-2000 ng/mL), with correlation coefficients (r2) of 0.99. Intra- and inter-assay precision values were within ± 10 % coefficient of variation. Recovery yields ranged from 78-85 %. The optimized method resolved isotopic cross-talk and exhibited no carryover. Comparison with an external laboratory showed strong correlation for both homologues (slopes of 0.979 and 1.049; r2 = 0.99).

Conclusion: We developed and validated a sensitive and specific LC-MS/MS method for the quantification of POPEth and PLPEth. The assay provides improved recovery, eliminates isotopic cross-talk, shows no carryover, and is suitable for high-throughput clinical laboratories. This method enables reliable and cost-effective monitoring of alcohol use in routine clinical practice.

Keywords
Alcohol biomarker; Analytical method validation; Isotopic cross-talk; PEth 16:0/18:1; PEth 16:0/18:2.
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