MSACL 2025 Abstract

Automated Liquid Chromatography Tandem Mass Spectrometry Analysis of Phosphatidylethanol (PEth)

Spencer Seely (1), Dawn Francisco (1), Robert Fitzgerald (1), Raymond T. Suhandynata (1,2)
(1) Department of Pathology, UC San Diego Health, San Diego, CA, USA, (2) Skaggs School of Pharmacy and Pharmaceutical Sciences, San Diego, CA, USA

Spencer Seely, PhD (Presenter) University of California, San Diego

Presenter Bio: I am a Clinical Chemistry fellow at the University of California, San Diego (2024-2026). My interests are the development of methods in clinical laboratories and quality improvement within the laboratory.
- Phd in Biochemistry and Molecular Biology (University of Miami, 2024)
- BS in Biology (Utah State University, 2015)
- Medical Laboratory Scientist since 2015 [MB(ASCP)cm]

Relevant Financial Disclosures (within past 24 months, reported on Sep 11, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Alcohol Abuse Disorder (AUD) or excessive ethanol consumption is detrimental to human health. Questionnaires regarding consumption can be inaccurate for the identification of at-risk patients. Indirect biomarkers, such as ethyl glucuronide (EtG) and ethyl sulfate (EtS), have short half-lives, limiting their effectiveness in the determination of chronic abuse. Phosphatidylethanols (PEths), phospholipids conjugated to ethanol, are biomarkers of ethanol use and are detectable 2-4 weeks post-consumption. Most recent published procedures for PEth analysis require time-consuming and complex extraction protocols. We describe an LC-MS/MS laboratory developed test (LDT) which employs a simple extraction process that can be performed manually or with an automated liquid handler to reduce overall turnaround time.

METHODS:
The measurement of two PEth homologues: 16:0/18:1 (POPEth) and 16:0/18:2 (PLPEth) was performed by isotope dilution LC-MS/MS in clinical whole blood specimens. Samples were extracted using an automated liquid handler using 0.1 M zinc sulfate solution and 50:50 acetonitrile: isopropanol. Extracts were vortexed for 5 minutes, followed by centrifugation at 2000 RCF for 5 minutes. Supernatants were collected and directly injected for analysis by LC-MS/MS. A retrospective analysis of 1847 patient results was performed to identify trends in the two measured homologues.

RESSULTS:
The analytical measurement range (AMR) for both POPEth and PLPEth was determined as 10 - 1,500 ng/mL, with strong linearity observed across the measure range (R2 >0.995). Calibrator and quality control biases were found to be within 15% of correlated values from an external reference laboratory. Biases were observed for the measurement of POPEth in a subset of externally correlated patient specimens. Isotope dilution studies were performed to assign the appropriate quantifier ion transition to resolve an unknown interferant. Retrospective patient data was used to evaluate PLPEth concentrations relative to established POPEth reference ranges; revealing similar ranges. The PLPEth:POPEth ratio was evaluated and found to be statistically higher in patients with light consumption (10-20 ng/mL POPEth) compared to moderate (20-200 ng/mL POPEth, p-value <0.0001), and heavy (>200 ng/mL POPEth, p-value= 0.0014) consumption; revealing additional utility for the measurement of PLPEth.

CONCLUSION:
This development of and LC-MS/MS LDT enables the accurate measure of PEth and compares well to external reference laboratory results. Reduction in turnaround time provides more actionable information for clinicians treating alcohol abuse and preventing organ damage during transplantation. More investigation is needed to establish reference ranges for PLPEth, which can provide additional insights to monitoring alcohol consumption and abuse.