MSACL 2025 Abstract

Reducing False-Positive Phosphatidylethanol (PEth) Quantification in Alcohol Testing via Isobaric Lipidome Analysis and Interference Correction

Ching-Hua Lee (1,2), Pei-Jer Chen (3), Ching-Hua Kuo (1,2,4)
(1) School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan (2) The Metabolomics Core Laboratory, Centers of Genomic and Precision Medicine, National Taiwan University, Taipei, Taiwan (3) Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan (4) Department of Pharmacy, National Taiwan University Hospital, Taipei, Taiwan

Ching-Hua Lee, Ph.D (Presenter) National Taiwan University >> POSTER (PDF)

Presenter Bio: I am a Postdoctoral Fellow at the Metabolomics Core Laboratory of National Taiwan University, specializing in the application of mass spectrometry to biochemical and clinical analysis. My research has a particular focus on lipidomics, especially the development of novel analytical methods for the ether-lipidome.

After graduating from National Taiwan University with a B.S. in Pharmacy and obtaining my pharmacist license in 2016, I completed my Ph.D. in 2023. I now continue my research as a postdoctoral fellow, dedicated to enhancing the accuracy of lipidomics analysis and its translational applications in clinical settings.

My work includes first-author publications on lipidomics method development in leading journals such as Analytical Chemistry (2023) and Analytica Chimica Acta (2021). I have also been recognized with several prestigious awards, including the Oral Presentation Award at the 2023 Taiwan Society for Mass Spectrometry (TSMS) Annual Conference, which led to a travel grant for an oral presentation at the 2024 Mass Spectrometry Society of Japan (MSSJ) conference. Additionally, I received the Outstanding Graduate Thesis Award from the NTU School of Pharmacy in 2023 and the Excellent Publication Award (Honorable Mention) from the NTU College of Medicine in both 2022 and 2023.

Relevant Financial Disclosures (within past 24 months, reported on Jul 17, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Phosphatidylethanol (PEth) is a widely used biomarker for alcohol consumption in clinical and forensic settings. A consensus establishes that a blood PEth(16:0/18:1) concentration >20 ng/mL strongly indicates alcohol use. However, studies show that single drinking events with blood alcohol concentrations (BAC) of 0.3–0.5 g/kg or daily consumption of ~16 g ethanol for three months typically do not result in median PEth levels exceeding 20 ng/mL in collected post-drinking. For stricter monitoring of alcohol consumption in patients with alcohol-related liver disease (ALD) or those awaiting liver transplantation – where stringent alcohol control is critical - lowering the cutoff for PEth using LC-MS/MS is necessary. However, reducing the cutoff often increases false-positive results, potentially causing patient conflicts or delaying surgeries. Given that PEth is biosynthesized only in the presence of ethanol, we hypothesize that co-eluting phospholipid isobars in commonly used LC-MS/MS configurations contribute to false positives and inaccurate quantification. In this study, we apply a lipidomics approach to identify interference sources and optimize PEth analysis strategy to lower the cutoff concentration without compromising specificity.

METHODS:
Fifteen participants were enrolled: six with no alcohol consumption history in recent months and nine who self-reported past drinking habits. Three pooled samples were analyzed: (1) pool blank (n=6), (2) pool drinker (n=9), and (3) pool-QC (1:1 mix of pools 1 and 2). Lipidomics analysis of whole blood was performed using an Agilent 6545XT QTOF with iterative data-dependent analysis (DDA). Agilent 6460 and 6495 QQQ instruments, operating in different scan modes, identified interference sources to complement QTOF data. Multiple reaction monitoring (MRM) was used to develop quantitative methods for PEth homologues and interfering lipids. Whole blood samples (50 μL) were extracted using a modified Folch method. Lipid separation was performed using an Agilent Eclipse Plus C18 RRHD and a Phenomenex Kinetex C8 column. Mobile phase and gradient settings were evaluated to investigate relative retention time (RT) shifts between PEth and lipid isobars. PEth identification criteria included: (1) mass accuracy <20 ppm by HRMS and (2) retention time alignment with PEth(16:0/18:1) synthetic standard and equivalent carbon number (ECN) estimation.

RESULTS:
We identified 11 PEth homologues and their interfering lipid isobars, which follow specific co-elution patterns: PEth(X:Y) co-elutes with PS(X+2:Y) and PA(X+2:Y+1), where X and Y represent the total carbon number and double bonds on the acyl chain, respectively. For instance, PEth(16:0/18:1) (m/z 701) is affected by in-source fragmentation (ISF) of PS(18:0_18:1) (m/z 788→701) and PA(36:2) isomers (m/z 699), including PA(18:1_18:1), PA(18:0_18:2), and PA(16:0_20:3). These interferences persist across various LC settings and are influenced by skimmer voltage and elution conditions.

To evaluate the impact of ISF from PS(18:0_18:1) on PEth(16:0/18:1) quantification, we used an the optimal skimmer voltage (170–190 V), determined with a synthetic standard. This resulted in ~3% ISF, leading to overestimation of MRM 701→281 to 40 ppb in pool blank samples under LC conditions with adequate separation (RT 5.3 min), demonstrating that ISF causes significant false-positive quantification.

Further evaluate the interferences other than ISF, we used an Agilent 6495 QQQ with an ion funnel to minimum ISF (< 0.5%) from PS(18:0_18:1). Pool blank spiked with 20 ppb PEth(16:0/18:1) yielded quantification result of 22 ppb (MRM 701→255) and 25 ppb (MRM 701→281) with adequate separation. Based on co-elution patterns and quantification results, we selected MRM 701→255 as the final quantifier to minimize interferences. However, high-throughput LC settings (RT 1.8 min) increased the quantification result to 24.7 ppb for MRM 701→255 due to compressed lipid elution times, causing co-elution of FA(16:0) fragments from multiple sources.

Further investigating the interferences suggested that lipids generating MRM 700→255 contribute approximately 24% of the [M+1] isotope to MRM 701→255. By mathematically subtracting isotopic interference yielded a final concentration with > 90 % accuracy. The optimized PEth quantification strategy - MRM 701→255 transition with isotope correction - enabled cutoff level to be lowered to 5 ppb with high specificity.

CONCLUSION:
Using a QTOF iterative-DDA lipidomics approach combined with QQQ instruments and various scan modes, we identified interference sources for PEth(16:0/18:1) analysis and developed an analytical strategy to minimize interference and improve quantification accuracy. These adjustments enable a lower PEth cutoff without compromising specificity, enhancing the reliability of alcohol consumption monitoring for ALD and liver transplant patients.