Munchelou M Gomonit, Markus Roman, Britni N Skillman, Michael T Truver, Robert Kronstrand
{"title":"Quantification of Phosphatidylethanol 16:0/18:1 in blood using Supercritical Fluid Chromatography-Tandem Mass Spectrometry.","authors":"Munchelou M Gomonit, Markus Roman, Britni N Skillman, Michael T Truver, Robert Kronstrand","doi":"10.1093/jat/bkaf007","DOIUrl":null,"url":null,"abstract":"<p><p>Phosphatidylethanol (PEth) consists of phospholipids synthesized in erythrocyte cell membranes in the presence of ethanol and serves as a sensitive and specific indicator of alcohol consumption. Further research on PEth formation, degradation, and stability in postmortem (PM) samples would support its routine application in forensic toxicology. A supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method was developed and validated to quantify PEth 16:0/18:1 in blood. PEth 16:0/18:1 was extracted from blood (0.25 g) using an 8:2 (v/v) heptane:2-propanol solvent mixture. Method validation results met ANSI/ASB 036 guidelines. Recovery was >48%, and matrix effects were <20%. The linear range was 10-2500 ng/g, and LLOQ was 10 ng/g. Bias was ±17.7%, and precision was <17.1% for all QC levels. Carryover, endogenous and exogenous interferences were negligible. Extracts were stable beyond 72 hours. In a proof-of-concept study reanalyzing 35 PM case samples, PEth concentrations ranged between 32.6-2476 ng/g. Short-term stability studies showed that fortified bovine blood (200 ng/g) preserved with 0.4% sodium fluoride (NaF) stored at room temperature had a 6.6% concentration drop after 48 hours, while blood stored at 4°C decreased by 13.5% over 14 days. Additionally, human PEth-positive blood preserved with 0.4% NaF showed a 6.7% decrease in in vivo PEth concentrations compared to a 17.5% decrease in heparin-preserved blood after 14 days at 4°C, supporting the use of 0.4% NaF in reducing PEth degradation over time. An in vitro model was also developed to simulate early PM PEth changes. Results found that PEth formation occurred in an ethanol concentration-dependent manner with minimal degradation, and considerations should be taken when interpreting PEth concentrations in cases with long PM interval, and if the decedent had a high BAC level and was left at elevated temperatures. This is the first SFC-MS/MS method successfully developed and validated for analysis of PEth in PM samples.</p>","PeriodicalId":14905,"journal":{"name":"Journal of analytical toxicology","volume":" ","pages":""},"PeriodicalIF":2.3000,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of analytical toxicology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1093/jat/bkaf007","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
引用次数: 0
Abstract
Phosphatidylethanol (PEth) consists of phospholipids synthesized in erythrocyte cell membranes in the presence of ethanol and serves as a sensitive and specific indicator of alcohol consumption. Further research on PEth formation, degradation, and stability in postmortem (PM) samples would support its routine application in forensic toxicology. A supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method was developed and validated to quantify PEth 16:0/18:1 in blood. PEth 16:0/18:1 was extracted from blood (0.25 g) using an 8:2 (v/v) heptane:2-propanol solvent mixture. Method validation results met ANSI/ASB 036 guidelines. Recovery was >48%, and matrix effects were <20%. The linear range was 10-2500 ng/g, and LLOQ was 10 ng/g. Bias was ±17.7%, and precision was <17.1% for all QC levels. Carryover, endogenous and exogenous interferences were negligible. Extracts were stable beyond 72 hours. In a proof-of-concept study reanalyzing 35 PM case samples, PEth concentrations ranged between 32.6-2476 ng/g. Short-term stability studies showed that fortified bovine blood (200 ng/g) preserved with 0.4% sodium fluoride (NaF) stored at room temperature had a 6.6% concentration drop after 48 hours, while blood stored at 4°C decreased by 13.5% over 14 days. Additionally, human PEth-positive blood preserved with 0.4% NaF showed a 6.7% decrease in in vivo PEth concentrations compared to a 17.5% decrease in heparin-preserved blood after 14 days at 4°C, supporting the use of 0.4% NaF in reducing PEth degradation over time. An in vitro model was also developed to simulate early PM PEth changes. Results found that PEth formation occurred in an ethanol concentration-dependent manner with minimal degradation, and considerations should be taken when interpreting PEth concentrations in cases with long PM interval, and if the decedent had a high BAC level and was left at elevated temperatures. This is the first SFC-MS/MS method successfully developed and validated for analysis of PEth in PM samples.
期刊介绍:
The Journal of Analytical Toxicology (JAT) is an international toxicology journal devoted to the timely dissemination of scientific communications concerning potentially toxic substances and drug identification, isolation, and quantitation.
Since its inception in 1977, the Journal of Analytical Toxicology has striven to present state-of-the-art techniques used in toxicology labs. The peer-review process provided by the distinguished members of the Editorial Advisory Board ensures the high-quality and integrity of articles published in the Journal of Analytical Toxicology. Timely presentation of the latest toxicology developments is ensured through Technical Notes, Case Reports, and Letters to the Editor.