Quantification of phosphatidylethanol 16:0/18:1 in blood using supercritical fluid chromatography–tandem mass spectrometry

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 mixture. Method validation results met American National Standards Institute/Academy Standards Board 036 guidelines. Recovery was >48%, and matrix effects were <20%. The linear range was 10-2500 ng/g, and lower limit of quantification was 10 ng/g. Bias was ±17.7%, and precision was <17.1% for all quality control 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 to 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 blood alcohol concentration level and was left at elevated temperatures. This is the first SFC-MS-MS method successfully developed and validated for the analysis of PEth in PM samples.