Quantitative Isomer Profiling of Synthetic Phosphatidylethanol by NMR and Advanced MS Techniques

Phosphatidylethanols (PEths) are direct alcohol biomarkers of clinical and forensic importance. Their accurate quantification depends critically on the regioisomeric purity of reference materials, as deviations from the natural isomer distribution can bias measurement outcomes. In this study, we systematically evaluated the relative isomer abundance of synthetic PEth 16:0/18:1 standards using nuclear magnetic resonance spectroscopy (NMR), ozone-induced dissociation (CID/OzID), collision-induced dissociation (CID), and electron-activated dissociation (EAD). Three commercial preparations were investigated: a mixed isomer standard and two "IsoPure" materials claimed to contain exclusively the PEth 16:0/18:1 or 18:1/16:0 regioisomer, respectively. Quantitative 13C NMR enabled unambiguous discrimination of the carbonyl resonances, revealing a mixture of ∼83/17% in the non-IsoPure material and <1% cross-contamination in the IsoPure standards. CID/OzID provided complementary structural information, with isomeric compositions consistent with NMR results. CID and EAD (sodiated, positive ionization) yielded characteristic product ion ratios and homologue-specific product ions, respectively, that distinguished isomers, although precise quantification remained limited. In contrast, EAD spectra in negative-ion mode were dominated by nondiagnostic product ions and did not allow isomer differentiation. Together, our results demonstrate that 13C NMR and CID/OzID are robust, quantitative approaches for assessing regioisomeric purity in synthetic PEth standards, while CID and EAD can support qualitative profiling. This multiplatform evaluation provides a framework for quality control of PEth reference materials and underlines the necessity of verifying regioisomeric composition to ensure reliable biomarker quantification.