Rapid Selective Detection of Phosphatidylethanols by Neutral Loss Scans in Tandem Mass Spectrometry to Evaluate Alcohol Consumption

Phosphatidylethanol (PEth) is a highly specific alcohol biomarker with increasing clinical and forensic utility. However, point-of-care (POC) detection in whole blood remains challenging due to complex LC-MS/MS systems, long run times, and poor selectivity in the negative ion mode. This study presents an alternative strategy for PEth detection in the positive-ion mode, enabled by cationization; neutral loss (NL)-based fragmentation and MS³ analyses were employed to achieve enhanced specificity. Aziridination by reactive ionization and adduct formation using Li⁺, Na⁺, and NH₄⁺ was systematically investigated to enhance ionization, with sodium adducts found to be the most effective. Notably, Na⁺ adduction at the fatty acyl moieties leads to a characteristic NL of 126 Da, corresponding to the ethyl phosphate headgroup. This unique fragmentation signature is absent in other phospholipids and enables the selective detection of PEths. To exploit this specificity, NL scanning was applied using a triple quadrupole, and product ion MS/MS and MS³ analyses were performed using a linear ion trap; both platforms enabled selective detection even in the presence of isomeric and structurally related lipids. For quantification, calibration curves for PEth 16:0-18:1 were established on both instruments, employing NL and product ion scans, and they demonstrated good linearity (<i>R</i>² = 0.98 and 0.97, respectively). Application to a DBS sample from a heavy alcohol user yielded consistent results, 1.23 ± 0.09 (linear ion trap) and 1.43 ± 0.12 ppm (triple quadrupole), validated by LC-MS. These results highlight the potential of NL scans with MS³ as a rapid, selective, onsite approach to PEth detection.