Determination of phosphatidylethanol 16:0/18:1 in whole blood by 96‐well supported liquid extraction and UHPLC ‐ MS / MS

Background Phosphatidylethanols ( PE ths) are specific, direct alcohol biomarkers that can be determined in human blood to distinguish between heavy and social drinking. PE th 16:0/18:1 is among the most predominant PE th homologues in human blood. The aim of the study was to develop a high throughput and sensitive UHPLC ‐ MS / MS method for the determination of PE th 16:0/18:1 in whole blood. Methods Whole blood samples were prepared by 96‐well supported liquid extraction ( SLE ). Extracted samples were analyzed for PE th 16:0/18:1 by reversed phase UHPLC ‐ MS / MS . Results The developed UHPLC ‐ MS / MS method was fully validated in whole blood with PE th 16:0/18:1‐D 5 as internal standard. Intermediate precision and intermediate accuracy were within ≤± 12% and ≤± 17%, respectively, at PE th 16:0/18:1 concentrations of 1.4‐2112 ng/ mL (2.0‐3004 nmol/L). Limit of quantification ( LOQ ) was 1.7 ng/ mL (2.4 nmol/L). Conclusion For the first time, 96‐well SLE was used for preparation of a PE th homologue in biological samples. A mixture of tert‐butyl methyl ether and 2‐propanol (5:1, v:v) was chosen as organic eluent based on an evaluation of extraction recovery, purity of extracts, and evaporation time. The developed UHPLC ‐ MS / MS method can be used for high throughput analyses and sensitive determinations of PE th 16:0/18:1 in whole blood.