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

Background: Phosphatidylethanols (PEths) are specific, direct alcohol biomarkers that can be determined in human blood to distinguish between heavy and social drinking. PEth 16:0/18:1 is among the most predominant PEth 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 PEth 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 PEth 16:0/18:1 by reversed phase UHPLC-MS/MS.

Results: The developed UHPLC-MS/MS method was fully validated in whole blood with PEth 16:0/18:1-D₅ as internal standard. Intermediate precision and intermediate accuracy were within ≤± 12% and ≤± 17%, respectively, at PEth 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 PEth 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 PEth 16:0/18:1 in whole blood.