Simultaneous determination of three phosphatidylethanol homologues, 12 drugs and metabolites in whole blood by LC–MS/MS

The use of alcohol, legal and illicit substances poses great negative consequences on health and economy worldwide. LC-MS/MS allow simultaneous determination of multiple compounds in biological matrices. The aim of this study was to develop a LC-MS/MS method for the determination of the alcohol biomarker phosphatidylethanol (PEth) – including three homologues (PEth 16:0/18:1, PEth 16:0/18:2, PEth 18:0/18:1) - cocaine and three metabolites, and 8 other drugs in whole blood. Whole blood in K2EDTA tubes was prepared by liquid-liquid extraction using heptane/ethyl acetate/2-propanol (16:64:20, v:v:v). Chromatographic separation was achieved on an Acquity BEH C 18 column (50 × 2.1 mm I.D., 1.7 µm particles). Mobile phase was 0.025% ammonia, pH 10.7 (Solvent A) and methanol (Solvent B). The method was fully validated with isotope-labelled internal standards for 10 compounds. Inter-assay precision and accuracy were within ± 16% for all analytes at five to seven tested concentrations. Recovery was within 42-79% for 14 compounds and 11% for benzoylecgonine. Matrix effects were within ± 25% for most analytes. Internal standards compensated for matrix effects for compounds that had their own internal standards. A robust, precise, and accurate LC-MS/MS method for the determinations of three PEth homologues and 12 drugs and metabolites was, developed and validated. The method is valuable, especially for detecting polydrug use and alcohol consumption. To the best of our knowledge, this is the first LC-MS/MS method for the simultaneous determination of three PEth homologues and different drugs and metabolites. • First method for simultaneous analysis of 3 PEths and 12 drugs and metabolites. • Three PEth homologues allow for better estimation of time of alcohol use. • LC-MS/MS method suited to detect alcohol consumption and polysubstance use. • Cocaethylene is included to monitor when cocaine and alcohol are taken together. • Extraction and LC-MS/MS method for analytes with different physicochemical properties.