New Phosphatidylethanol (PEth) reference materials by chemical synthetic pathway

Abstract

Phosphatidylethanol (PEth) is a widely used alcohol biomarker comprising a group of homologous phospholipids with a common polar phosphoethanol head onto which two fatty acid moieties are attached at position sn-1 and sn-2is. PEth are formed in the presence of ethanol by conversion of phosphatidylcholine (PC) to PEth via the catalytic action of the enzyme phospholipase D (PLD). Compared to traditional, indirect biomarkers, PEth is a direct biomarker, is more specific, has wider detection windows in toxicological samples and is not influenced by age, gender, other ingested substances or other pathological conditions. The accurate identification and quantification of PEth homologs in toxicology casework is analytically demanding, made more challenging by the limited number of reference standards available and their cost. Aims: to develop a novel chemical synthesis method to increase production volumes of PEth reference materials; to produce stable isotopically labelled internal standards; to improve the quality/purity of reference materials; and to address the possible impact of regio-isomers and other impurities in PEth reference materials on routine toxicological analysis. Chemical synthetic methods were developed for the synthesis of individual PEth homologs and stable isotope labelled PEth Internal standards. Synthetic methods with levulinyl protection in one of the crucial steps were chosen especially for PEth having one or more double bonds in the fatty acid at sn-2 position, and for PEth internal standards having deuterium or 13C-isotopic labelling in the glycerol moiety. Regio-chemical control has been addressed in the chemical synthesis to avoid or minimize the acyl migration of the two fatty acid moieties. Nuclear magnetic resonance (1H and 13C-NMR) and liquid chromatography mass spectrometry (LC-MS) analysis was used for product characterization and purity assessment. 13C-NMR was also used for identification of PEth regioisomers and the data have been externally validated using LC-MS/MS. PEth (16:0/18:1), penta-deuterium labelled PEth(16:0/18:1)-d5 (ethyl-d5) and PEth (16:0/18:1)-d5 (glycerol-d5) and 13C-labelled PEth (16:0/18:1)-13C3 (glycerol-13C3) as the free acid or as ammonium salt have been synthesized by new chemical synthetic pathways. The PEth reference materials were obtained in high chemical purity and regio-isomeric purity. Reversed position isomer PEth (18:1/16:0) was also synthesized for comparation and identification of regio-isomers and regio-isomeric purity. Analytical differentiation of the regio-isomers by 600 MHz and 800 MHz NMR was performed and different 13C-NMR signals of the carbonyl groups were observed and identified. Compared to the enzymatic route, chemical synthesis of PEth may provide higher quality, and more reproducible reference standards. The regio-isomers of PEth have an identical molecular weight and similar chromatographic and spectral properties making them difficult to differentiate. They can be detected only using NMR and ion ratio evaluation in LC-MS/MS analysis. Chemically and isomerically pure reference standards have been produced using our new synthesis methods. Additionally, PEth-d5 (glycerol-d5) and 13C-labelled PEth-13C3 (glycerol-13C3) both have the isotopic labelling inside the glycerol moiety, different from existing PEth-d5 (ethyl-d5) reference standards. The isotopic labelling in the glycidyl backbone of the molecule is more stable than in the ethyl side chain, whilst by-products were observed by loss of the entire phosphate head group (including the ethyl-d5) from PEth-d5. 13C-labelled internal standards are superior than the deuterium labelled ones due to better ion-suspension effect in MS analysis. Chemical synthetic pathways have been developed for the synthesis of PEth homologues. With the synthetic methods developed it is possible to prepare highly pure PEth homologues without unwanted regio-isomers and without other chemical impurities being present. Similarly, PEth with deuterium and 13C-labelled glycerol moieties have also been prepared. These labelled PEth standards can be used as the new isotopically internal standards for quantitative analysis of PEth.