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.

Since 2008, Chiron has been continuously running toxicology and new psychoactive substance (NPS) reference standard-focused synthesis research projects. This work has been carried out in collaboration with the German rapid test provider nal-von-minden and a variety of Nordic institutions: St. Olavs Hospital, The Norwegian Public Health Institute (now Oslo University Hospital), and, more recently, with Linköping University, Sweden. A considerable knowledge-base on systematic NPS drug trend analysis, synthesis of emerging and prophetic NPS reference standards and NPS metabolite reference standards has been accumulated through the EUROSTARS-funded projects QUANTUM SPICE (2013–2016), PSYCHOMICS (2016–2019), and the current NPS REFORM (2019–2022). The aim of this presentation is to provide an overview of what has been learnt and how the NPS market is expected to develop in future. The outcomes of the current and previous EUROSTARS projects will be presented and the future expected developments in the field will be discussed. The synthesis approach developed and applied in these projects is denoted “P4”, a Predictive Parallel Production Platform. It uses our knowledge-base to predict which new NPS compounds will arrive on the illicit or grey market and to produce as many analogues and isomers as possible of those chemicals by applying key intermediates and parallel synthesis and analytical instrumental characterisation. It also considers the most likely analogues to appear on the market based on precursor availability and price. An extensive portfolio of NPS reference materials (including NPS metabolites) has been made available to forensic chemists and clinical and forensic toxicologists across the scientific community. This community is operating within the context of a rapidly evolving, and often transient, illicit drugs market. To date, the EUROSTARs-funded projects have produced approximately 600 new native NPS compounds and NPS metabolites, in addition to approximately 50 labelled internal standards, characterised by GC-MS, LC-MS, NMR and other methodologies. The current project, NPS-REFORM, supplies emerging, often highly potent synthetic opioid reference materials (e.g., nitazenes and other emerging chemical classes); new, emerging and prophetic classes of synthetic cannabinoids appearing on the illicit market; as well as cathinones and other NPS compounds entering the marketplace. Not only do the EUROSTARs projects provide reference materials to the forensic and clinical scientific communities, they support a wide range of in vitro metabolism studies, which have identified and synthesised suitable metabolites and biomarkers to support the detection of NPS in biological fluids, an overview of which is provided. The NPS-focused EUROSTARs projects have greatly increased the breadth and availability of the NPS and NPS reference standards that are a fundamental cornerstone of drug detection in analytical laboratories. A fourth EUROSTARS project is in planning, again in collaboration with Linköping University and with the focus on various new synthetic variations of THC and other phytocannabinoids, and on the new synthetic cannabinoids emerging in response to recent legislative changes in the People's Republic of China and beyond.