Phospholipid metabolites of GHB as potential biomarkers in whole blood: Synthesis, analytics, and in vitro formation of homolog 16:0/18:1

Abstract

Gamma-hydroxybutyric acid (GHB) is a common drug of abuse, and the detection of a consumption or administration is a longstanding research objective in clinical and forensic toxicology. However, until now, the short detection window of GHB could not be enlarged by the use of GHB metabolites. Therefore, new biomarkers for the detection of a GHB intake are needed. In analogy to phosphatidylethanols as long-time biomarkers of ethanol, phospholipids with GHB might represent a promising compound class. While the availability of reference compounds often represents a bottleneck in clinical and forensic toxicological research, two phospholipids—phosphatidyl-GHB (16:0/18:1) and its isomer phosphatidyl beta-hydroxybutyric acid (16:0/18:1)—were successfully synthesized by a new highly versatile synthetic route. Structural characterization data, together with 1H-, 13C-, and 31P-NMR and high-resolution mass spectrometry (HRMS) spectra, are reported. Subsequently, a HPLC–MS/MS method was established for the determination of both compounds (limits of detection [LOD] ≤ 2 ng/ml), and the formation of these metabolites was investigated in two in vitro experiments. The formation of phosphatidyl-GHB (16:0/18:1) was observed in an incubation experiment by converting phosphatidylcholine (16:0/18:1) and GHB with phospholipase D and in whole blood samples spiked with 50 mM GHB, respectively. Therefore, phosphatidyl-GHB (16:0/18:1) might represent a valuable new metabolite of GHB with the potential for an extension of the detection window as GHB biomarker.