Easy and convenient millimole‐scale synthesis of new, potential biomarkers for gamma‐hydroxybutyric acid (GHB) intake: Feasible for analytical laboratories

Abstract

New biomarkers indicating the abuse of drugs and alcohol are still of major interest for clinical and forensic sciences. The endogenous neurotransmitter and approved drug, gamma-hydroxybutyric acid (GHB), is often illegally used for drug-facilitated crimes by spiking GHB into alcoholic beverages. Analytical detection windows of only 6 h in blood and 12 h in urine are often too short to provide reliable proof of GHB ingestion. Therefore, new biomarkers are needed to prove exogenous GHB administration. Previously, amino acid GHB conjugates were discovered in an untargeted metabolomics screening and fatty acid esters with GHB were recently discussed as promising biomarkers to enlarge the analytical detection time windows. However, the development of analytical methods is still slowed down since reference compounds for targeted screenings are still missing. In this paper, we describe simple procedures for the rapid synthesis and purification of amino acid GHB conjugates as well as fatty acid esters, which can be adopted in analytical and clinical/forensic laboratories. Structural characterization data, together with IR, 1 H-nuclear magnetic resonance (NMR), 13 C-NMR, high-resolution mass spectra (MS), and MS/MS spectra in positive and negative ionization mode are reported for all obtained GHB conjugates and GHB conjugate precursors.

New gamma‐hydroxybutyric acid (GHB) biomarkers: Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of GHB amino acid, carnitine, and fatty acid conjugates in urine

“…Kraemer et al described the lack of a second specific transition in ESI negative mode already for GHB C16 given the predominant release of the fatty acid 41 . Switching to positive ionization, which yielded at least two potential fragment ions for each FA conjugate, 35 would only theoretically circumvent this problem, as sensitivity in ESI positive mode was far below that of ESI negative 41 . Organic acids were previously successfully screened for by classic GC–MS analysis after TMS derivatization, monitoring two selected ions per compound 28,31 .…”

“…GHB carnitine was obtained from Toronto Research Chemicals (Ontario, Canada) after custom‐made synthesis. The GHB AA and FA conjugates GHB glycine, GHB glutamate, GHB phenylalanine, GHB caprylic acid (GHB C8), GHB capric acid (GHB C10), GHB lauric acid (GHB C12), GHB C14, GHB C16, and GHB C18 were synthesized at ETH Zurich as described in detail elsewhere 35 . GHB C18:1 and GHB taurine were later synthesized similarly to the described unsaturated FA conjugates.…”

“…The GHB AA and FA conjugates GHB glycine, GHB glutamate, GHB phenylalanine, GHB caprylic acid (GHB C8), GHB capric acid (GHB C10), GHB lauric acid (GHB C12), GHB C14, GHB C16, and GHB C18 were synthesized at ETH Zurich as described in detail elsewhere. 35 GHB C18:1 and GHB taurine were later synthesized similarly to the described unsaturated FA conjugates. GHB isomers 2-hydroxy butyric acid (alpha-hydroxy butyric acid, AHB) and 3-hydroxy butyric acid (beta-hydroxy butyric acid, BHB), 2,4-DHB and 3,4-DHB, GA, SA and succinylcarnitine were obtained from Sigma Aldrich (Buchs, Switzerland).…”

“…An important prerequisite for comprehensive evaluation of expected concentrations in different matrices and over different time intervals is the availability of reference material for method development and validated quantification. For that purpose, AA and FA conjugate synthesis was recently performed for GHB glycine, glutamate, and seven FA esters 35 …”

“…For that purpose, AA and FA conjugate synthesis was recently performed for GHB glycine, glutamate, and seven FA esters. 35 The aim of the present study was the development and validation of a simple LC-MS/MS method that should particularly cover GHB and its novel detected conjugates GHB carnitine, GHB glycine, GHB glutamate, GHB phenylalanine, GHB taurine, and seven GHB FA esters (C8-C18, C18:1) in urine. In addition, already known and evaluated organic acids 2,4-DHB, 3,4-DHB, GA, SA, succinylcarnitine, and GHB glucuronide should be included.…”