Identification of new urinary gamma‐hydroxybutyric acid markers applying untargeted metabolomics analysis following placebo‐controlled administration to humans

Abstract

Gamma‐hydroxybutyrate (GHB) is a short‐chain fatty acid that occurs naturally in the mammalian brain and is prescribed as a medication against narcolepsy or used as a drug of abuse. Particularly, its use as a knock‐out drug in cases of drug‐facilitated crimes is of major importance in forensic toxicology. Because of its rapid metabolism and resulting narrow detection windows (<12 hours in urine), detection of GHB remains challenging. Thus, there is an urgent call for new markers to improve the reliable detection of GHB use. In the framework of a randomized, placebo‐controlled, crossover study in 20 healthy male volunteers, urine samples obtained 4.5 hours post‐administration were submitted to untargeted mass spectrometry [MS, quadrupole time of flight (QTOF)] analysis to identify possible new markers of GHB intake. MS data from four different analytical methods (reversed phase and hydrophilic interaction liquid chromatography; positive and negative electrospray ionization) were filtered for significantly changed features applying univariate and multivariate statistics. From the resulting 42 compounds of interest, 8 were finally identified including conjugates of GHB with carnitine, glutamate, and glycine as well as the endogenous compounds glycolate and succinylcarnitine. While GHB conjugates were only detectable in the GHB, but not in the placebo group, glycolate and succinylcarnitine were present in both groups albeit significantly increased through GHB intake. Untargeted metabolomics proved as a suitable tool for the non‐hypothesis driven identification of new GHB markers. However, more studies on actual concentrations, detection windows, and stability will be necessary to assess the suitability of these markers for routine application.

Comparative Untargeted Metabolomics Analysis of the Psychostimulants 3,4-Methylenedioxy-Methamphetamine (MDMA), Amphetamine, and the Novel Psychoactive Substance Mephedrone after Controlled Drug Administration to Humans

“…Only few studies on drugs of abuse and particularly NPS have been performed until now [12,16,21], the majority of them in rodents. Most valuable (controlled) human data are limited due to ethical restrictions and so far only available for MDMA [22][23][24], cocaine [25,26], and gamma-hydroxybutyric acid (GHB) [27][28][29].…”

Metabolomic Strategies in Biomarker Research–New Approach for Indirect Identification of Drug Consumption and Sample Manipulation in Clinical and Forensic Toxicology?

“…Analysis of urine samples collected 4.5 h after GHB or placebo intake of a randomized, double-blind, placebo-controlled crossover study in 20 men allowed identification of novel GHB metabolites GHB carnitine, GHB glycine, and GHB glutamate as exemplified in Figure 3. However, more studies addressing quantitative values, pharmacokinetics, and stability are demanded for a final conclusion on the routine applicability of these markers (Steuer et al, 2018c). Mollerup et al performed an interesting omics-based retrospective analysis to identify potential markers of valproic acid in blood that should allow the detection of valproic acid intake using the commonly applied positive ESI-MS mode.…”

“…Statistical evaluation was carried out using a paired t -test ( p < 0.05; **** p < 0.0001). Reprinted (adapted) with permission from Steuer et al (2018c). Copyright 2018, Wiley.…”

“…New strategies for GHB detection and differentiation between endogenous and exogenously consumed or administered GHB is still of high interest in forensics. Also, metabolomic studies were recently performed in order to find endogenous markers that might be able to prolong the detection window of GHB (Palomino-Schatzlein et al, 2017; Steuer et al, 2018c). Palomino-Schatzlein et al used an NMR-based metabolomics approach to identify changes caused by GHB in a controlled administration study in human urine.…”

Gamma-hydroxybutyric acid, gamma-butyrolactone, and 1,4-butanediol addiction: a serious health threat

“…The development of hyphenated analytical techniques (17,18) such as ultra-high performance liquid chromatography Beck R, Mimica Matanović S, Zibar L. Gamma-hydroxybutyric acid, gamma-butyrolactone, and 1,4-butanediol addiction: a serious health threat Arh Hig Rada Toksikol 2019;70:149-150 tandem mass spectrometry (UHPLC-MS/MS) and gas chromatography tandem mass spectrometry (GC-MS/MS) is therefore crucial if we want to be able to identify GHB and its precursors in conventional and unconventional biological matrices, bearing in mind the current proposed cut-offs to distinguish between endogenous and exogenous GHB (22). In samples taken from living subjects these are 5 mg/L for blood and 10 mg/L for urine, whereas in post-mortem specimens they are 30 mg/L and 50 mg/L for peripheral and central blood, respectively and 10 mg/L for urine (22).…”