Phase I metabolites (organic acids) of gamma‐hydroxybutyric acid–validated quantification using GC–MS and description of endogenous concentration ranges

Abstract

Gamma‐hydroxybutyric acid (GHB) is a sedative drug used in drug‐facilitated crimes. Its detection window is very short. GHB undergoes intensive phase I metabolism to organic acids (glycolic acid, succinic acid, dihydroxybutyric acids). These could be potential analytical targets to broaden the detection window. The aim of the present study was to enable the detection of endogenous levels of these metabolites in biological samples (blood and urine). A gas chromatographic–mass spectrometric method using liquid–liquid extraction and derivatization with N‐methyl‐N‐tri‐methylsilyltrifluoracetamide was developed for the quantification. Validation results were consistent with international guidelines, and the method was able to quantify endogenous levels of the substances in both urine and blood. Endogenous concentrations were shown to be <0.03–4.92 mg/L for glycolic acid, <0.03–1.28 mg/L for GHB, <0.28–18.1 mg/L for succinic acid, <0.12–1.38 mg/L for 2,4‐dihydroxybutyric acid, and <0.13–2.59 mg/L for 3,4‐dihydroxybutyric acid in serum samples of 101 volunteers. Urinary endogenous concentrations were shown to be 1.30–400 mg/L for glycolic acid, <0.03–1.94 mg/L for GHB, 1.17–2.73 mg/L for succinic acid, 0.72–26.2 mg/L for 2,4‐dihydroxybutyric acid, and 1.88–122 mg/L for 3,4‐dihydroxybutyric acid in urine samples of 132 volunteers. These endogenous concentrations represent a basis to which concentrations after the intake of GHB can be compared to in order to prove the intake of this substance.

Towards Extending the Detection Window of Gamma-Hydroxybutyric Acid—An Untargeted Metabolomics Study in Serum and Urine Following Controlled Administration in Healthy Men

“…Still, without final structural characterization, it cannot be unambiguously confirmed, that it is actually a derivative of glycolic acid and whether it might be useful as such to extent the detection window of GHB. As already discussed in former studies, using glycolic acid alone most likely lacks specificity as a proof for GHB intake given its presence in all samples and possible other sources for increased concentration, such as e.g., ethylene glycol poisoning [13,15]. In our data set, dihydroxybutyric acid, the oxidation product formed through alpha-and beta-oxidation of GHB was indicated as statistically significant after GHB intake.…”

“…The current analytical method was not optimized for chromatographic separation of GHB or its metabolites and was unable to separate the two isomers 2,4-and 3,4-dihydroxybutyric acid. Previous studies though indicated different concentrations of both isomers in humans, with higher concentrations of the 3,4-isomer in blood and urine [15].…”

“…While the aforementioned conjugates of GHB were largely undetectable in the placebo session samples, organic acids or follow-up metabolites of those, could be detected in both sessions, but with significant differences. Organic acids have already been discussed by different authors as elevated after GHB intake [13][14][15][16][17][18]. Succinylcarnitine and glycolate were already determined in the initial biomarker search investigation [13].…”

“…Anyhow, to date reliable methods discriminating whether GHB-glucuronide or GHB-sulfate are derived from exogenous or endogenous sources are lacking [9][10][11][12]. Recently, research interest moved to targeted and untargeted evaluation of endogenous metabolites of GHB resulting either from GHB beta-oxidation or its initial formation to SSA [13][14][15][16]. Based on former findings of elevated levels 2,4-and 3,4-dihydroxy butyric acid [17,18], Jarsiah et al recently developed a method to quantify these metabolites together with other organic dicarboxylic acids glycolate and succinate in blood and urine samples of GHB users.…”

“…Based on former findings of elevated levels 2,4-and 3,4-dihydroxy butyric acid [17,18], Jarsiah et al recently developed a method to quantify these metabolites together with other organic dicarboxylic acids glycolate and succinate in blood and urine samples of GHB users. However, the actual influence of GHB on the organic acids in real consumer samples has not yet been published [15]. Luca et al reported up to sevenfold higher levels of glycolate in mice brain and liver samples [16].…”