GHB related acids are useful in routine casework of suspected GHB intoxication cases.

GHB related acids (3,4-dihydroxy butyric acid, 2,4-dihydroxy butyric acid and glycolic acid) are produced through oxidative GHB metabolism. These analytes could be potential biomarkers to ensure the diagnosis of a GHB intoxication and even prolong the detection window. Within this study, forensic routine cases were measured to consider the potential of additional gas chromatographic mass spectrometric analysis on these acids. 17 GHB positive real cases (10 serum samples and 7 urine samples) and 40 cases with suspicion of drugging in DFC cases and negative GHB results (21 serum samples and 19 urine samples) were evaluated. Increased GHB related acid concentrations were detected in all serum and most urine samples positive on GHB. In some GHB negative cases, especially in serum samples, concentrations of GHB related acids gave hints that GHB actually was taken. We recommend to use the following cut-offs for a more reliable interpretation of potential GHB intoxication cases: 3,4-OH-BA:>3 mg/L in serum and>50 mg/L in urine; 2,4-OH-BA:>2 mg/L in serum and>25 mg/L in urine; GA:>5 mg/L in serum and>400 mg/L in urine.

Detection of γ-hydroxybutyric acid-related acids in blood plasma and urine: Extending the detection window of an exogenous γ-hydroxybutyric acid intake?

In crimes facilitated by γ-hydroxybutyric acid (GHB) administration, the frequent occurrence of anterograde amnesia of the victims as well as the short detection window and variations of endogenous GHB concentrations complicate obtaining analytical proof of GHB administration. Because elevated endogenous organic acid concentrations have been found in the urine of patients with succinic semialdehyde deficiency (leading to accumulation of GHB in human specimens) and after GHB ingestion, we searched for an alternative way to prove GHB administration via detection of elevated organic acid concentrations in blood plasma and urine. We collected blood and urine samples from narcolepsy patients (n = 5) treated with pharmaceuticals containing GHB sodium salt (1.86-3.72 g GHB as free acid per dose). Although GHB was detectable only up to 4 h in concentrations greater than the commonly used cutoff levels in blood plasma, 3,4-dihydroxybutyric acid (3,4-DHB) could be detected up to 12 h in blood plasma in concentrations exceeding initial concentrations of the same patient before GHB ingestion. Furthermore, four of the five patients showed an increase above endogenous levels described in the scientific literature. In urine, GHB concentrations above commonly used cutoff levels could be observed 4.5-9.5 h after GHB intake. Creatinine standardized initial concentrations were reached again for glycolic acid (GA), 3,4-DHB, and 2,4-dihydroxybutyric (2,4-DHB) acid at 6.5-22, 11.5-22, and 8.5-70 h after GHB intake, respectively. Therefore, 2,4-DHB, 3,4-DHB, and GA are promising and should be further investigated as potential biomarkers to prolong the detection window of GHB intake.

GHB related acids (dihydroxy butyric acids, glycolic acid) can help in the interpretation of post mortem GHB results.

Post mortem gamma hydroxy butyric acid (GHB) concentrations should be interpreted with caution since GHB concentrations can increase after death. Post mortem concentrations after the intake of GHB ante mortem do overlap with concentration ranges in cases without known exposure to GHB and make an interpretation challenging. GHB is known to undergo intensive metabolism to related acids (glycolic acid (GA), succinic acid (SA), 2,4- and 3,4-dihydroxy butyric acid (2,4-OH-BA and 3,4-OH-BA)). GHB and these related acids were analyzed using a validated gas chromatographic mass spectrometric (GC-MS) method after liquid liquid extraction and trimethylsilylation. SA concentrations were not usable post mortem due to instability. Concentrations in cases without known exposure to GHB (urine: n = 80; femoral blood: n = 103) were: for GA 4.6-121 mg/L in urine and 1.6-11.2 mg/L in blood, for 2,4-OH-BA < LoD-25,3 mg/L in urine and < LoD-3.7 mg/L in blood and for 3,4-OH-BA < LoD-54,3 mg/L in urine and < LoD-5.3 mg/L in blood. In death cases involving GHB (n = 11) concentrations of GHB related acids were increased compared to these levels (for GA in 7/10 cases and up to 391 mg/L in urine, in 6/11 cases and up to 34 mg/L in blood; for 2,4-OH-BA in 9/10 cases and up to 144 mg/L in urine, in 11/11 cases and up to 9.1 mg/L in blood; for 3,4-OH-BA in 7/10 cases and up to 665 mg/L in urine, in 11/11 cases and up to 19 mg/L in blood). Therefore, the concentrations of these GHB related acids can aid in a more reliable differentiation of GHB exposure in post mortem toxicology. We recommend to add the analysis of 2,4-OH-BA, 3,4-OH-BA and GA in femoral blood for the diagnosis of a GHB intake post mortem. Post mortem femoral blood concentrations > 4 mg/L for 2,4-OH-BA, > 5 mg/L for 3,4-OH-BA and > 12 mg/L for GA give hints for a GHB intake.

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

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.