GHB Concentrations Research Articles

Cardiorespiratory effects of Gamma-Hydroxybutyric Acid (GHB) during isoflurane anaesthesia in pigs

ObjectiveTo investigate the haemodynamic effects of gamma-hydroxybutyric acid (GHB) in isoflurane anaesthetised pigs. Study DesignExperimental, randomized, non-blinded, cross-over study. AnimalsA group of six stress-resistant Landrace pigs (approximately 3 months old; three male, three female; bodyweight 39.2 ± 4 kg (mean ± standard deviation). MethodsAfter premedication (midazolam 0.5 mg kg-1 and ketamine 10 mg kg-1 intramuscularly), and induction [propofol 0.25–0.5 mg kg-1 intravenously IV)], anaesthesia was maintained with isoflurane in oxygen, and either GHB 250 mg kg-1 IV or an equal volume of saline was administered (minimum washout period 1 week). Systolic (SAP), diastolic (DAP), and mean (MAP) arterial pressures, heart rate and rhythm, respiratory rate, were recorded every 5 minutes for 2 hours. Arterial samples were collected for blood gas and pharmacokinetic analyses. Relative changes from baseline were calculated and compared between treatments using a mixed model with time, period, and treatment as variables (α < 0.05). ResultsChanges from baseline differed significantly between treatments (p < 0.001) for SAP (GHB -1.6 ± 10.7; saline -5.9 ± 14.8 mmHg), DAP (GHB +2.9 ± 9.6; saline -6.5 ± 10.7 mmHg) and MAP (GHB +2.2 ± 10.5; saline -5.7 ± 9.6 mmHg). Statistical analysis of secondary outcomes suggested effects on PaO2 (GHB -45.2 ± 29.8 mmHg [-6.03 ± 3.97 kPa]; saline +24.5 ± 32.4 mmHg [+3.27 ± 4.32 kPa]; p < 0.001) and PaCO2 (GHB -2 ± 10 mmHg [-0.27 ± 1.33 kPa]; saline -9 ± 8 mmHg [-1.20 ± 1.07 kPa]; p < 0.001). Mean maximum plasma concentration of GHB was 1171.1 ± 229.3 μg mL-1, with volume of distribution 335.3 ± 68.5 mL kg-1, clearance 77.2 ± 19.12 mL kg-1 hour-1 and elimination half-life 3.10 ± 0.80 hours. Conclusions and clinical relevanceGHB did not cause severe physiological side effects and may reduce cardiovascular depression.

Sex and Cross-Sex Testosterone Treatment Alters Gamma-Hydroxybutyrate Acid Toxicokinetics and Toxicodynamics in Rats.

Γ-hydroxybutyric acid (GHB) is widely abused due to its sedative/hypnotic and euphoric effects. In recent years, GHB use has witnessed a notable rise within the LGBTQ+ community. GHB is a substrate of monocarboxylate transporters (MCTs) and exhibits nonlinear toxicokinetics, characterized by saturable metabolism, absorption, and renal reabsorption. This study investigates the impact of exogenous testosterone administration on GHB toxicokinetics and toxicodynamics, exploring the potential of MCT1 inhibition as a strategy to counteract toxicity. Ovariectomized (OVX) females and castrated (CST) male Sprague Dawley rats were treated with testosterone or placebo for 21 days. GHB was administered at two doses (1000 mg/kg or 1500 mg/kg i.v.), and the MCT1 inhibitor AR-C 155858 (1 mg/kg i.v.) was administered 5 min after GHB (1500 mg/kg i.v.) administration. Plasma and urine were collected up to 8 h post-dose, and GHB concentrations were quantified via a validated LC/MS/MS assay. Sleep time (sedative/hypnotic effect) was utilized as the toxicodynamic endpoint. Testosterone treatment significantly affected GHB toxicokinetics and toxicodynamics. Testosterone-treated CST rats exhibited significantly lower renal clearance, higher AUC, and increased sedative effect, while testosterone-treated OVX rats demonstrated higher metabolic clearance. AR-C 155858 treatment led to an increase in GHB renal and total clearance together with an improvement in sedative/hypnotic effect. In conclusion, exogenous testosterone treatment induces significant alterations in GHB toxicokinetics and toxicodynamics, and MCT inhibition can serve as a potential therapeutic strategy for GHB overdose in both cisgender and transgender male populations.

The interest of using vitreous humor for γ-hydroxybutyrate (GHB) quantification in related fatalities: Stability evaluation, case report and literature review

Analysis and interpretation of the findings for γ-hydroxybutyrate (GHB) in related fatalities remains problematic. Indeed, GHB is a naturally occurring compound present in both the mammalian central nervous system and peripheral tissue. Moreover, a postmortem increase in endogenous GHB concentration has been observed, especially in blood. Facing this issue, the use of an alternative matrix such as vitreous humor (VH) can thus be particularly interesting for GHB testing and quantification. VH is considered to be less prone to postmortem redistribution, is easy to collect, and has relatively few interfering compounds for the analytical process. In this context, the authors report the case of a GHB-related fatality involving 22-year-old male. In this case, GHB femoral blood (FB) (790 mg/L) and vitreous (750 mg/L) concentrations appeared similar with a FB to VH (FB/VH) ratio of 1.05. In addition, other similar cases with both GHB blood and vitreous concentrations were reviewed. Five cases were identified. The blood to VH ratios ranging from 0.13 to 2.58. Finally, GHB stability was documented in postmortem blood and VH, in order to address the reliability of VH as an alternative matrix for GHB quantitation at postmortem. GHB appeared relatively stable in postmortem blood specimens (at 50 mg/L) over a period of 28 days when stored at +4 °C or −20 °C. The same results were observed in VH specimens.

GHB toxicokinetics and renal monocarboxylate transporter expression are influenced by the estrus cycle in rats

BackgroundThe illicit use and abuse of gamma-hydroxybutyric acid (GHB) occurs due to its sedative/hypnotic and euphoric effects. Currently, there are no clinically available therapies to treat GHB overdose, and care focuses on symptom treatment until the drug is eliminated from the body. Proton- and sodium-dependent monocarboxylate transporters (MCTs (SLC16A) and SMCTs (SLC5A)) transport and mediate the renal clearance and distribution of GHB. Previously, it has been shown that MCT expression is regulated by sex hormones in the liver, skeletal muscle and Sertoli cells. The focus of the current study is to evaluate GHB toxicokinetics and renal monocarboxylate transporter expression over the estrus cycle in females, and in the absence of male and female sex hormones.MethodsGHB toxicokinetics and renal transporter expression of MCT1, SMCT1 and CD147 were evaluated in females over the estrus cycle, and in ovariectomized (OVX) female, male and castrated (CST) male rats. GHB was administered iv bolus (600 and 1000 mg/kg) and plasma and urine samples were collected for six hours post-dose. GHB concentrations were quantified using a validated LC/MS/MS assay. Transporter mRNA and protein expression was quantified by qPCR and Western Blot.ResultsGHB renal clearance and AUC varied between sexes and over the estrus cycle in females with higher renal clearance and a lower AUC in proestrus females as compared to males (intact and CST), and OVX females. We demonstrated that renal MCT1 membrane expression varies over the estrus cycle, with the lowest expression observed in proestrus females, which is consistent with the observed changes in GHB renal clearance.ConclusionsOur results suggest that females may be less susceptible to GHB-induced toxicity due to decreased exposure resulting from increased renal clearance, as a result of decreased renal MCT1 expression.

Alcohol perturbed locomotor behavior, metabolism, and pharmacokinetics of gamma-hydroxybutyric acid in rats

Gamma-hydroxybutyric acid (GHB), both a metabolic precursor and product of gamma-aminobutyric acid (GABA), is a central nervous system depressant used for the treatment of narcolepsy-associated cataplexy and alcohol withdrawal. However, administration of GHB with alcohol (ethanol) is a major cause of hospitalizations related to GHB intoxication. In this study, we investigated locomotor behavior as well as metabolic and pharmacokinetic interactions following co-administration of GHB and ethanol in rats. The locomotor behavior of rats was evaluated following the intraperitoneal administration of GHB (sodium salt, 500 mg/kg) and/or ethanol (2 g/kg). Further, time-course urinary metabolic profiling of GHB and its biomarker metabolites glutamic acid, GABA, succinic acid, 2,4-dihydroxybutyric acid (OH-BA), 3,4-OH-BA, and glycolic acid as well as pharmacokinetic analysis were performed. GHB/ethanol co-administration significantly reduced locomotor activity, compared to the individual administration of GHB or ethanol. The urinary and plasma concentrations of GHB and other target compounds, except for 2,4-OH-BA, were significantly higher in the GHB/ethanol co-administration group than the group administered only GHB. The pharmacokinetic analysis results showed that the co-administration of GHB and ethanol significantly increased the half-life of GHB while the total clearance decreased. Moreover, a comparison of the metabolite-to-parent drug area under the curve ratios demonstrated that the metabolic pathways of GHB, such α- and β-oxidation, were inhibited by ethanol. Consequently, the co-administration of GHB and ethanol aggravated the metabolism and elimination of GHB and enhanced its sedative effect. These findings will contribute to clinical interpretation of GHB intoxication.

Concise analysis of γ-hydroxybutyric acid in beverages and urine by capillary electrophoresis with capacitively coupled contactless conductivity detection using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid as background electrolyte

γ-Hydroxybutyric acid (GHB), a neurotransmitter or neuromodulator in the human central nervous system, is often abused in drug-facilitated sexual assaults due to its euphoric and sedative effects. While the analysis of GHB has received continuous attention, its inherent characteristics pose challenges. In the current study, capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4D) was built, and Good's buffers were evaluated as the background electrolytes for CE separation and C4D detection. On this basis, a simple and efficient CE-C4D method was developed for GHB analysis. Through theoretical discussion and experimental optimization, the separation of GHB and related positional isomers α-hydroxybutyric acid (AHB) and β-hydroxybutyric acid (BHB) was achieved within 4 min using 150 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as the running buffer. Under the optimized condition, the relative standard deviations of migration time and peak area were less than 1.1% and 4.5%, indicating good precision. The C4D signal of GHB showed a good linear relationship with GHB concentration in the range of 3-300 µM with a determination coefficient of 0.9997, and the detection limit was calculated to be 0.37 µM based on the signal-to-noise ratio of three. Furthermore, liquid-liquid extraction (LLE) and solid-phase extraction (SPE) were comparatively studied for sample matrix purification. Combined with the optimized SPE procedure, the developed CE-C4D method has been successfully applied for the determination of exogenous GHB in spiked beverages and endogenous GHB in human urine.

The study of postmortem blood gamma-hydroxybutyric acid (GHB) concentrations in Thai dead bodies unrelated to GHB use

Background: Gamma-hydroxybutyric acid (GHB) is classified as a category I psychotropic substance in Thai legislation. It can be exogenously consumed or endogenously produced and cut-off concentrations are required. Baseline GHB concentration ranges in Thai postmortem cases are still not available. Objectives: To determine baseline GHB concentrations and factors influencing GHB concentrations in Thai postmortem cases. Materials and methods: Postmortem femoral and cardiac blood samples were collected from Thai cadavers aged 18 years old or older sent for medico-legal autopsies at the Department of Forensic Medicine, Siriraj Hospital, Mahidol University between 18th August 2021 and 25th January 2022 with postmortem interval (PMI) not greater than 24 hours. Case data including sex, age, PMI, sites of collection and causes of death were recorded. Blood GHB concentrations were extracted using protein precipitation and analyzed by gas chromatography-mass spectrometry (GC-MS). Descriptive statistics, paired samples Wilcoxon signed-rank test, Spearman's correlation, Mann-Whitney U test and Kruskal-Wallis H test were analyzed where appropriate. Results: A total of 150 subjects were recruited; 63 (42%) were female with mean age 44.59 years old (range 18-75 years old). Femoral and cardiac GHB concentration ranges were &lt;0.5-20.81 µg/mL and 1.12-39.04 µg/mL, respectively while median GHB concentrations in femoral and cardiac blood were 6.38 and 8.41 µg/mL. Femoral GHB concentrations were significantly lower than cardiac GHB concentrations (p&lt;0.001). Both femoral and cardiac blood showed a positive correlation between GHB concentrations and PMI (Spearman’s correlation =0.52 and 0.32, p&lt;0.001). Most GHB concentrations in femoral blood were less than 10 µg/mL and almost all samples from both sources had GHB concentrations less than 30 µg/mL. Conclusion: GHB concentrations in Thai postmortem cases were &lt;0.5-20.81 µg/mL in femoral blood and 1.12-39.04 µg/mL in cardiac blood, respectively. GHB concentrations in femoral blood were significantly lower than in cardiac blood. GHB concentrations significantly increased with longer PMI

Method validation of gamma-Hydroxybutyric acid detection upon Herpes Simplex Virus-Type 1 infection using LC-MRM-MS with 3-nitrophenylhydrazine derivatization

Volatile organic compounds (VOCs) release triggered by infection of DNA virus has not been studied extensively. Previously, we reported that gamma-butyrolactone (GBL), a VOC, was released upon Herpes Simplex Virus Type-1 (HSV-1) acute infection. Based on the metabolic pathway and chemical conversion of GBL, we hypothesized that infected cells produce gamma-Hydroxybutyric acid (GHB) as a key pathway intermediate for the subsequent production of GBL. An analytical technique for the rapid detection of GHB is crucial for further understanding its role in the cellular response to HSV-1 infection. To address this, we developed a sensitive, reliable, and specific method for the detection and quantification of GHB in mammalian cell culture using a pre-column derivatization approach. Our data showed that the carboxylic acid functional group of GHB could be derivatized with 3-nitrophenylhydrazine hydrochloride (3-NPH) to produce its hydrazineyl derivative. Unlike GHB, the derivative could be detected seamlessly in HPLC-MS. We also demonstrate quantitive conversion of GHB into the derivative with over 95% yield at a range of 1 μg/mL- 6 μg/mL GHB concentration. This method offers a rapid quantification of GHB in aqueous mixtures, especially in cultured extracts.

Absence of drug-drug interactions between γ-hydroxybutyric acid (GHB) and cobicistat.

Potential interactions between CYP3A4 inhibitors and γ-hydroxybutyric acid (GHB) have been suggested as a possible explanation for cases of GHB overdose in recent years among people living with HIV engaged in chemsex. Our objective was to assess the effect of cobicistat on the pharmacokinetics of GHB. Fifteen healthy adults were enrolled in this randomized, double-blind, placebo-controlled, two-arm, crossover clinical trial. Participants underwent two 5 day treatment periods with at least a 1 week washout period between them. In each treatment period, participants received cobicistat (150 mg q24h orally) or matched placebo. On day 5 of each treatment period, participants were given a single oral dose of GHB (25 mg/kg). Plasma concentrations of GHB, subjective effects, blood pressure, heart rate and oxygen saturation were monitored for 5 h after dosing. GHB pharmacokinetic and pharmacodynamic parameters were calculated for each participant during each study period by non-compartmental analysis and were compared using linear mixed-effects models. The study was registered at https://www.clinicaltrialsregister.eu (Eudra-CT number 2019-002122-71) and at https://clinicaltrials.gov (NCT04322214). Ten participants completed the two study periods. No drug-related adverse events that necessitated subject withdrawal or medical intervention occurred during the study. Compared with placebo, none of the primary pharmacokinetic parameters of GHB was substantially changed by the administration of GHB with cobicistat. Similarly, no differences regarding subjective or physiological effects were observed when GHB was administered alone or with cobicistat. Neither pharmacokinetic nor pharmacodynamic drug-drug interactions between cobicistat and GHB were identified in this study.

Drug-drug interaction between diclofenac and gamma-hydroxybutyric acid.

Gamma hydroxybutyric acid (GHB) has been approved clinically to treat excessive daytime sleepiness and cataplexy in patients with narcolepsy, alcohol and opioid withdrawal, and as an anesthetic. The use of GHB clinically is limited due to its high abuse potential. The absorption, clearance and tissue uptake of GHB is mediated by proton-dependent and sodium-coupled monocarboxylate transporters (MCTs and SMCTs) and inhibition of these transporters may result in a change in GHB pharmacokinetics and pharmacodynamics. Previous studies have reported that non-steroidal anti-inflammatory drugs (NSAIDs) may inhibit these monocarboxylate transporters. Therefore, the purpose of this work was to analyze the interaction between GHB (at a dose of 600 mg/kg i. v.) and the NSAID, diclofenac, by examining the effects of this drug on the in vivo pharmacokinetics and pharmacodynamics in rat studies. The pharmacodynamic effect evaluated was respiratory depression, a measure of toxicity observed by GHB at this dose. There was an improvement in the respiratory rate with diclofenac administration suggesting an effect of diclofenac on GHB toxicity. In vitro studies with rat blood brain endothelial cells (RBE4) that express MCT1 indicated that diclofenac can inhibit GHB transport with an IC50 of 10.6 μM at pH 7.4. In vivo studies found a decrease in brain GHB concentrations and a decrease in the brain-to-plasma concentration ratio following diclofenac treatment. With this study we can conclude that diclofenac and potentially other NSAIDs can inhibit the transport of GHB into the brain, therefore decreasing GHB's pharmacodynamic effects and toxicity.

Determination of endogenous GHB levels in chest and pubic hair

Endogenous nature of GHB represents a critical issue for forensic toxicologists, especially in alleged sexual assaults. Therefore, discrimination between physiologically and additional amounts from exogenous sources of such a substance must be effective and reliable in order to avoid severe misinterpretation. This study aimed to quantify the GHB baseline concentrations in chest and pubic hairs collected from 105 healthy volunteers, non-consumers of any drugs of abuse. The final scope was to investigate if these keratin matrices could represent valid alternative to scalp hair when not available. Moreover, we also evaluated the age and gender influences on the GHB baseline levels. 25 mg of hair were incubated overnight with NaOH at 56 °C. After acidification with H2SO4, the solution was liquid-liquid extracted with ethyl acetate and a trimethylsilyl derivatization was then achieved. Analysis was performed in gas chromatography-mass spectrometry in single ion monitoring mode (m/z 233, 234, 147 for GHB; m/z 239, 240 and 147 for GHB-d6). The endogenous amount in “blank” hair was estimated by the standard addition method (0.301 for chest hair and 0.235 ng/mg for pubic hair). GHB concentration ranged from 0.205 to 1.511 ng/mg for chest hair and from 0.310 to 1.913 ng/mg for pubic hair. These values were consistent with previous studies on scalp hair and on pubic hair. Unfortunately, research on chest hair is not available in literature. T-Test and Linear Regression highlighted no statistically significant differences for the two matrices and for all age/gender sub-groups. However, further studies are required to estimate a reliable cut-off value for these keratin matrices. For the first time, we demonstrated the suitability of chest and pubic hair to detect endogenous levels of GHB.

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 4h 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 4.5-9.5h 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.

Potential application of BC3 nanotubes as a gamma-hydroxybutyric acid drug sensor: A DFT study

Using density functional theory (DFT) computations, the application of a BC3 nanotube was scrutinized to detect gamma-hydroxybutyric acid (GHB). After the adsorption of GHB the HOMO-LUMO gap reduces from 2.358 to 2.199 eV. The electrical conductance of GHB/BC3 nanotube complex is 486 times higher compared to that of the bare nanotube. Hence, the BC3 nanotube can yield an electronic sign after the adsorption of GHB, showing that it is a viable electronic sensor. The sensing response for the BC3 nanotube is 485, representing a high sensitivity. The sensing response is increased to 639, 746, and 906 for 2, 3, and 4 GHB adsorbed nanotube, respectively, indicating that the sensor response depends on the GHB concentration. The interaction between GHB and the BC3 nanotube in the ethanol solvent is strengthened.

γ-Hydroxybutyric Acid-Ethanol Drug-Drug Interaction: Reversal of Toxicity with Monocarboxylate Transporter 1 Inhibitors.

The drug of abuse, γ-hydroxybutyric acid (GHB), is commonly co-ingested with ethanol, resulting in a high incidence of toxicity and death. Our laboratory has previously reported that GHB is a substrate for the monocarboxylate transporters (MCTs), necessary for its absorption, renal clearance, and tissue distribution, including across the blood-brain barrier. Our goal was to investigate the drug-drug interaction (DDI) between GHB and ethanol and to evaluate MCT1 inhibition as a strategy to reverse toxicity. The toxicokinetics of this DDI were investigated, including brain-to-plasma concentration ratios, in the presence and absence of ethanol. The toxicodynamic parameters examined were respiratory depression (breathing frequency, tidal volume) and sedation (time of return-of-righting reflex). Ethanol was administered (2 g/kg i.v.) 5 minutes before the intravenous or oral administration of GHB, and MCT1 inhibitors AZD-3965 and AR-C155858 (5 mg/kg i.v.) were administered 60 minutes after GHB administration. Ethanol administration did not alter the toxicokinetics or respiratory depression caused by GHB after intravenous or oral administration; however, it significantly increased the sedation effect, measured by return-to-righting time. AZD-3965 or AR-C155858 significantly decreased the effects of the co-administration of GHB and ethanol on respiratory depression and sedation of this DDI and decreased brain concentrations and the brain-to-plasma concentration ratio of GHB. The results indicate that ethanol co-administered with GHB increases toxicity and that MCT1 inhibition is effective in reversing toxicity by inhibiting GHB brain uptake when given after GHB-ethanol administration. SIGNIFICANCE STATEMENT: These studies investigated the enhanced toxicity observed clinically when γ-hydroxybutyric acid (GHB) is co-ingested with alcohol and evaluated strategies to reverse this toxicity. The effects of the novel monocarboxylate transporter 1 (MCT1) inhibitors AR-C155858 and AZD-3965 on this drug-drug interaction have not been studied before, and these preclinical studies indicate that MCT1 inhibitors can decrease brain concentrations of GHB by inhibiting brain uptake, even when administered at times after GHB-ethanol. AZD-3965 represents a potential treatment strategy for GHB-ethanol overdoses.

Comparative Study: Postmortem Long-Term Stability of Endogenous GHB in Cardiac Blood, Femoral Blood, Vitreous Humor, Cerebrospinal Fluid and Urine with and without Sodium Fluoride Stabilization.

The interpretation of postmortem γ-hydroxybutyric acid (GHB) concentrations is challenging due to endogenous existence and postmortem GHB production in body tissues and fluids. As an additional complication, formation of GHB was also described in stored postmortem samples. We examined cardiac blood, femoral blood, vitreous humor, cerebrospinal fluid and urine of eight different corpses (male/female 5/3, aged 33-92 years, postmortem interval 1-6 days) where no intake of GHB or one of its precursors was assumed. All samples were collected during autopsy and divided into two aliquots. To one of the aliquots, sodium fluoride (NaF, 1% w/v) was added. Both aliquots were vortexed, further divided into seven aliquots and stored at -20°C. GHB concentrations were measured immediately and subsequently 1 day, 7 days, 2 weeks, 4 weeks, 3 months and 6 months, after sample collection using trimethylsilyl derivatization and gas chromatography, coupled to single quadrupole mass spectrometry. Similar progression curves of GHB concentrations were obtained for the different matrices in the individual corpses. Femoral and cardiac blood GHB concentrations were always found to be higher than in vitreous humor, cerebrospinal fluid, and urine irrespective of stabilization and storage time. None of the obtained GHB concentrations exceed the cutoff values for postmortem matrices commonly used for the identification of an exogenous GHB intake (urine, venous blood and cerebrospinal fluid: 30 mg/L, cardiac blood and vitreous humor 50 mg/L). No significant differences were found for the GHB concentrations measured immediately and 6 months after autopsy. However, we found a significant increase for the GHB concentrations 4 weeks as well as 3 months after sample collection, which was followed by a decrease nearly to initial values. There were no significant differences between samples with and without NaF addition. The data presented are useful for the interpretation of GHB concentrations in upcoming death cases, with special attention to storage conditions and different postmortem matrices.

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.

Methyl-4-Hydroxybutyrate and Ethyl-4-Hydroxybutyrate as Potential Markers for Simultaneous Consumption of GHB/GBL and Alcohol: Preliminary Investigations.

γ-Hydroxybutyric acid (GHB) and its corresponding lactone γ-butyrolactone (GBL) are misused as knock out (k.o.) drugs. The short detection window and the major inter- and intra-individual variations of endogenous GHB concentrations in commonly used matrices such as blood and urine complicate the analytical proof of an exogenous GHB/GBL administration. We searched for an alternative way to prove an exogenous GHB/GBL administration via detection of methyl- and ethyl-4-hydroxybutyrate, which could arise in alcoholic solutions after spiking with GHB/GBL. A liquid chromatographic-triple quadrupole mass spectrometric method was developed and validated to quantitatively determine methyl- and ethyl-4-hydroxybutyrate in alcoholic beverages (limit of detection [LoD]: 5.8 and 3.4 ng/mL, respectively). A sample collective of alcoholic beverages (n = 47) revealed natural occurring amounts of ethyl-4-hydroxybutyrate (<LoD-approx. 3980 ng/mL) with higher concentrations particularly found in wine samples. Nearly no ethyl-4-hydroxybutyrate was observable in spirits/liqueurs and no methyl-4-hydroxybutyrate was detectable at all. A moderate correlation was shown between the ethyl-4-hydroxybutyrate concentration and the pH-value in wine samples (pH 2.9-3.7, n = 29) as well as between the ethyl-4-hydroxybutyrate concentration and the GHB concentration in all measured beverages (GHB: < limit of quantification [LoQ]-11.4 µg/mL, n = 47). A dependency on alcohol content could not be observed. A voluntary intake (n = 1) of 750-mL wine naturally containing high amounts of ethyl-4-hydroxybutyrate (approx. 2010 ng/mL) revealed no observable GHB-ester concentrations in blood and urine. Furthermore, an experiment simulating a beverage that could potentially be used in a drug-facilitated crime (DFC) case showed ethyl-4-hydroxybutyrate concentrations exceeding the concentrations naturally observed in beverage samples. However, in order to evaluate whether ethyl-4-hydroxybutyrate could be useful as marker for the co-consumption of GHB/GBL and alcohol and to prolong the detection window of unintended GHB/GBL intake, further experiments have to be performed.

Endogenous GHB in Segmented Hair Part I: Inter-individual Variation for Group Comparisons.

While earlier studies have attempted to resolve the challenges encountered when interpreting gamma-hydroxybutyric acid (GHB) concentrations in hair (primarily due to its endogenous presence), few have had large sample sizes. The first objective of this study was to evaluate the inter-individual variation of endogenous GHB concentrations. The second objective, to be detailed in another report, was to assess intra-individual variation and the impact on exogenous GHB discrimination. Over 2,000 hair segments from 141 women and 73 men (all processed hair 3-12cm long) were analyzed in this study. The raw calculated range of endogenous GHB concentrations was <0.40-5.47ng/mg with 97.5% of the segmental results calculated less than 2.00ng/mg. Imputation, assuming a lognormal distribution, was applied to the data to include non-detect (ND) data (<LOQ), which led to an estimated endogenous GHB range of 0.16-5.47ng/mg. Kruskal-Wallis tests were employed on a segmental basis for group comparisons. This test was applied to the male and female segmental medians and subsequently indicated that these groups were different at the α=0.05 level of significance. Additionally, female hair samples appeared to have a trend comprising higher endogenous GHB concentrations close to the scalp and a mean net decrease of ~0.2-0.3ng/mg distally. Male hair samples displayed the opposite trend, with a mean net increase of ~0.5-0.6ng/mg from the proximal to the distal end of the hair shaft. It was also concluded that differences exist between the median GHB concentrations of the 'treated' and 'untreated' hair in the female group at the α=0.05 level of significance. Age groups and races were analyzed, but none of the observed differences in median concentration were significant at α=0.05. This is the largest endogenous GHB hair population study to date and provides substantial new data on inter-individual variation and chronological trends of GHB concentrations in hair.

Rate of elimination of γ-hydroxybutyrate from blood determined by analysis of two consecutive samples from apprehended drivers in Norway

AimGamma-hydroxybutyrate (GHB) is a common drug of abuse with an elimination half-life of 20−45 min. However, there is some evidence that GHB might exhibit saturation kinetics after ingesting high recreational doses. The aim of this study was to investigate the elimination kinetics of GHB from blood in people apprehended by the police for impaired driving and secondary to describe concentrations in all GHB-positive drivers. MethodsTwo consecutive blood samples were taken about 30−40 min apart from N = 16 apprehended drivers in Norway. GHB was determined in blood by an Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method. The changes in GHB between the two consecutive blood samples allowed estimating GHB’s elimination half-life, assuming first-order and zero-order elimination kinetics. GHB concentrations are also reported for N = 1276 apprehended drivers with GHB in blood. ResultsThe median time interval between collecting the two blood samples was 36 min (range 20−56 min). The median concentration of GHB in the first blood sample was 56.5 mg/L (range 14.1−142 mg/L) compared with 47.8 mg/L in the second sample (range 9.75−113 mg/L). The median elimination half-life was 103 min (range 21−187 min), and GHB’s median zero-order elimination rate constant was 21.0 mg/L/h (range 6.71–45.4 mg/L/h). Back-calculation to the time of driving resulted in GHB concentrations up to 820 mg/L assuming first-order kinetics and up to 242 mg/L assuming zero-order kinetics. In all drivers (N = 1276), the median GHB concentration was 73.7 mg/L and highest was 484 mg/L. ConclusionThe elimination half-life of GHB in blood samples from apprehended drivers was longer than expected compared with results of controlled dosing studies. Zero-order kinetics seems a more appropriate model for GHB when concentrations are back-calculated, and the median elimination rate was 21 mg/L/h.

The health threat of γ-butyrolactone (GBL): Hair analysis in a case of intoxication

GHB is currently under legal control in a number of countries. Because of that, pushers and users have switched to two GHB precursors, GBL and 1,4-BD, which can still be legally purchased. GBL has become a growing public health issue because it is relatively cheap and easy to obtain, due to its many industrial uses. Here, we report a case involving a 31-year-old male, who consumed, during a sexual intercourse with more partners (chemsex), a colorless liquid sold as “G”, and shortly after experienced adverse effects never felt before. Segmental hair analysis highlighted exogenous GHB concentration in six segments of 1 cm each (range: 27.2–23.9 ng/mg). Classic drugs of abuse and some new psychoactive substances were also found in te fist and last hair segment. A toxicological analysis of the liquid was performed and it highlighted the presence of GBL (982 g/L) and GHB (0.9 g/L), slightly higher than those reported in literature. The high variability of concentration of GBL illegal liquid preparations increases the risk of severe intoxication as in the case here presented.