GHB In Blood Research Articles

Determination of endogenous GHB in ante-mortem whole blood, urine, and oral fluid by LC–MS/MS: The effect of different additives and storage conditions on the stability of GHB in blood

Two challenges in detecting γ-hydroxybutyric acid (GHB) intake are its endogenous presence and in vitro production after sampling. This study developed an LC–MS/MS method for selective GHB determination in human antemortem blood, urine, and oral fluid at endogenous concentrations. Furthermore, the stability of GHB in blood samples and its endogenous concentrations in samples taken under controlled circumstances were investigated. Samples were extracted in methanol/acetonitrile and processed by anion exchange solid-phase extraction. GHB was separated from structural isomers using a reversed–phase LC column with anion properties. The validated limit of quantification was 0.005 µg/mL in blood and 0.010 µg/mL in urine and oral fluid, at which the relative reproducibility standard deviation and bias were <15 %. The mean extraction recovery was ≥90 %. The average GHB concentration increased by 1.2 µg/mL in fluoride/citrate- preserved blood after 28 days of storage at 4°C; however, in fluoride/oxalate (FX)-preserved blood, the mean concentration increased by only 0.055 µg/mL. No change was observed at −20°C. In 105 randomly selected samples of FX-preserved blood collected for forensic antemortem toxicological analysis, all concentrations were <0.066 µg/mL, even after long-term storage at −20°C. In blood, urine, and oral fluid samples from a clinical study of GHB intake, endogenous baseline levels from 30 participants ranged from 0.0069–0.050, 0.024–0.38, and 0.034–0.93 µg/mL, respectively. These results demonstrate that the current cut-off level of 5 µg/mL for discriminating between endogenous and exogenous GHB in antemortem blood could be considerably lower for FX-preserved blood stored at −20°C.

Occurrence of GHB in blood and urine specimens from victims of alleged sexual assault or blackout in Western Switzerland

Although much has been written on the subject of drug facilitated sexual assault (DFSA) or victim of blackout, no epidemiological study has yet been conducted in Switzerland to evaluate the occurrence of GHB and other drugs in this context. GHB is frequently mentioned on social networks and in the media as the most regularly used substance in DFSA, but often without being based on scientific studies. In order to have an assessment of the situation, political authorities asked our laboratory to systematically screen for GHB in samples obtained in case of DSFA or blackout, and driving under the influence of drugs (DUID), with the aim to assess the occurrence of voluntary or involuntary GHB use in Western Switzerland. All DFSA, blackout and DUID cases received in 2021 in our laboratory were routinely tested for the presence of GHB at non-physiological concentrations by enzymatic assay with a urine cut-off of 10 mg equivalent GHB/L. All positive cases were confirmed by gas chromatography coupled to mass spectrometry (GC-MS) using a cut-off of 6 mg/L in urine and 1.5 mg/L in whole blood. All DFSA and blackout cases were also analyzed by GC-MS, to ensure the absence of false negative cases. When requested by the justice, other substances such as ethanol, benzodiazepines, cannabinoids, cocaine, amphetamines, opioids, antidepressant, and other psychoactive substances were screening in urine and/or blood, by immunoassays, head-space gas chromatography coupled to flame ionization detector (HS-GC-FID), GC-MS, and/or liquid chromatography coupled to tandem mass spectrometry or high resolution mass spectrometry (LC-MS/MS; LC-HRMS (orbitrap)). 60 DFSA and blackout cases and 633 DUID cases were included in the study. Women predominated over men in the DFSA and blackout group (77%), while men predominated over women in the DUID group (90%). Median age in the DFSA and blackout group was 23, and median age in the DUID group was 30. The median delay to obtain urine after the event was 12 hours in case of DFSA and blackout, and 2 hours for DUID cases. The median delay to obtain whole blood after the event was 12 hours in case of DFSA and blackout, and 1.5 hours for DUID cases. The presence of GHB at concentration above the cut-off was detected in 1 DFSA and blackout case (130 mg/L in blood and 730 mg/L in urine), and in 3 DUID cases (78 mg/L, not detected, and not detected in blood, and 660 mg/L, 8.6 mg/L, and 25 mg/L in urine, respectively). These cases concerned only men. For these 4 cases, other substances were also detected. Thus, for the DFSA and blackout case, methamphetamine, methylphenidate, zolpidem and pregabaline were detected, and in the DUID cases, methamphetamine was detected in one case, ethanol, cocaine and cannabis were detected in another case, and 3-MMC was detected in the last case. When analyzed, ethanol was detected in 48% of DFSA and blackout cases, cannabinoids in 17%, benzodiazepines and Z-drugs in 10%, antidepressant in 9.8%, cocaine in 6.5%, amphetamines in 6.5%, methylphenidate in 4.8%, and opioids in 2.1%. Median concentration of ethanol after back calculation was 2.07 g/kg in DFSA and blackout cases. The results obtained, showing a very limited voluntary or involuntary use of GHB, are comparable to those obtained in other international studies. For the first time in Switzerland, a study was conducted for the evaluation of the occurrence of GHB in blood and urine specimens from victims of alleged sexual assault or blackout, and DUID cases. The results showed that GHB is very rarely consumed and used in DSFA or blackout cases, in comparison to other psychoactive drugs.

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.

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.

Gammahydroxybutyrate in hair of non-GHB and repeated GHB users: A new and optimized method

Gamma-hydroxybutyric acid (GHB) is a short-chain fatty acid used recreationally as a drug of abuse due its strong suppressive effect on the central nervous system. The detection window of GHB in blood and urine is very narrow (t1/2=30min) but can be substantially prolonged using alternative matrices such as hair. We here present a newly developed and limited validated method with a solid phase extraction (SPE) using GC–MS/MS to determine concentrations of GHB in hair samples. The soft extraction technique (water and 90min ultrasonic bath) preserves GHB with a high yield and clean extracts. In addition, endogenous GHB can be detected in hair of non-GHB users. However, little is known about GHB concentrations in hair of abstinent, frequent and chronic GHB users. Therefore, we present data from hair samples of healthy volunteers to evaluate the proposed endogenous GHB ranges, and from GHB-dependent patients to address GHB concentrations in hair with GHB intake. In 20 non-GHB users, a mean endogenous concentration of 1.1±0.6ng/mg hair (range of 0.3–2ng/mg) was found. In GHB-dependent patients, concentrations between 6.3–239.6ng/mg hair were found, with no correlation between concentrations in hair and dose of GHB intake. In summary, we present a new and limited validated method, adequately sensitive for the detection of GHB in hair, as well as first-time measurements of GHB concentrations in dependent patients in order to better understand the relationship between the frequency of use/dose and concentrations observed in hair samples.

Stability of endogenous GHB in vitreous humor vs peripheral blood in dead bodies

For the first time, the stability of GHB was tested in post-mortem peripheral blood and vitreous humor samples, collected from 22 dead bodies at two different times: at the external body examination at the place of death and then during autopsy.An ad hoc method for the detection and quantification of GHB in vitreous humor by gas chromatography coupled to mass spectrometry (GC–MS) was developed and validated, with a good linearity between 0.1 and 50μg/mL (r2=0.991) and a precision and accuracy always better than 10% and an analytical recovery higher than 90%.The geometric mean of GHB concentration in the 22 peripheral blood samples at t0 was: 3.6μg/mL (95% CI: 2.3–5.9μg/mL) and at t1 it was 7.4μg/mL (95% CI: 5.0–10.9μg/mL); that of GHB in the 22 vitreous humor at t0 was: 2.5μg/mL (95% CI: 1.5–4.1μg/mL) and at t1 it was 3.0μg/mL (95% CI: 1.9–4.8μg/mL). There was no significant difference between the GHB concentrations in vitreous humor and peripheral blood at t0 in all the samples (p>0.10). Conversely at t1, the increase of GHB in the peripheral blood was significantly increased by a 102% (range: 86–120%) (p<0.001 vs t0), while in the vitreous humor only a slight increase by 19% was observed (range: 16–21%) (p>0.05 vs t0). Finally at t1, GHB values in the two matrices were statistically different, being that of peripheral blood higher (p<0.01).This study demonstrated the usefulness of vitreous humor as a more stable alternative matrix in comparison to peripheral blood for the post-mortem determination of endogenous GHB.

Determination of GHB levels in breast milk and correlation with blood concentrations

The sodium salt of GHB or sodium oxybate is approved and registered in some countries as a therapeutic substance (Xyrem®) for the treatment of narcolepsy-associated cataplexy. This study was designed to measure the GHB endogenous levels in blood and breast milk of 20 breastfeeding women. In addition, blood and breast milk samples of a 32-year-old narcoleptic nursing mother, who was on sodium oxybate treatment, were simultaneously collected at 0.5, 1, 3, 4 and 5h following a 4.5g GHB dose and analyzed, in order to establish the safety interval of time to breastfeed. A GC–MS method for the detection and quantification of GHB in blood and breast milk was developed and fully validated. The geometric mean of endogenous GHB levels in blood and breast milk detected at time 0 were 0.57mg/L; 95% Reference Interval (RI): 0.21–1.52mg/L and 0.36mg/L; 95% RI: 0.13–1.03mg/L, respectively. The geometric mean of the concentration of GHB in milk was 37% less (95% RI: from 14 to 53%) compared to that found in the blood.The analysis of blood and breast milk samples collected from the 32 years-old female showed the following results: GHB blood concentration 0.5h after medication intake was 80.10mg/L, reaching the peak 1h after the drug administration (108.34mg/L) and it steadily decreased to reach a level of 1.75mg/L, 5h after the medication intake. The GHB concentration found in breast milk followed the same pattern as for the blood, with the highest concentration being 23.19mg/L, 1h after sodium oxybate administration and the lowest 0.99mg/L, 5h after the medication's intake. The comparison between blood and breast milk GHB levels in the 32-year-old woman, showed significant lower GHB levels in milk at 0.5, 1 and 3h, ranging from 71 to 80% less. It is interesting to note that only at 4 and 5h the difference between blood and breast milk GHB levels fell within the 95% RI (14–53%) of endogenous levels.Taking into consideration the absence of reference values for endogenous GHB in milk, we suggest the following reference interval: 0.13–1.03mg/L.We would recommend, following these preliminary data, that nursing mothers under sodium oxybate treatment should breastfeed at least 5h after the last GHB administration. However, further studies are necessary in order to confirm these findings.

Stability of -Hydroxybutyrate in Blood Samples from Impaired Drivers after Storage at 4 C and Comparison of GC-FID-GBL and LC-MS-MS Methods of Analysis

The stability of γ-hydroxybutyrate (GHB) was determined in 50 blood samples from impaired drivers after storage at 4 °: C for up to 12 months. GHB was determined in whole blood by gas chromatography-flame ionization detector (GC-FID) after conversion into γ-butyrolactone (GBL) and results were compared with LC-MS-MS. Both analytical methods showed a linear response (R(2) > 0.99) to GHB concentrations from 2 to 250 mg/kg. The mean decrease in concentration after storage was 4.8 mg/kg, with extreme changes of +13 mg/kg or -29 mg/kg. Results by the GC-FID-GBL method (y-variate) and the LC-MS-MS method (x-variate) were highly correlated (R(2) = 0.974). The regression equation was y = 0.85x + 2.2 and residual standard deviation (SD) was 7.8 mg/kg. The y-intercept (2.2 mg/kg) was not significantly different from zero (P > 0.05), although the slope of the regression line (0.85) differed from unity (P < 0.001), indicating a proportional bias of 15%. The LC-MS-MS method tended to give higher results than the GC-FID-GBL method. The mean difference (bias) was 12 mg/kg (P < 0.001). The SD of individual differences was 11.3 mg/kg and 95% limits of agreement were -11 to +33 mg/kg. The results of this study show that concentrations of GHB in whole blood are stable during storage at 4 °: C for up to 6 months.

A surrogate analyte-based LC–MS/MS method for the determination of γ-hydroxybutyrate (GHB) in human urine and variation of endogenous urinary concentrations of GHB

γ-Hydroxybutyrate (GHB) is a drug of abuse with a strong anesthetic effect; however, proving its ingestion through the quantification of GHB in biological specimens is not straightforward due to the endogenous presence of GHB in human blood, urine, saliva, etc. In the present study, a surrogate analyte approach was applied to accurate quantitative determination of GHB in human urine using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in order to overcome this issue. For this, 2H6-GHB and 13C2-dl-3-hydroxybutyrate were used as a surrogate standard and as an internal standard, respectively, and parallelism between the surrogate analyte approach and standard addition was investigated at the initial step. The validation results proved the method to be selective, accurate, and precise, with acceptable linearity within calibration ranges (0.1–1μg/ml). The limit of detection and the limit of quantification of 2H6-GHB were 0.05 and 0.1μg/ml, respectively. No significant variations were observed among urine matrices from different sources. The stability of 2H6-GHB was satisfactory under sample storage and in-process conditions. However, in vitro production of endogenous GHB was observed when the urine sample was kept under the in-process condition for 4h and under the storage conditions of 4 and −20°C. In order to facilitate the practical interpretation of urinary GHB, endogenous GHB was accurately measured in urine samples from 79 healthy volunteers using the surrogate analyte-based LC–MS/MS method developed in the present study. The unadjusted and creatinine-adjusted GHB concentrations in 74 urine samples with quantitative results ranged from 0.09 to 1.8μg/ml and from 4.5 to 530μg/mmol creatinine, respectively. No significant correlation was observed between the unadjusted and creatinine-adjusted GHB concentrations. The urinary endogenous GHB concentrations were affected by gender and age while they were not significantly influenced by habitual smoking, alcohol drinking, or caffeine-containing beverage drinking.

Pharmacokinetics of GHB and detection window in serum and urine after single uptake of a low dose of GBL – an experiment with two volunteers

During the last few years γ-hydroxybutyric acid (GHB) and γ-butyrolactone (GBL) have attracted much interest as recreational drugs and knock-out drops in drug-facilitated sexual assaults. This experiment aims at getting an insight into the pharmacokinetics of GHB after intake of GBL. Therefore Two volunteers took a single dose of 1.5 ml GBL, which had been spiked to a soft drink. Assuming that GBL was completely metabolized to GHB, the corresponding amount of GHB was 2.1 g. Blood and urine samples were collected 5 h and 24 h after ingestion, respectively. Additionally, hair samples (head hair and beard hair) were taken within four to five weeks after intake of GBL. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after protein precipitation with acetonitrile. The following observations were made: spiked to a soft drink, GBL, which tastes very bitter, formed a liquid layer at the bottom of the glass, only disappearing when stirring. Both volunteers reported weak central effects after approximately 15 min, which disappeared completely half an hour later. Maximum concentrations of GHB in serum were measured after 20 min (95 µg/ml and 106 µg/ml). Already after 4-5 h the GHB concentrations in serum decreased below 1 µg/ml. In urine maximum GHB concentrations (140 µg/ml and 120 µg/ml) were measured after 1-2 h, and decreased to less than 1 µg/ml within 8-10 h. The ratio of GHB in serum versus blood was 1.2 and 1.6.

Evaluation of the glycemic control in neonates: a novel technical approach for measuring fetal-glycated hemoglobin

and r ¼0.54, P<0.001, respectively); and (2) nearly significant differences between gestational diabetes mellitus offspring and controls (a-chain: 2.35±0.21 vs 2.20±0.18%, P ¼0.049; g-chain: 2.43±0.30 vs 2.34±0.26%, P ¼0.35), whereas in mothers, A1c was slightly different (5.8±0.4 vs 5.5±0.3%, P ¼0.004). Measurement of glycation of the a-chain only might be a suggestion, as a-chain is produced throughout pregnancy. However, despite the fact that the chemical structure of the a-chain is identical in HbA and HbF, the rate of glycation of the a-chain in HbF and HbA may be different, considering that the activity of the glycation site is influenced by its three-dimensional structure rather than by the amino-acid sequences of the polypeptide. 5 Therefore, notwithstanding the views expressed by Koga et al., 1 we have shown that the determination of GHb in cord blood is feasible using EI-TOF-MS technology, and could be used as a potential glycemic control marker in neonatal diabetes mellitus.

In vitro production of GHB in blood and serum samples under various storage conditions

The in vitro production of GHB was observed in freshly collected, untreated whole blood samples using glass BD-Vacutainers and polypropylene S-monovettes. GHB concentrations were determined daily over a period of one week and after 3, 6 and 9 weeks again. Furthermore, the GHB concentration in 40 untreated random whole blood samples stored at 4°C for a longer period of time (10 samples 12 month, 10 samples 24 month and 20 samples 36 month) was also determined. For comparison, the in vitro production of GHB in freshly collected and prepared serum samples was observed. GHB serum concentrations were determined three times over a period of one week and once again after six weeks. Sample preparation was performed by means of methanolic extraction following the precipitation of whole blood and serum samples. A methanolic standard calibration was done in a low range of 0.005-0.1 μg/mL (LOD: 0.004, LLOQ: 0.013). For quantification a spiked blood bank serum with a determined GHB concentration of 0.09 μg/mL was used. Corrected calibrations in the range of 0.09-5.09 μg/mL were used (LOD: 0.08 μg/mL, LLOQ: 0.30 μg/mL), recovery: 91.3% (high level: 4.09 μg/mL) 50.5% (low level: 0.19 μg/mL). Relevant elevation of GHB was observed in all whole blood samples stored in liquid form (4°C or room temperature). In two of the 40 whole blood samples stored over a longer period of time at 4°C, GHB concentrations in the range of 13 μg/mL were even determined. These findings constitute grounds for caution. Even a GHB cut-off level of 5 μg/mL cannot be considered as "absolutely positive" proof of a case of exogenous administration, at least in untreated liquid blood samples in long time storage. However, no significant elevations of GHB were otherwise observed in any of the serum samples independently of storage temperature nor in the whole blood samples that were frozen for storage. The results suggest that the cut-off for exogenous GHB of 5 μg/mL could be lowered significantly, with the consequence of winning valuable time for the potential victim, but only if serum is collected for GHB determination or if the whole blood sample is frozen immediately after collection and the procedure well documented.

Effects of 1,4-butanediol administration on oxidative stress in rat brain: Study of the neurotoxicity of γ-hydroxybutyric acid in vivo

gamma-Hydroxybutyric acid (GHB) is a naturally occurring compound in the central nervous system (CNS) whose tissue concentration are highly increased in the neurometabolic-inherited deficiency of succinic semialdehyde dehydrogenase (SSADH) activity or due to intoxication. SSADH deficiency is biochemically characterized by increased concentrations of GHB in tissues, cerebrospinal fluid, blood and urine of affected patients. Clinical manifestations are variable and include retardation of mental, motor, and language development along with other neurological symptoms, such as hypotonia, ataxia and seizures, whose underlying mechanisms are practically unknown. The precursor of GHB, 1,4-butanediol (1,4-BD) has been used to study the mechanisms of in vivo GHB neurotoxicity. Therefore, in the present work, the effect of acute administration of 20 or 120 mg/Kg 1,4-BD was investigated on various parameters of oxidative stress, such as spontaneous chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), total antioxidant reactivity (TAR), sulfhydryl and protein carbonyl contents, as well as the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in homogenates from cerebral cortex of 14-day-old Wistar rats. Acute administration of 120 mg/Kg 1,4-BD significantly increased spontaneous chemiluminescence and TBA-RS levels, while TAR measurement was markedly diminished, whereas injection of a lower dose (20 mg/Kg) did not change the parameters examined. Other parameters of oxidative stress evaluated were not affected by administration of 1,4-BD. These results indicate that 1,4-BD induces in vivo oxidative stress by stimulating lipid peroxidation and decreasing the non-enzymatic antioxidant defenses in cerebral cortex of young rats. If these effects also occur in humans, it is possible that they might contribute to the brain damage found in SSADH-deficient patients and possibly in individuals intoxicated by GHB or its prodrugs (gamma-butyrolactone or 1,4-BD).

Liquid Ecstasy – A Significant Drug Problem

Gamma-hydroxybutyric acid (GHB, "liquid ecstasy") and its legal pro-drugs gamma-butyrolactone and 1,4-butanediol are gaining in importance as recreational drugs in Germany. The effects of these substances are comparable with those of alcohol or benzodiazepines. Because of the wide availability of GHB physicians are increasingly being confronted with cases of intoxication. This review is based on a selective literature search as well as on the authors' own experience and on information provided by the GIZ-Nord Poisons Centre, Göttingen, Germany. Consumption of a high dose of GHB or its prodrugs leads to severe intoxication with respiratory depression and coma. Only supportive therapy can be offered; no antidote is available. In any patient with impaired consciousness of unknown cause, the possibility of intoxication with GHB must be considered. Chemical detection of GHB in blood or urine is possible only using specific analytical methods and only within a short time frame (<12 h). Because of the short half-life of GHB, intoxications treated in intensive care units rarely show any complications. However, a number of fatalities have occurred. The potential abuse of GHB as a date rape drug must be borne in mind.

Determination of γ-hydroxybutyrate (GHB) and its precursors in blood and urine samples: A salting-out approach

γ-Hydroxybutyrate (GHB) is an increasingly popular drug of abuse that causes stimulation, euphoria, anxiolysis or hypnosis, depending on the dose used. Low doses of the drug are used recreationally, and also implicated in drug-facilitated sexual assaults. Because of the unusually steep dose–response curves, accidental GHB overdosing, leading to coma, seizures or death can occur. Being a controlled substance, GHB is often substituted with its non-scheduled precursors γ-butyrolactone (GBL) and 1,4-butanediol (BD), which are rapidly metabolized into GHB in the body. Here we describe an assay for GHB, GBL and BD in blood and/or urine samples. GHB and BD were extracted from diluted 200 μL aliquots of samples with t-butylmethylether (plus internal standard benzyl alcohol) in test tubes preloaded with NaCl. After acidification and centrifugation the solvent phase was transferred to a test tube preloaded with Na 2SO 4, incubated for 30 min, centrifuged again, and evaporated in vacuum. The residue was mixed with N-methyl- N-trimethylsilyl-trifluoroacetamide (MSTFA) in acetonitrile, and injected into a GC–MS. When analyzing GBL, the salting-out step was omitted, and analysis was performed with a GC–FID apparatus. As revealed by the validation data this procedure is suitable for quantitative determination of GHB and its precursors in blood and/or urine samples.

Endogenous Level and in Vitro Production of GHB in Blood from Healthy Humans, and the Interpretation of GHB Levels Detected in Antemortem Blood Samples

The endogenous level of γ-hydroxybutyric acid (GHB) and the in vitro production of GHB in blood from healthy humans have been investigated. The endogenous GHB concentrations in aseptically collected whole blood samples ranged from 5 to 10 ng/ml, which were far below the previously-reported “endogenous” levels . Also, the levels of in vitro GHB production during storage for 16 months at 4°C were lower than 0.4 μg/ml, which were much lower than those in postmortem samples previously reported. Based on the results of this investigation, the authors concluded that an interpretative cutoff of 1.0 μg/ml would be appropriate for differentiating exogenous from endogenous GHB, if only limited to in-life blood specimens that were collected aseptically, stored at 4°C or lower, examined within two weeks, and excludes the possibility of GHB aciduria.

γ-Hydroxybutyrate Concentrations in Pre- and Postmortem Blood and Urine

With γ-hydroxybutyrate (GHB) becoming popular as a drug of abuse in the US and elsewhere (1), we are receiving increasing requests for the analytical determination of GHB in blood or urine in criminal investigations, especially in sexual assault cases. In a recent report of a fatal poisoning with GHB, the victim had a postmortem blood GHB concentration of 27 mg/L (2), and another three GHB-related fatalities were reported with postmortem …