ChemInform Abstract: Screening and Confirmation Methods for GHB Determination in Biological Fluids
Abstract
The purpose of this review is to provide a comprehensive overview of reported methods for screening and confirmation of the low-molecular-weight compound and drug of abuse gamma-hydroxybutyric acid (GHB) in biological fluids. The polarity of the compound, its endogenous presence, its rapid metabolism after ingestion, and its instability during storage (de novo formation and interconversion between GHB and its lactone form gamma-butyrolactone) are challenges for the analyst and for interpretation of a positive result. First, possible screening procedures for GHB are discussed, including colorimetric, enzymatic, and chromatography-based procedures. Confirmation methods for clinical and forensic cases mostly involve gas chromatography (coupled to mass spectrometry), although liquid chromatography and capillary zone electrophoresis have also been used. Before injection, sample-preparation techniques include (a combination of) liquid-liquid, solid-phase, or headspace extraction, and chemical modification of the polar compound. Also simple "dilute-and-shoot" may be sufficient for urine or serum. Advantages, limitations, and trends are discussed.
... Studies are currently underway to identify markers and metabolites that extend the detection window or detect exposure in various matrices. Although phase I and II metabolites do not appear to contribute, GHB-derived acids, such as glycolic acid and 2,4 or 3,4dihydroxybutyric acids, may be used to aid the postmortem interpretation in cases of intoxication ( ). -Formation of GHB/GBL due to de novo synthesis (especially postmortem) and interconversion (Ingels et al., 2014). Therefore, the storage conditions are decisive (Ingels et al., 2014). ...
... Although phase I and II metabolites do not appear to contribute, GHB-derived acids, such as glycolic acid and 2,4 or 3,4dihydroxybutyric acids, may be used to aid the postmortem interpretation in cases of intoxication ( ). -Formation of GHB/GBL due to de novo synthesis (especially postmortem) and interconversion (Ingels et al., 2014). Therefore, the storage conditions are decisive (Ingels et al., 2014). The samples should be stored at − 20 • C if analysis is not immediately performed (Ingels et al., 2014;Busardò et al., , 2017aThai et al., 2006;Gallart-Mateu et al., 2021). ...
... Therefore, the storage conditions are decisive (Ingels et al., 2014). The samples should be stored at − 20 • C if analysis is not immediately performed (Ingels et al., 2014;Busardò et al., , 2017aThai et al., 2006;Gallart-Mateu et al., 2021). The preservatives used also impact the stability of the samples (Ingels et al., 2014;Kugelberg et al., 2010;Jarsiah et al., 2020b;Busardò et al., 2014;Kietzerow et al., 2020;Busardò and Jones, 2019). ...
γ-hydroxybutyrate (GHB) is synthesized endogenously from γ-aminobutyric acid (GABA) or exogenously from 1,4-butanediol (butane-1,4-diol; 1,4-BD) or γ-butyrolactone (GBL). GBL, and 1,4-BD are rapidly converted to GHB. The gastric absorption time, volume of distribution, and half-life of GHB are between 5 and 45 min, 0.49 ± 0.9 L/kg, and between 20 and 60 min, respectively. GHB and its analogues have a dose-dependent effect on the activation of GHB receptor, GABA-B, and GABA localized to the central nervous system. After ingestion, most patients present transient neurological disorders (lethal dose: 60 mg/kg). Chronic GHB consumption is associated with disorders of use and a withdrawal syndrome when the consumption is discontinued. GHB, GBL, and 1,4-BD are classified as narcotics but only the use of GHB is controlled internationally. They are used for drug facilitated (sexual) assault, recreational purposes, slamsex, and chemsex. To confirm an exogenous intake or administration of GHB, GBL, or 1-4-BD, the pre-analytical conservation is crucial. The antemortem cutoff doses for detection are 5 and 5-15 mg/L, with detection windows of 6 and 10 h in the blood and urine, respectively Control of GHB is essential to limit the number of users, abuse, associated risks, and death related to their consumption.
... Sur un plan médical et médico-légal, il est évident que ce panorama de substances dessine un profil de pratiquant de chemsex.23,24]. Le GHB est cependant connu pour son instabilité in vitro. ...
... Exogenous GHB was initially used as an anaesthetic before being abandoned due to side effects that include myoclonus, unpredictable periods of apnoea, and emergence delirium. Currently, the sodium salt of GHB is licensed in only a few countries for either the treatment of narcolepsy or alcohol withdrawal [1,2]. In Australia it is a restricted substance with no listed therapeutic use, but is used illicitly with the target effects including euphoria, disinhibition, relaxation, and sexual arousal. ...
Background:
Gamma-hydroxybutyrate is a potent central nervous system depressant with a narrow recreational dose window and analytical detection time. We describe data relating to intoxicated patients presenting to emergency departments across metropolitan Adelaide who tested positive for gamma-hydroxybutyrate. This work was part of the Emergency Department Admission Blood Psychoactive Testing study.
Methods:
Over a 15-month period, patients presenting to four metropolitan emergency departments with symptoms of drug intoxication were enrolled in the study. The methodology involved the collection of demographic and clinical data and a de-identified blood sample which underwent comprehensive toxicological analysis. Gamma-hydroxybutyrate was determined using an acid-catalysed cyclisation followed by liquid-liquid extraction and gas chromatography-mass spectrometry. Data relating to samples positive for gamma-hydroxybutyrate were examined.
Results and discussion:
A total of 1120 patients were enrolled between March 2019 and May 2020, 309 of whom were positive for gamma-hydroxybutyrate (27.6%). Of these, 256 (83%) were also positive for metamfetamine (methamphetamine). The most common clinical observation in gamma-hydroxybutyrate-positive patients was central nervous system depression (89%). There was a significant relationship between gamma-hydroxybutyrate status and sex; although males outnumbered females in absolute terms, a higher proportion of females (32%) tested positive for gamma-hydroxybutyrate than males (25%, P = 0.0155). Blood gamma-hydroxybutyrate concentrations ranged from 10 to 651 mg/L (0.096-6.2 mmol/L) and increasing gamma-hydroxybutyrate concentration correlated with severe toxicity. The presence of gamma-hydroxybutyrate had a significant impact on the patient discharge destination: the majority (69.2%) of gamma-hydroxybutyrate-positive patients were managed and discharged from the emergency department or their attached short stay wards. A significantly higher proportion of gamma-hydroxybutyrate-positive patients were admitted to the intensive care unit (28.2%) compared with gamma-hydroxybutyrate-negative patients (12.7%, chi-squared = 36.85, P <0 .001). Gamma-hydroxybutyrate positive cases accounted for 45.8% of all study-related intensive care unit admissions.
Conclusions:
Gamma-hydroxybutyrate is commonly detected in illicit drug-related emergency department presentations and is detected disproportionately in the patient cohort who require intensive care unit level care.
... A rapid elimination, short detection times in common matrices and endogenous presence are some of the characteristics making GHB an analytical challenge (17). In addition, when using LC-MS, GHB needs to be analyzed in a negative ion mode. ...
Gamma-hydroxybutyrate (GHB) is a central nervous system depressant that has gained popularity as an illicit recreational drug. We describe a case of an elderly woman who was found unconscious in her home. The paramedics initially suspected an intracranial incident. A head computed tomography (CT) was negative, as was the initial urinary drug screening. The diagnosis of GHB intoxication was made with detection of GHB in a urine sample obtained 28-29 hours after assumed time of intake. Our case underscores the importance of considering drug testing in a broad range of patients, and shows that elderly patients may have an extended detection window of GHB.
Prodrugs are an important strategy for the development and design of therapeutic drugs. They involve the addition of an easily removable group (such as an ester) to a molecule in order to chemically protect it from metabolism, hydrolysis, or interaction with tissues. In the therapeutic drug market, prodrugs may reduce adverse effects, lead to more accurate tissue delivery, increase bioavailability and duration of effects. The same strategy has, however, been used in the illicit drug market. Internationally controlled drugs such as LSD, MDMA and benzodiazepines have been detected in the market with the addition of alkyl chain that can be removed in physiological conditions. Cyclical analogues may also be used, as is the case for 1cP‐LSD and GBL (a prodrug of GHB). Adequate detection requires increased care with extraction and analytical conditions—as the parent drugs may be formed as artifacts during these processes—and an in‐depth knowledge of metabolic pathways to avoid erroneous detection and allow for adequate identification of the drug used. Legislative updates may also be necessary to ensure prodrugs can be controlled at the same level as other illicit drugs.
This article is categorized under: Toxicology > New Psychoactive Substances
Toxicology > Drug Analysis
Toxicology > Analytical
Gamma-hydroxybutyric acid (GHB) represents an important drug in clinical and forensic toxicology, particularly in the context of drug-facilitated crimes. Analytically, GHB remains a major challenge given its endogenous occurrence and short detection window. Previous studies identified a number of potential interesting novel conjugates of GHB with carnitine, amino acids (AA, glutamate, glycine, and taurine) or fatty acids. As a basis for comprehensive studies on the suitability of these novel biomarkers, we developed and validated a liquid chromatography - tandem mass spectrometry (LC-MS/MS) method in human urine. Additionally, already knowns markers 2,4-dihydroxy butyric acid (2,4-DHB), 3,4-DHB, glycolic acid, succinic acid, succinylcarnitine, and GHB glucuronide were included. The method was fully validated according to (inter)national guidelines. Synthetic urine proved suitable as a surrogate matrix for calibration. Matrix effects were observed for all analytes with suppression effects of about 50% at QC LOW, and approximately 20 to 40% at QC HIGH, but with consistent standard deviation of < 25% at QC LOW and <15% at QC HIGH, respectively. All analytes showed acceptable intra- and inter-day imprecision of below 20%, except for inter-day variation of GHB taurine and FA conjugates at the lowest QC. Preliminary applicability studies proved the usefulness of the method and pointed towards GHB glycine, followed by other AA conjugates as the most promising candidates to improve GHB detection. FA conjugates were not detected in urine samples yet. The method can be used now for comprehensive sample analysis on (controlled) GHB administration to prove the usefulness of the novel GHB biomarkers.
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 endog- enous GHB, if only limited to in-life blood specimens that were collected aseptically, stored at 4°C or lower, exam- ined within two weeks, and excludes the possibility of GHB aciduria.
Study Objective: We describe seven patients presenting with combination substance abuse involving γ-Hydroxybutyric acid (GHB). Methods: During a 3-month period, we identified consecutive patients with GHB ingestion confirmed by urine mass spectrometry presenting to a high-volume urban emergency department. Results: All patients presented with acute delirium and transient but severe respiratory depression. With supportive care, including intubation and mechanical ventilation in four cases, normal mentation and respiratory function returned within 2 to 6 hours. None of these patients had documented seizures, and none of the four patients who received naloxone had a reversal response. This clinical observation supports previous experimental work in GHB-intoxicated human subjects demonstrating neither epileptiform changes on electroencephalography nor reversal with naloxone. Two findings are remarkable in this series. The first is the observation of a peculiar state of violent aggression present on stimulation of the GHB-intoxicated patient despite near or total apnea. The fact that patients fully recovered from this state may be the result of a previously demonstrated GHB hypoxia-sparing effect. The second is the observation of ECG abnormalities in several cases, including U waves in five patients. Conclusion: Emergency physicians should be alerted to this agent, its characteristic effects, and its potential for serious sequelae including respiratory arrest and death. [Li J, Stokes SA, Woeckener A: A tale of novel intoxication: Seven cases of γ-Hydroxybutyric acid overdose. Ann Emerg Med June 1998;31:723-728.]
In this work, the interconversion of GHB and GBL in a variety of aqueous media was studied. The effects of solution pH and time were determined by spiking GHB or GBL into pure water and buffered aqueous solutions, and determining the GHB and GBL contents at various time intervals. The degree of GBL hydrolysis to GHB was determined for several commercial aqueous-based GBL products, and further studied as a function of time. The effects of temperature and time were also determined for five commercial beverages spiked with GHB or GBL. GHB and GBL contents were determined using high performance liquid chromatography (HPLC). GHB and/or GBL confirmations were made using gas chromatography-mass spectrometry (GC-MS) and/or infrared spectroscopy (IR).
Solution pH, time, and storage temperature were determined to be important factors affecting the rate and extent of GBL hydrolysis to GHB. Under strongly alkaline conditions (pH 12.0), GBL was completely converted to GHB within minutes. In pure water, GBL reacted to form an equilibrium mixture comprising ca. 2:1 GBL:GHB over a period of months. This same equilibrium mixture was established from either GHB or GBL in strongly acidic solution (pH 2.0) within days. A substantial portion of GBL (ca. ⅓) was hydrolyzed to GHB in aqueous-based GBL products, and in spiked commercial beverages, after ambient storage for a period ranging from several weeks to several months. Heat increased and refrigeration decreased the rate of GBL hydrolysis relative to ambient conditions. These studies show that hydrolysis of GBL to GHB does occur in aqueous-based solutions, with samples and time frames that are relevant to forensic testing. Implications for forensic testing and recommendations are discussed.
In this work, the interconversion of GHB and GBL in a variety of aqueous media was studied, The effects of solution pH and time were determined by spiking GHB or GBL into pure water and buffered aqueous solutions, and determining the GHB and GBL contents at various time intervals. The degree of GBL hydrolysis to GHB was determined for several commercial aqueous-based GBL products, and further studied as a function of time. The effects of temperature and time were also determined for five commercial beverages spiked with GHB or GBL. GHB and GBL contents were determined using high performance liquid chromatography (HPLC). GHB and/or GBL confirmations were made using gas chromatography-mass spectrometry (GC-MS) and/or infrared Spectroscopy (IR). Solution pH, time, and storage temperature were determined to be important factors affecting the rate and extent of GBL hydrolysis to GHB. Under strongly alkaline conditions (pH 12.0), GBL was completely converted to GHB within minutes. In pure water, GBL reacted to form an equilibrium mixture comprising ca. 2:1 GBL:GHB over a period of months. This same equilibrium mixture was established from either GHB or GBL in strongly acidic solution (pH 2.0) within days. A substantial portion of GBL (ca. 1/3) was hydrolyzed to GHB in aqueous-based GBL products, and in spiked commercial beverages, after ambient storage for a period ranging from several weeks to several months. Heat increased and refrigeration decreased the rate of GBL hydrolysis relative to ambient conditions. These studies show that hydrolysis of GBL to GHB does occur in aqueous-based solutions, with samples and time frames that are relevant to forensic testing. Implications for forensic testing and recommendations are discussed.
Laboratory detection of γ-hydroxybutyrate (GHB) has been published as early as the 1960s. However, wide-scale use of GHB during the 1990s has led to the development of current analytic methods to test for GHB and related compounds. Detection of GHB and related compounds can be clinically useful in confirming the cause of coma in an overdose patient, determining its potential role in a postmortem victim, as well as evaluating its use in a drug-facilitated sexual assault victim. Analytical method sensitivity must be known in order to determine the usefulness and clinical application. Most laboratory cut-off levels are based on instrument sensitivity and will not establish endogenous versus exogenous GHB levels. Interpretation of GHB levels must include a knowledge base of endogenous GHB, metabolism of GHB and related compounds, as well as postmortem generation. Due to potential analytical limitations in various GHB methods, it is clinically relevant to specifically request for GHB as well as related GHB compounds if they are also in question. Various storage conditions (collection time, types of containers, use of preservatives, storage temperature) can also affect the analysis and interpretation of GHB and related compounds.
Objective:
SSADH deficiency, the most prevalent autosomal recessive disorder of GABA degradation, is characterized by elevated gamma-hydroxybutyric acid (GHB). Neurological outcomes may be improved with early intervention and anticipatory guidance. Morbidity has been compounded by complications, e.g. hypotonia, in undiagnosed infants with otherwise routine childhood illnesses. We report pilot methodology on the feasibility of newborn screening for SSADH deficiency.
Method:
Dried blood spot (DBS) cards from patients affected with SSADH deficiency were compared with 2831 archival DBS cards for gamma-hydroxybutyric acid content. Following extraction with methanol, GHB in DBS was separated and analyzed using ultra high-performance liquid chromatography tandem mass spectrometry.
Results:
Methodology was validated to meet satisfactory accuracy and reproducibility criteria, including intra-day and inter-day validation. Archival refrigerated dried blood spot samples of babies, infants and children (N = 2831) were screened for GHB, yielding a mean +/- S.D. of 8 ± 5 nM (99.9%-tile 63 nM) (Min 0.0 Max 78 nM). The measured mean and median concentrations in blood spots derived from seven SSADH deficient patients were 1182 nM and 699 nM respectively (Min 124, Max 4851 nM).
Conclusions:
GHB concentration in all 2831 dried blood spot cards was well below the lowest concentration of affected children. These data provide proof-of-principle for screening methodology to detect SSADH deficiency with applicability to newborn screening and earlier diagnosis.
To avoid the detection of small fragmentation products of γ-hydroxybutyrate (GHB), a liquid chromatography-tandem mass spectrometry GHB quantification method in human serum supported by adduct formation was developed and validated. The continuous infusion of GHB/GHB-D6 made the identification of two adducts possible and GHB/GHB-D6 sodium acetate adduct fragmentation was used as target mass transition. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column and elution with a programmed flow of the mobile phase consisting of 10 % A (H2O/methanol = 95/5, v/v) and 90 % B (H2O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and 0.1 % acetic acid (pH = 3.2), were used. Protein precipitation with 1 mL of the mobile phase B was used as the sample preparation. The calculated limit of detection/quantification was 1 μg/mL. The presented study shows that the fragmentation of GHB sodium acetate adducts is an effective way of quantification of this small molecule and is an interesting alternative to other methods based on the detection of ions smaller than 85 Da. This fact together with the short analysis time of 3 min and the fast sample preparation make this method very attractive for forensic/clinical application.