Report on the analysis of common beverages spiked with gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE solvent-suppression technique

In forensic evidence, the identification and quantitation of gamma-hydroxybutyric acid (GHB) in "spiked" beverages is challenging. In this report, we present the analysis of common alcoholic beverages found in clubs and bars spiked with gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL). Our analysis of the spiked beverages consisted of using (1)H NMR with a water suppression method called Presaturation Utilizing Relaxation Gradients and Echoes (PURGE). The following beverages were analyzed: water, 10% ethanol in water, vodka-cranberry juice, rum and coke, gin and tonic, whisky and diet coke, white wine, red wine, and beer. The PURGE method allowed for the direct identification and quantitation of both compounds in all beverages except red and white wine where small interferences prevented accurate quantitation. The NMR method presented in this paper utilizes PURGE water suppression. Thanks to the use of a capillary internal standard, the method is fast, non-destructive, sensitive and requires no sample preparation which could disrupt the equilibrium between GHB and GBL.     

The reactivity of gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL) in alcoholic solutions

In this work the stability of GBL (gamma-butyrolactone) and GHB (gamma-hydroxybutyric acid) in alcoholic media was studied. Under acidic conditions the GBL will react with ethanol or methanol to give the corresponding ethyl and methyl esters of GHB. It can be seen that ester formation is dependent on the type of alcohol, the alcohol content of the solution, and the pH of the solution. Under the same conditions it was shown that GHB does not give rise directly to the corresponding ester when merely in the presence of an alcohol; however the ester will be formed if the conditions are present for conversion of GHB to GBL followed by subsequent reaction with alcohol. In alcoholic beverage samples spiked with GBL the expected conversion to GHB occurred, and the formation of the ethyl ester of GHB was also seen in some samples. Wine samples were analyzed for the presence of the ethyl ester of GHB, and the effect of adding GHB/GBL to hot beverages was studied.     

化学显色法快速筛选饮料及尿液中γ-羟基丁酸和γ-丁内酯

目的建立化学显色法快速筛选饮料及尿液中γ-羟基丁酸(GHB)及其前体γ-丁内酯(GBL)的方法。方法在酸性条件下GHB转化为GBL,GBL和盐酸羟胺在碱性条件下生成γ-羟基丁酰羟胺,γ-羟基丁酰羟胺在酸性条件下和三氯化铁反应,生成紫红色的络合物。结果饮料中GHB最低检出浓度为0.5～2mg/mL,低于常见滥用质量浓度。该方法也可以用于尿液分析,最低检出质量浓度为0.5mg/mL。考察了常见有机溶剂和麻醉镇静药物的干扰。结论该方法简单、安全、快速,为临床和法庭科学实验室快速筛选GHB和GBL提供了便利。     

The chemical interconversion of GHB and GBL: forensic issues and implications.

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.     

Analysis of gamma-hydroxybutyric acid (GHB) in spiked water and beverage samples using solid phase microextraction (SPME) on fiber derivatization/gas chromatography-mass spectrometry (GC/MS)

Gamma-Hydroxybutyric acid (GHB) is a CNS depressant that has been abused recreationally for its purported euphoric and relaxation effects and for the purposes of drug facilitated sexual assault due to its sedative and amnesic effects at higher doses. The dramatic increase in the abuse of GHB and association in criminal investigations over the past decade has created the need for forensic laboratories to develop analytical methods to detect GHB in a variety of matrices. The method developed in this work used solid-phase microextraction (SPME) to extract GHB from aqueous samples followed by on-fiber derivatization and analysis by gas chromatography/mass spectrometry (GC/MS). This method detected GHB in aqueous matrices with good sensitivity, high precision, excellent linearity from 0.01 mg/mL to 0.25 mg/mL, and without the need for sample manipulation that could cause interconversion between GHB and its lactone, GBL. The method was successfully applied for detection of GHB in spiked water and beverage samples.     

A hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry method for the simultaneous determination of γ-hydroxybutyrate and its precursors in forensic whole blood

A liquid-chromatography-tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of γ-hydroxybutyric acid (GHB), γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in human ante-mortem and post-mortem whole blood. The blood proteins were precipitated using a mixture of methanol and acetonitrile, and the extract was cleaned-up by passage through a polymeric strong cation exchange sorbent. Separation of the analytes and their structural isomers was obtained using a column with a zwitterionic stationary phase. Matrix-matched calibrants, combined with isotope dilution, were used for quantitative analysis. GHB was determined in both positive and negative ion modes. The relative intra-laboratory reproducibility standard deviations were better than 10% and 6% for blood samples at concentrations of 2mg/L and 20-150mg/L, respectively. The mean true extraction recoveries were 80% for GHB and greater than 90% for GBL and 1,4-BD at concentration levels of 20-50mg/L. The limits of detection were approximately 0.5mg/L for GHB and GBL, and 0.02mg/L for 1,4-BD in ante-mortem blood. The corresponding lower limits of quantification were less than 1mg/L for GHB and GBL, and less than 0.1mg/L for 1,4-BD. GBL was unstable in whole blood freshly preserved with a sodium fluoride oxalate mixture, but the stability could be improved significantly by preservation with a sodium fluoride citrate EDTA mixture.     

GHB free acid: I. Solution formation studies and spectroscopic characterization by 1HNMR and FT-IR

In forensic evidence, gamma-hydroxybutyric acid (GHB) has frequently been encountered in one of its salt forms (gamma-hydroxybutyrate), but has also been encountered in its free acid form (GHB). Owing to the physical properties, encounters of the free acid have been largely restricted to forensic exhibits comprising aqueous solutions, such as acidic beverages that have been "spiked" or formulated with GHB salts or gamma-butyrolactone (GBL). The analysis of GHB free acid presents particular difficulties including the potential for altering the original proportions of GHB free acid, GHB carboxylate, and GBL in the course of analysis, and discrimination between GHB free acid and carboxylate forms. In this work, the formation of GHB free acid in aqueous solutions (water and/or D2O) was studied as a function of solution pH. Proton nuclear magnetic resonance (1HNMR) and Fourier-transform infrared spectrometry (FT-IR) measurements were obtained on freshly prepared mixtures of NaGHB and HCl stock solutions representing a series of points along the GHB titration curve. Both 1HNMR and FT-IR were shown to track the changing proportions of GHB free acid and carboxylate forms as a function of pH, while simultaneously monitoring for the formation of the lactone (GBL). The results were consistent with acid-base conversion behavior for a carboxylic acid. 1HNMR was shown to provide an ideal means for analysis of aqueous-based GHB/GBL forensic exhibits based on simple dilution of the neat liquid exhibit, without altering the original proportions of GHB free acid, carboxylate, and GBL in the samples.     

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

Gamma-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 gamma-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 microL 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(2)SO(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.     

1H NMR analysis of GHB and GBL: further findings on the interconversion and a preliminary report on the analysis of GHB in serum and urine

A 1H nuclear magnetic resonance (1H NMR) method for the determination of gamma-hydroxybutyric acid (GHB) and gamma-hydroxybutyrolactone (GBL) in human serum and urine using spiked samples has been developed. The method gives linear responses (correlation coefficients of 0.99 or greater) over the concentration range 0.01 mg/mL to 4.0 mg/mL in urine and 0.3 mg/mL to 2.0 mg/mL in serum. No sample pretreatment is required. Studies of the chemical interconversion of GBL and GHB showed hydrolysis of GBL to be rapid at pH 11.54, slower and less complete (30% hydrolysis) at pH 2.54 and slowest at pH 7.0, reaching 30% hydrolysis in about 40 days. No esterification of GHB was observed at any pH.     

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 Na₂SO₄, 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.     

Identification and quantitation of gamma-hydroxybutyrate (NaGHB) by nuclear magnetic resonance spectroscopy

The most common means of identification of gamma-hydroxybutyrate (NaGHB) involves using Fourier transform infrared spectroscopy (FTIR) or gas chromatography-mass spectrometry (GC-MS) of a suitable derivative. However, these methods may be complicated by possible shifts in chemical equilibrium between gamma-hydroxybutyric acid (GHB), GHB salts and the precursor lactone, gamma-butyrolactone (GBL). This paper addresses the technique of proton and carbon nuclear magnetic resonance spectroscopy (1H and 13C NMR) for the direct and accurate identification of GHB and GBL. The application of 1H NMR for GHB quantitation is also discussed.     

Gamma hydroxybutyric acid (GHB) concentrations in humans and factors affecting endogenous production

The endogenous nature of the drug of abuse gamma hydroxybutyric acid (GHB) has caused various interpretative problems for toxicologists. In order to obtain data for the presence of endogenous GHB in humans and to investigate any factors that may affect this, a volunteer study was undertaken. The GHB concentrations in 119 urine specimens from GHB-free subjects and 25 urine specimens submitted for toxicological analysis showed maximal urinary GHB concentrations of 3mg/l. Analysis of 15 plasma specimens submitted for toxicological analysis detected no measurable GHB (less than 2.5mg/l). Studies in a male and female volunteer in which different dietary food groups were ingested at weekly intervals, showed significant creatinine-independent intra-individual fluctuation with overall urine GHB concentrations between 0 and 2.55, and 0 and 2.74mg/l, respectively. Urinary concentrations did not appear to be affected by the particular dietary groups studied.The concentrations measured by gas chromatography with flame ionisation detection (GC-FID) and gas chromatography with mass spectrometry (GC-MS) lend further support to the proposed urinary and plasma interpretative cut-offs of 10 and 4mg/l, respectively, where below this it is not possible to determine whether any GHB detected is endogenous or exogenous in nature.     

GHB free acid: II. Isolation and spectroscopic characterization for forensic analysis

A reference standard for gamma-hydroxybutyric acid (GHB) free acid is not commercially available, making its analysis in forensic exhibits more difficult. GHB free acid is typically encountered in aqueous solution and in the presence of the lactone, gamma-butyrolactone (GBL), presenting difficulty in Fourier transform infrared (FT-IR) analysis. The strong infrared (IR) absorptivity of the GBL carbonyl band, the shifting of the GBL carbonyl band in aqueous solutions, and the position of the O-H bend for water can mask the main carbonyl band for GHB free acid. Model solutions of beta-hydroxybutyric acid (BHB) and GBL were studied in order to further understand the masking of the GHB free acid carbonyl band in FT-IR analysis. The use of second derivative FT-IR spectroscopy was shown to provide resolution of the free acid carbonyl band, and a presumptive test for GHB free acid was developed and applied. An extension of this work included preparing, for use as a standard reference material, small amounts (< or = 10 mg) of GHB free acid. Preparation was based on the instantaneous reaction of GHB's sodium salt with a stoichiometric amount of hydrochloric acid in aqueous solution, and subsequent isolation of the free acid in neat liquid form. Both FT-IR and proton nuclear magnetic resonance spectra of the neat reference material were obtained and used to verify its identity. The isolation of GHB free acid from actual forensic exhibits is also presented, with identity confirmation using FT-IR.     

Detection of gamma-hydroxybutyrate (GHB) in beverages

OBJECTIVE: To establish an analytical method for the determination of GHB in beverages using GC/MS and LC/MS/MS. METHODS: After beverage samples with GHB-d6 as the internal standard were extracted with ethyl acetate, then the extracts were derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), at last the derivateized extracts analyzed by gas chromatography- mass spectrometry. After beverage samples with GHB-d6 as the internal standard were diluted by mobile phase then directly analyzed by LC/MS/MS. Results The limit of detection was 0.2 microg/mL and both relative standard deviations for between-day and within-day assays were < 8.54% in GC/MS. The limit of detection was 2 microg/mL and both relative standard deviations for between-day and within-day assays were <8.62% in LC/MS/MS. Conclusion These methods of qualitative and quantitative analysis were found to be sensitive, accurate, rapid and suitable for the forensic toxicology to test of GHB in real cases.     

Enzymatic detection of γ-hydroxybutyrate using aldo-keto reductase 7A2

Gamma-hydroxybutyrate (GHB) is a prescribed medication as well as a drug of abuse. Its detection in various matrices for in-field forensic scientists remains a challenge. We have developed an assay that uses aldo-keto reductase 7A2 (AKR7A2) for the specific determination of GHB in various drinks. AKR7A2 was purified using Ni-affinity chromatography. The Michaelis-Menten constant for the GHB oxidation reaction was 10 mM, and the minimum detection limit was 4 mM. Ethanol was not a substrate for AKR7A2. In a coupled reaction with NADP(+), phenazine methosulfate (PMS), and 2,6-dichlorophenolindophenol, various beverages (orange juice, milk, soda, and numerous alcoholic drinks) containing GHB turned from blue to light yellow. In a second coupled reaction where diaphorase replaced PMS, the presence of GHB also caused the expected change of color in various beers.     

Spontaneous formation of γ-hydroxybutyric acid from γ-butyrolactone in tap water solutions

The spontaneous conversion of γ-butyrolactone (GBL) to γ-hydroxybutyric acid (GHB) in seven different Swedish tap waters was investigated. The waters used in the study were selected to represent the diversity among Swedish tap waters as well as possible, which was enabled by principal component analysis (PCA) of a number of water quality parameters. GBL solutions (5, 25 and 50% v/v) were prepared in each of the tap waters and in deionized water and the formation of GHB was followed over time. The GHB quantifications were made using a CZE method, employing a carrier electrolyte consisting of 25mM benzoic acid, 54mM tris(hydroxymethyl)aminomethane (Tris) and 1.7mM tetradecyltrimethylammonium bromide (TTAB), which was developed as a part of the current study. Data evaluation showed that the formation of GHB was largely dependent on the type of tap water. For example, there was a negative correlation between the kinetics of the GHB formation and the alkalinity of the tap waters (r(2)=0.990). This could be explained by a faster decrease in pH in the waters with low buffering capacity (i.e. low alkalinity), which catalysed the hydrolysis of GBL. Equilibrium was reached after 40-250 days depending on the initial GBL concentration and the type of tap water. The level of the equilibrium appeared to be dependent on the initial GBL concentration and ranged from 26 to 37%. Gained knowledge on the levels of the GHB/GBL equilibrium and the kinetics of the formation of GHB in tap water solutions of GBL, including the influence of the tap water quality, may be useful information for casework with the GHB/GBL problem in focus.     

Enhancement of microcrystalline identification of gamma-hydroxybutyrate

An enhancement of the microcrystalline test for the detection of gamma-hydroxybutyrate (GHB) is described. The original test used a silver/copper reagent which consisted of 0.1 g of silver nitrate and 0.1 g of copper nitrate in 10 mL water. The enhanced test utilizes lanthanum nitrate in place of copper nitrate. A detection limit of 0.5 mg/mL was achieved and the visual discrimination was improved because of larger sized crystals. Transient crystals were observed between 0.1 and 0.4 mg/mL. Silver nitrate alone appeared to be suitable for GHB detection but was not specific as other hydroxyl acids, such as glycolic acid, produced a similar crystal pattern. Tests conducted on chemical precursors of GHB and substances with similar biological activity highlight the specificity of the enhanced test. The reagent is therefore selective and sensitive for GHB in aqueous solutions. However, in beverage testing, crystal formation appeared to be inhibited for some drinks. Citric acid was identified as a possible interference depending on its concentration relative to GHB.     

尿液、血液中γ-羟丁酸的气质联用法分析

目的为尿液、血液中γ-羟丁酸(gamma-hydroxybutyricacid,GHB),γ-羟丁酸内酯(gamma-butyrolactone,GBL)和1,4-丁二醇(1,4-butanediol,1,4-BD)的鉴定提供方法和依据。方法100μl尿液或血液以GHBd6为内标,经乙酸乙酯提取、BSTFA衍生化后,用GC／MS法分析。结果测尿液中内源性GHB的线性范围是20-800ng／ml,R2=0.9995,最低检出限为10ng／ml(S／N≥3);测尿液、血液中外源性GHB的线性范围为5-60μg／ml,R2分别为0.9999和0.9928。相对回收率为99％-104％。以所建方法测定了健康志愿者尿液中内源性GHB含量,并考察了健康受试者外源性GHB的代谢情况。结论所建方法准确、便捷、省时、选择性好,适用于法医毒物学鉴定。     

Simultaneous analysis of gamma-hydroxybutyric acid, gamma-butyrolactone, and 1,4-butanediol by micellar electrokinetic chromatography

A micellar electrokinetic chromatography (MEKC) method was optimised for simultaneous analysis of gamma-hydroxybutyric acid (GHB), gamma-butyrolactone (GBL), and 1,4-butanediol (BD). Best conditions for separation and baseline stability were achieved using a carrier electrolyte comprising 30.0mM sodium barbital and 150.0mM sodium dodecyl sulphate (SDS) at pH 10.2. Calibration functions were linear, giving correlation coefficients (r(2)) >0.998 for the three target compounds. Limits of detection (LOD) defined as three times the noise, were 5.1mg/l, 0.34 and 0.25g/l for GHB, GBL and BD, respectively. The repeatability of migration times and peak areas, expressed as the R.S.D. (n = 9) was better than 0.41 and 3.05%, respectively. Some casework samples were analysed using the optimised conditions.     

GHB and driving impairment.

Gamma hydroxybutyrate (GHB) was identified in the blood of 13 subjects arrested for impaired driving. GHB concentrations ranged from 26 to 155 mg/L (mean 87 mg/L, median 95 mg/L). In eight cases, GHB was the only drug detected, and signs of impairment were consistent with those of a CNS depressant, including erratic driving (weaving, swerving, ignoring road signs), confusion, incoherent speech, unresponsiveness, lack of balance, unsteady coordination, poor performances on field sobriety tests, and varying states of wakefulness. Given the ability of GHB to induce sleep and unconsciousness, it is evident from these cases that recreational use of the drug has the potential to impair a person's driving ability.