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.     

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.     

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.     

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.     

The presence of gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL) in alcoholic and non-alcoholic beverages

Gamma-hydroxybutyric acid (GHB) and its precursor gamma-butyrolactone (GBL) are regularly implicated in instances of surreptitious drug administration, particularly in beverages (so-called "spiked drinks"). In order to assist in the interpretation of cases where analysis of the actual beverage is required, over 50 beverages purchased in the UK were analysed for the presence of GHB and GBL. It was found that naturally occurring GHB and GBL were detected in those beverages involving the fermentation of white and particularly red grapes. No GHB or GBL was detected in other drinks such as beer, juice, spirits or liqueurs. GHB/GBL was detected in red wine vermouth (8.2 mg/L), sherry (9.7 mg/L), port (GBL), red wine (4.1-21.4 mg/L) and white wine (<3-9.6 mg/L). The presence of GHB/GBL did not appear to be influenced by the alcohol content or the pH of the beverage. In addition, the concentration in wines did not appear to be related to the geographical origin of the grape type. This is believed to be the first published data concerning the endogenous presence of GHB and GBL in the beverages described.     

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.     

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.     

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.     

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。     

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.     

Determination of gamma-hydroxybutyrate in water and human urine by solid phase microextraction-gas chromatography/quadrupole ion trap spectrometry

A simple method of detection was developed for gamma-hydroxybutyrate (GHB). The method involves the derivatization of GHB using a hexyl-chloroformate procedure in aqueous media (such as water or urine), extraction of the derivatization product directly from the sample using solid-phase microextraction, and subsequent separation and detection with gas chromatography quadrupole ion trap mass spectrometry. The deuterated form of GHB (GHB-D6) is used as an internal standard for quantitation. The method was linear for GHB-spiked pure water samples from 2 to 150 microg/mL GHB with a detection limit of 0.2 microg/mL. Spiked urine samples showed linearity from 5 to 500 microg/mL GHB with a detection limit of 2 microg/mL. The SPME-GC/MS method is applied to actual case samples, and the results are compared to those values obtained using a conventional GC/MS method. Sensitivity and linearity are comparable to those seen using traditional methods of separation, yet the SPME method is superior due to the simplicity, speed of analysis, reduction in solvent waste, and ability to differentiate between GHB and gamma-butyrolactone (GBL).     

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。     

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.     

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.     

The extraction and infrared identification of gamma-hydroxybutyric acid (GHB) from aqueous solutions

The chemical analysis of gamma-hydroxybutyric acid (GHB) in most forensic laboratories is complicated by the highly polar nature of the GHB molecule, which makes it unsuitable for direct analysis by gas chromatography (GC). Consequently, a popular analytical approach is to convert GHB into the corresponding lactone or a derivative compound that is then identified by mass spectrometry employed in conjunction with GC (GC/MS). An alternative approach is presented here where GHB may be isolated as a free acid specie from complex aqueous solutions employing a liquid-liquid extraction technique. This approach can yield a relatively pure residue of GHB that presents an infrared transmission spectrum that is sufficiently distinct for identification purposes. Infrared spectroscopy (IR) is a very popular technique that is available to most crime laboratories. The liquid-liquid extraction behavior of GHB is examined in detail and the uniqueness of the infrared spectrum is discussed.     

Detection of Gamma-Hydroxybutyric Acid in Various Drink Matrices via AccuTOF-DART*

Abstract: A new screening method for detecting gamma-hydroxybutyric acid (GHB) in drink matrices, using the IonSense, Inc. (Saugus, MA) direct analysis in real time (DART) ion source coupled to a JEOL exact mass time-of-flight mass spectrometer (AccuTOF), was validated and compared with the current screening methodology. The DART ion source allows for analysis of samples under ambient conditions with little to no sample preparation. Fifty drink specimens were spiked at levels of 1, 2, 3, and 4 mg/mL GHB, and analyzed on the AccuTOF-DART. Positive detection of GHB occurred for each of the samples at each concentration level, giving 100% accuracy for the samples tested. Twenty-five of the 50 drink specimens were spiked at 1 mg/mL GHB and tested using a color test known as the GHB Color Test #3. Only two of these 25 specimens tested positive for the presence of GHB, giving only 8% accuracy. Implementation of this new methodology as a screening tool for GHB analysis will quickly eliminate negative specimens allowing the examiner to focus analysis time on those that screened positive.     

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.     

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.     

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). RESULTS: 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. CONCLUSIONS: 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.     

毛发中GHB的检测及评价

目的探索毛发中外源性GHB的检测及判断的可行性,为涉GHB的鉴定提供方法和依据。方法建立毛发中GHB的GC/MS分析方法,并通过动物实验,考察毛发中内源性GHB的质量分数范围、外源性GHB在毛发中的时间过程以及给药剂量、毛发颜色与毛发中GHB的质量分数关系。结果豚鼠和中国人黑色毛发中内源性GHB质量分数分别为(3.01±1.41)ng/mg(n=28)和(1.02±0.27)ng/mg(n=20);摄GHB后毛发中GHB质量分数明显增加且与给药剂量呈正相关性;GHB在毛干中呈窄带分布;深色毛发中GHB质量分数高于浅色毛发。结论毛发中GHB的检测适用于GHB滥用和中毒的法医毒物学鉴定;根据毛发中的GHB质量分数和毛发分段分析可判断GHB的来源。