GC-MS determination of gamma-hydroxybutyric acid (GHB) in blood

A fast and efficient procedure has been developed for the analysis of total mercury in human tissues and blood using a hydride vapor generator system coupled to an atomic absorption spectrometer (HVG-AA). Tissue and blood samples were digested in a pressurized microwave decomposition system and the digest diluted prior to formation of free mercury vapor and analysis by atomic absorption. Recovery studies performed on 10 spiked/unspiked pairs of human liver and on 10 spiked/unspiked pairs of human blood samples yielded average recoveries of 99.7% (CV=0.4%) and 101.2% (CV=0.5%), respectively. The method detection limit for liver and blood was 50 microg Hg/kg and 12.5 microg Hg/l, respectively. The "normal" concentrations of mercury in human liver and blood are 33-490 microg Hg/kg and 0.6-59 microg Hg/l, respectively [1]. This method is able to determine mercury poisoning levels and may also be applied to detect mercury near the lower levels of these "normal" ranges, using the standard addition method approach.     

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 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.     

Determination of endogenous gamma-hydroxybutyric acid (GHB) levels in antemortem urine and blood

Gamma-hydroxybutyric acid's (GHB's) natural presence in the body has made the interpretation of its levels a challenging task for the forensic toxicologist. This study was designed to measure endogenous GHB levels in antemortem urine and blood samples. The range detected in urine was from 34 to 575 microg/dl and in blood from 17 to 151microg/dl. The results indicate that the concentration of endogenous GHB in urine and blood concur with the suggested cut-off levels at 1000 and 500 microg/dl, respectively.     

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.     

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.     

Recordkeeping and reporting requirements for drug products containing gamma-hydroxybutyric acid (GHB). Final rule

DEA is amending its regulations to require additional recordkeeping and reporting requirements for drug products containing gamma-hydroxybutyric acid (GHB) for which an application has been approved under the Federal Food, Drug, and Cosmetic Act. DEA makes these changes under section 4 of the "Hillory J. Farias and Samantha Reid Date-Rape Drug Prohibition Act of 2000." These additional requirements are necessary to protect against the diversion of GHB for illicit purposes.     

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.     

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.     

From alcohol to liquid ecstasy (GHB)--a survey of old and modern knockout agents. Part 3: gamma-hydroxybutyric acid ("liquid ecstasy")

Currently, gamma-hydroxybutyric acid (GHB/"liquid ecstasy") is frequently abused as a knockout substance. Its detection and the interpretation of the results present numerous problems which are illustrated by case reports. In this context, hair analysis and the increasing significance of gamma-butyrolactone (GBL) are also discussed.     

Rapid colorimetric screening test for gamma-hydroxybutyric acid (liquid X) in human urine

A rapid colorimetric test for the detection of gamma-hydroxybutyric acid (GHB) is described. The ferric hydroxamate test for ester detection has been adapted to detect GHB in human urine samples from a healthy female and a healthy male subject. The assay can be performed within 5 min and with a GHB detection limit of 0.5 mg/ml when 0.3 ml of human urine is used and a GHB detection limit of 0.1 mg/ml when 1 ml of human urine is used. The colored complex indicating the presence of GHB is purple according to the assay conditions. Test results are free from the interference by alcohol, phenolic compounds and other biological chemicals under the assay conditions. In addition, the colorimetric test is free from the potential false-positive test result that could result from physiological concentrations of GHB.     

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.     

Chemical composition and structure of the microcrystals formed between silver(I) and gamma-hydroxybutyric acid and gamma-hydroxyvaleric acid

This study examined microcrystals formed by silver with gamma-hydroxybutyric acid (GHB) and gamma-hydroxyvaleric acid (GHV), the five-carbon analog of GHB, in the presence of silver, copper, and lanthanide nitrates. Distinct microcrystals formed with silver (+1) and lanthanum (+3) ions but not with the copper (+2) ions. The crystals formed with GHB were distinctly different than those formed with GHV and in all cases, the drug microcrystals were easily distinguishable from reagent crystals. X-ray diffraction analysis provided definitive structure for the microcrystals. The morphological differences between the silver-GHB and silver-GHV crystals were characterized using simple measurements such as size and angles provided by image recognition software. The utility of the test for casework was demonstrated using spiked beverage samples.     

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 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.     

Prevalence of gamma-hydroxybutyrate (GHB) in serum samples of amphetamine, metamphetamine and ecstasy impaired drivers

Two hundred and forty-seven serum samples which have been collected by police during roadside testing and have been found positive for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and/or 3,4-methylenedioxyethamphetamine (MDE) were analyzed for gamma-hydroxybutyrate (GHB). Serum samples were spiked with deuterated GHB as internal standard and acetonitrile was added to achieve dilution and protein precipitation. Samples were analyzed with a LC-MS/MS system operated in the multiple reaction monitoring mode (MRM) using a TurboIonSpray source. Chromatographic separation was achieved using a Synergi Polar RP column applying a gradient elution with a runtime of 15 min. To differentiate between endogenous and exogenously administered GHB a cut-off concentration of 10 microg/mL was applied. Five samples exceeded this concentration and were found positive for GHB. These samples were only found positive for amphetamine but no other amphetamine derivatives were detected, while in three samples THC and in one sample cocaine, benzoylecgonine and ethanol were found.     

Investigations concerning the threshold value between endogenous and exogenous GHB (liquid ecstasy)

The article describes critical investigations concerning the threshold value between endogenous and exogenous concentrations of gamma-hydroxybutyric acid (GHB/"liquid ecstasy") in human blood. The values of GHB in the blood samples of 50 blood donors and 50 postmortem cases were measured with a validated gas-chromatographic/mass-spectrometric procedure according to the guidelines of the GTFCh (Society of Toxicological and Forensic Chemistry). GHB-concentrations were found to range between 0.11 and 1.56 mg/L (mean value 0.54 mg/L/standard deviation 0.37 mg/L/coefficient of variation 68.4 %) in the donors' blood, and between 2.2 and 116 mg/L (mean value 32.4 mg/L/standard deviation 25.6 mg/L/coefficient of variation 79 %) in the postmortem samples, respectively.     

胶体金免疫层析一步法快速检测Glm(3)因子

建立一种简便快速的胶体金免疫层析一步法,用于检测Glm(3)因子.采用柠檬酸三钠还原法制备胶体金颗粒,标记抗人Glm(3)单克隆抗体,研制出抗人Glm(3)因子免疫层析检测试剂盒.样品的Glm(3)因子,与测试条上的金标记抗体结合后沿着反应膜移动,再与膜上固相抗体结合形成肉跟可见的红色反应带.使用该方法可检出10万倍稀释的血清样品,整个试验只需5min完成.对常见的23种动物血(痕)检验,未出现交叉反应.在100例样品的检测中,本方法的检测结果,与Dot-ELISA的检测结果的符合率为100%.该方法适用于法医物证快速检验.     

Estimation of gamma-hydroxybutyrate (GHB) co-consumption in serum samples of drivers positive for amphetamine or ecstasy

There is no toxicological analysis of gamma-hydroxybutyrate (GHB) applied routinely in cases of driving under influence (DUI); therefore the extent of consumption of this drug might be underestimated. Its consumption is described as occurring often concurrently with amphetamine or ecstasy. This study examines 196 serum samples which were collected by police during road side testing for GHB. The samples subject to this study have already been found to be positive for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and/or 3,4-methylenedioxyethamphetamine (MDEA). Analysis has been performed by LC/MS/MS in the multiple reaction monitoring (MRM) mode. Due to its polarity, chromatographic separation of GHB was achieved by a HILIC column. To differentiate endogenous and exogenous levels of GHB, a cut-off concentration of 4μg/ml was applied. Of the 196 samples, two have been found to be positive for GHB. Of these samples, one sample was also positive for amphetamine and one for MDMA. Whilst other amphetamine derivates were not detected in these samples, both samples were found to be positive for cannabinoids. These results suggest that co-consumption of GHB with amphetamine or ecstasy is relatively low (1%) for the collective of this study.     

胶体金免疫层析一步法快速检测G1m(3)因子

建立一种简便快速的胶体金免疫层析一步法 ,用于检测 G1m(3)因子。采用柠檬酸三钠还原法制备胶体金颗粒 ,标记抗人 G1m(3)单克隆抗体 ,研制出抗人 G1m(3)因子免疫层析检测试剂盒。样品的 G1m(3)因子 ,与测试条上的金标记抗体结合后沿着反应膜移动 ,再与膜上固相抗体结合形成肉眼可见的红色反应带。使用该方法可检出 10万倍稀释的血清样品 ,整个试验只需 5 min完成。对常见的 2 3种动物血 (痕 )检验 ,未出现交叉反应。在 10 0例样品的检测中 ,本方法的检测结果 ,与 Dot- EL ISA的检测结果的符合率为 10 0 %。该方法适用于法医物证快速检验。