Effect of storage temperature on endogenous GHB levels in urine

Because gamma-hydroxybutyrate (GHB) is an endogenous substance present in the body and is rapidly eliminated after ingestion, toxicologists investigating drug-facilitated sexual assault cases are often asked to differentiate between endogenous and exogenous levels of GHB in urine samples.This study was designed to determine the effects of storage temperature on endogenous GHB levels in urine. Specifically, it was designed to ascertain whether endogenous levels can be elevated to a range considered indicative of GHB ingestion.Urine specimens from two subjects that had not been administered exogenous GHB were collected during a 24h period and individually pooled. The pooled specimens were separated into standard sample cups and divided into three storage groups: room temperature ( approximately 25 degrees C), refrigerated (5 degrees C), and frozen (-10 degrees C). Additionally, some specimens were put through numerous freeze/thaw cycles to mimic situations that may occur if multiple laboratories analyze the same specimen. Periodic analysis of the samples revealed increases in the levels of endogenous GHB over a 6-month period. The greatest increase (up to 404%) was observed in the samples maintained at room temperature. The refrigerated specimens showed increases of 140-208%, while the frozen specimens showed smaller changes (88-116%). The specimens subjected to multiple freeze/thaw cycles mirrored specimens that had been thawed only once. None of the stored urine specimens demonstrated increases in GHB concentrations that would be consistent with exogenous GHB ingestion.     

Gamma-hydroxybutyric acid stability and formation in blood and urine

Gamma-hydroxybutyric acid (GHB) can cause problems in interpretation of toxicological findings due to its endogenous nature, significant production in tissues after death and potential formation in stored samples. Our study was designed to determine the influence of storage conditions on GHB levels and its possible in vitro formation in blood and urine in cases where no exogenous use of GHB or its precursors was suspected. The samples were prepared by validated method based on liquid-liquid reextraction with adipic acid internal standard and MSTFA derivatization and assayed on a GC-MS operating in EI SIM mode. The first part of the study was performed with pooled blood and urine samples obtained from living and deceased subjects stored with and without NaF (1% w/v) at 4 and -20 degrees C over 8 months. In ante-mortem samples (both blood and urine) no significant GHB production was found. After 4 months of storage, the substantial GHB rise up to 100 mg/Lwas observed in post-mortem blood stored at 4 degrees C without NaF with subsequent gradual decrease in following months. The inhibition of GHB production was apparent during storage in NaF treated frozen blood samples. In post-mortem urine only slight temporary GHB levels were ascertained (up to 8 mg/L). The second part of our study was aimed to analyse 20 individual post-mortem blood samples stored at 4 degrees C for 16-27 days between autopsy and analysis without preservation followed by storage at 4 degrees C with NaF for 4 months. The temporary GHB production with maximum of 28 mg/Lwas detected in some samples.     

The possible influence of micro-organisms and putrefaction in the production of GHB in post-mortem biological fluid

In recent years, the post-mortem production of the drug of abuse gamma-hydroxybutyric acid (GHB) in biological fluids (e.g. blood and urine) has caused various interpretative problems for toxicologists. Previously, other researchers have shown certain microbial species (Pseudomonas spp. and Clostridium aminobutyricum) possess the necessary enzymes to convert GABA to GHB. A preliminary investigation involving putrefied post-mortem blood indicated there was no observed relationship between "endogenous" GHB concentrations and concentrations of common putrefactive markers (tryptamine and phenyl-2-ethylamine). Microbiological analysis identified the presence of various micro-organisms: Clostridia spp., Escherichia coli, Proteus vulgaris, Enterococcus faecalis and Aeromonoas spp. Equine plasma, human blood and urine samples were inoculated with these and an additional micro-organism (Pseudomonas aeruginosa) and incubated at 22 degrees C for 1 month. Following comparison with control samples and pre-inoculation concentrations, the data indicated an apparent production of GHB in unpreserved P. aeruginosa inoculated blood (2.3 mg/l). All other fluoride-preserved and unpreserved samples (including controls) had GHB concentrations <1mg/l. Although this concentration is lower than is typically associated with "endogenous" post-mortem GHB concentrations, this paper proposes a potential microbial production of GHB with time.     

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.     

Site-dependent production of gamma-hydroxybutyric acid in the early postmortem period

This study compared endogenous gamma-hydroxybutyric acid (GHB) concentrations in various postmortem fluid samples of 25 autopsy cases. All bodies were stored between 10-20 degrees C until autopsy, and the intervals between death and autopsy were less than 2 days (6-48 h). GHB concentrations were measured by headspace gas chromatography after GHB was converted to gamma-butyrolactone. Endogenous GHB concentrations were significantly higher in femoral venous blood (4.6+/-3.4 microg/ml, n=23) than in cerebrospinal fluid (1.8+/-1.5 microg/ml, n=9), vitreous humor (0.9+/-1.7 microg/ml, n=8), bile (1.0+/-1.1 microg/ml, n=9) and urine (0.6+/-1.2 microg/ml, n=12). GHB concentrations were similar in blood samples taken from different sites. Cut-off limits of 30 and 10 microg/ml are proposed for blood and urine, respectively, to discriminate between exogenous and endogenous GHB in decedents showing no or little putrefaction (postmortem intervals usually 48 h or less). The criterion established for endogenous GHB in postmortem urine may also be applicable to analytical results in cerebrospinal fluid, vitreous humor and bile from deceased persons.     

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。     

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.     

Fatal Overdose of Gamma‐hydroxybutyrate Acid After Ingestion of 1,4‐Butanediol

We report a case of fatal intoxication from 1,4‐butanediol (1,4‐BD), which was ingested by a young and “naïve” gamma‐hydroxybutyrate (GHB) consumer during a party with the co‐ingestion of alcohol, cannabis, and methylene‐dioxy‐methamphetamine. The following drug concentrations were found using gas chromatography coupled with mass spectrometry on autopsy samples and on a cup and a glass found at the scene: 20,350 mg/L (bottle) for 1,4‐BD; 1020 mg/L (femoral blood), 3380 mg/L (cardiac blood), 47,280 mg/L (gastric content), and 570 mg/L (vitreous humor) for GHB. The concentration of GHB is difficult to interpret in forensic cases due to the possibility of an endogenous production of GHB. The variable tolerance of the user may also modify the peri‐ and postmortem GHB concentrations. This case underscores the need to have many different sources of toxicology samples analyzed to avoid the hypothesis of endogenous production of GHB.     

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.     

Urinary endogenous concentrations of GHB and its isomers in healthy humans and diabetics

Urinary endogenous concentrations of gamma-hydroxybutyric acid (GHB), alpha-hydroxybutyric acid (AHB) and beta-hydroxybutyric acid (BHB) have been investigated for both healthy humans and diabetics by using a newly optimized GC-MS procedure. The endogenous concentrations in healthy volunteers' urine ranged 0.16-2.14 microg/ml for GHB, 0.10-2.68 microg/ml for AHB and 8.51-34.7 microg/ml for BHB. In diabetics, the concentrations ranged 0.17-3.03 microg/ml for GHB, 0.14-124 microg/ml for AHB and 4.94-4520 microg/ml for BHB. Although notably elevated BHB and AHB concentrations were observed for severely uncontrolled diabetics, their GHB concentrations ranged within or near the range seen in healthy humans. The results of this study confirm the previously suggested 10 microg/ml cutoff concentration of urinary GHB to distinguish exogenous GHB, even for uncontrolled diabetic patients suffering severe ketoacidosis.     

GHB. Club drug or confusing artifact?

GHB can be produced either as a pre- or postmortem artifact. The authors describe two cases in which GHB was detected and discuss the problem of determining the role of GHB in each case. In both cases, NaF-preserved blood and urine were analyzed using gas chromatography. The first decedent, a known methamphetamine abuser, had GHB concentrations similar to those observed with subanesthetic doses (femoral blood, 159 microg/ml; urine, 1100 microg/ml). Myocardial fibrosis, in the pattern associated with stimulant abuse, was also evident. The second decedent had a normal heart but higher concentrations of GHB (femoral blood, 1.4 mg/ml; right heart, 1.1 mg/ml; urine, 6.0 mg/ml). Blood cocaine and MDMA levels were 420 and 730 ng/ml, respectively. Both decedents had been drinking and were in a postabsorptive state, with blood to vitreous ratios of less than 0.90. If NaF is not used as a preservative, GHB is produced as an artifact. Therefore, the mere demonstration of GHB does not prove causality or even necessarily that GHB was ingested. Blood and urine GHB concentrations in case 1 can be produced by a therapeutic dose of 100 mg, and myocardial fibrosis may have had more to do with the cause of death than GHB. The history in case 2 is consistent with the substantial GHB ingestion, but other drugs, including ethanol, were also detected. Ethanol interferes with GHB metabolism, preventing GHB breakdown, raising blood concentrations, and making respiratory arrest more likely. Combined investigational, autopsy, and toxicology data suggest that GHB was the cause of death in case 2 but not case 1. Given the recent discovery that postmortem GHB production occurs even in stored antemortem blood samples (provided they were preserved with citrate) and the earlier observations that de novo GHB production in urine does not occur, it is unwise to draw any inferences about causality unless (1) blood and urine are both analyzed and found to be elevated; (2) blood is collected in NaF-containing tubes; and (3) a detailed case history is obtained.     

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。     

Forensic cases involving the use of GHB in The Netherlands

In this study, forensic cases involving the use of Gamma Hydroxy Butyric acid (GHB) from the second half of 1999 through the second half of 2001 in The Netherlands (blood >5mg/l and urine >10mg/l) are described. GHB was analysed by GC-MS after lactone formation and using GHB-d6 as internal standard. The results are divided into three groups: cases of chemical submission, cases of driving under the influence and cases of unknown causes of death.GHB was found in six cases of possible chemical submission. In these cases, relatively low concentrations of GHB were found. The results show that in cases of chemical submission, urine should be analyzed, because GHB is present longer in urine than in blood. The police should collect the samples in containers that do not contain citrate as anticoagulant. Especially at low levels of GHB, the formation of GHB in these tubes hampers an interpretation of the results.GHB was found in 13 cases of driving under the influence. In contrast to the cases of chemical submission, high concentrations of GHB were found, corresponding with observations of extreme sleepiness or temporary loss of consciousness.GHB was found in 16 cases of unexplained death: the measured range of GHB concentrations in blood might correspond to effects such as drowsiness, but not to serious toxicity of GHB. In 4 of these 16 cases, the role of GHB could be excluded. In the remaining cases, the role of GHB remains unclear; more research into "background" concentrations of GHB in post-mortem material is required.The incidence of the use of GHB in The Netherlands cannot be derived from these toxicological data. As GHB is not routinely found during systematical toxicological analyses, these data may seriously underestimate the use of GHB. Therefore, information from the police to the forensic institute is essential.     

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.     

A novel method for GHB detection in urine and its application in drug-facilitated sexual assaults

A confirmation procedure for the identification and quantification of gamma-hydroxybutyric acid (GHB) in urine is presented. This method is unique in that it does not involve the conversion of GHB to the gamma-butyrolactone (GBL). The urine samples were extracted using ethyl acetate, evaporated and derivatized with N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS), and analyzed by gas chromatography-mass spectrometry. Quantification was performed using selective ion monitoring (SIM), using GHB-d(6) as the internal standard. This method is simple and provides excellent linearity and sensitivity for GHB in urine.     

Drug-facilitated sexual assault involving gamma-hydroxybutyric acid

The first case involving an alleged sexual assault linked to the use of gamma-hydroxybutyric acid (GHB) in Oklahoma is reported. A-48-year-old Caucasian woman taking amitriptyline was known to have voluntarily ingested a sports drink containing a relaxing health product. She purportedly experienced unconsciousness that persisted for approximately 4 h. The toxicological testing on urine identified GHB, amitriptyline, and nortriptyline using a capillary Hewlett-Packard 6890 gas chromatograph coupled to a Hewlett-Packard 5973 mass selective detector (MSD). The GHB concentration in urine was 26.9 microg/mL. Urine concentrations of amitriptyline and nortriptyline were not determined. The analytical method used for identifying and quantitating GHB can be applied to matters of forensic interests.     

Testing for GHB in hair by GC/MS/MS after a single exposure. Application to document sexual assault

Gamma-hydroxybutyric acid, or GHB, is a substance naturally present within mammal species. Properties of neurotransmitter or neuromodulator are generally given to this substance. GHB is therapeutically used as an anesthetic, but can be used for criminal offenses (date-rape drug). It appears that the window of detection of GHB is very short in both blood and urine, and therefore its presence is very difficult to prove after a rape case. In order to document single exposure, we investigated the use of hair. Hair was collected one month after the allegated event in order to sample the corresponding period after regular growing. After rapid (2 min) decontamination with dichloromethane, the hair shaft was cut into 3-mm segments. They were overnight incubated in 0.01 N NaOH in the presence of GHB-d6, followed by neutralization and extraction in ethyl acetate under acidic conditions. GHB (precursor ion m/z 233, product ions m/z 147 and 148) was tested by GC/MS/MS (Finnigan TSQ 700) after derivatization with BSTFA + 1% TMCS. Physiological concentrations (n = 24) were in the range 0.5 to 12.0 ng/mg, with no influence due to hair color. No variation of concentrations was observed along the hair shaft in controlled subjects, except for the proximal segment, due to an incorporation through sweat. This demonstrates that endogenous levels for each single subject are constant during hair growth. A controlled human administration of 25 mg/kg to a volunteer demonstrated that a single exposure to GHB is detectable in hair after segmentation. In a case of rape under influence, a clear increase of the corresponding segment (about 2.4 ng/mg) in time was observed, in comparison with the other segments (0.6 to 0.8 ng/mg). This study demonstrates that a single exposure to GHB in a case of sexual assault can be documented by hair analysis when collected about one month after the crime.     

Fatal intoxication with 1,4-butanediol: Case report and comprehensive review of the literature

A fatal case of 1,4-butanediol (1,4-BD) oral ingestion is reported here, in which a 51-year-old man was found dead in his bed. According to the police report, the deceased was a known drug user. A glass bottle labeled (and later confirmed to be) “Butandiol 1,4” (1,4-BD) was found in the kitchen. Furthermore, the deceased's friend stated that he consumed 1,4-BD on a regular basis. The autopsy and histological examination of postmortem parenchymatous organ specimens did not revealed a clear cause of death. Chemical-toxicological investigations revealed gammahydroxybutyrat (GHB) in body fluids and tissues in the following quantities: femoral blood 390 mg/L, heart blood 420 mg/L, cerebrospinal fluid 420 mg/L, vitreous humor 640 mg/L, urine 1600 mg/L, and head hair 26.7 ng/mg. In addition, 1,4-BD was qualitatively detected in the head hair, urine, stomach contents, and the bottle. No other substances, including alcohol, were detected at pharmacologically relevant concentrations. 1,4-BD is known as precursor substance that is converted in vivo into GHB. In the synoptic assessment of toxicological findings, the police investigations and having excluded other causes of death, a lethal GHB-intoxication following ingestion of 1,4-BD, can be assumed in this case. Fatal intoxications with 1,4-BD have seldom been reported due to a very rapid conversion to GHB and, among other things, non-specific symptoms after ingestion. This case report aims to give an overview to the published of fatal 1,4-BD-intoxications and to discuss the problems associated with detection of 1,4-BD in (postmortem) specimens.     

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.     

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.