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。     

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.     

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.     

Further evidence of in vitro production of gamma-hydroxybutyrate (GHB) in urine samples

This study was designed to supplement previous studies that documented in vitro production of gamma-hydroxybutyrate (GHB) in urine samples. Urine samples were provided by subjects who reported that they had never used GHB (n=31). The specimens were stored under standard conditions of refrigeration (5 degrees C) without any preservatives added. All specimens were repeatedly analyzed for the presence of endogenous GHB over a 6-month period using a previously reported headspace GC-MS method. Significant elevations in GHB were observed in many of the urine samples as storage time increased. As a result, the in vitro production of GHB may increase the apparent GHB concentrations in urine during storage. This potential for an artificial increase in GHB concentration must be appreciated when establishing the threshold between endogenous and exogenous concentrations of GHB.     

Intoxication due to 1,4-butanediol

We report a case of intoxication resulting from the ingestion of a liquid, sold in the illicit market as "liquid ecstasy," which was found to contain 1,4-butanediol, a metabolic precursor of gamma-hydroxybutiric acid (GHB). Identification of the substance in the liquid was performed by gas chromatography-mass spectrometry (GC-MS).The toxicological analysis of blood, urine and gastric content of the victim was performed by immunoassay and gas chromatography with nitrogen-phosphorus detection as screening techniques and by means of GC-MS for confirmation and quantitation of 1,4-butanediol and GHB. The following drug concentrations were found: 82 microg/ml (blood), 401 microg/ml (urine) and 7.4 microg/ml (gastric content) for 1,4-butanediol and 103 microg/ml (blood), 430.0 microg/ml (urine) for GHB. In addition to these, other drugs detected and their blood concentration found in this case were methylenedioxymethylamphetamine (MDMA) 0.23 microg/ml and its metabolite methylenedioxyphenylamphetamine (MDA) 0.10 microg/ml. In the urine, a concentration of 0.10 microg/ml of benzoylecgonine was also found.     

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.     

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.     

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.     

An unusual fatality in a child due to oxycodone

An unusual fatality secondary to oxycodone in a child is reported. A 2-year-old female child was conveyed to a local hospital after exhibiting signs of rubbing of the mouth and staggering. A hospital toxicological immunoassay screen for drugs of abuse and tricyclic antidepressants was performed on a urine sample and reported as negative. She was discharged and found unresponsive the next morning. She was conveyed to a second hospital in full cardiopulmonary arrest and despite resuscitative efforts, was pronounced dead upon arrival. An autopsy was performed and postmortem specimens were submitted and screened for drugs using mainly chromatographic techniques. Quantitation was achieved by gas chromatography with nitrogen phosphorus detection. Confirmation was performed by gas chromatography/mass spectrometry. Oxycodone was the only drug detected in the following concentrations: heart blood, 1.36 mg/L; gastric contents, 7.33 mg in 33 mL (222.34 mg/L); liver, 0.2 mg/kg; and urine, 47.23 mg/L (47,230 ng/mL). In addition, immunoassay testing of the urine was positive for the opiate class of drugs. This case report demonstrates an unusual cause of death in a young child with emphasis on potential limitation in hospital urine screening tests and the importance of complete forensic toxicological testing in all child deaths.     

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.     

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.     

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.     

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.     

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.     

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.     

Blood, brain, and hair GHB concentrations following fatal ingestion

Despite the increasing incidence of illicit use of gamma-hydroxybutyrate (GHB), little information is available documenting levels of the drug in GHB fatalities. We measured GHB levels in postmortem blood, brain and hair specimens from a suspected overdose case by gas chromatography/mass spectrometry (GC/MS) following solid phase extraction (SPE) and derivatization with bis(trimethyl-silyl) trifluoroacetamide (BSTFA). Examination found 330 microg/mL GHB in femoral blood and 221 ng/mg GHB in frontal cortex brain tissue, values higher than those typically reported in the literature. The hair shaft was negative for GHB whereas the plucked root bulbs with outer root sheath attached (2,221 ng/mg) and root bulbs after washing and removal of the outer root sheath (47 ng/mg) contained the drug. Our results are consistent with an acute single dose of GHB and, as the toxicology screen was negative for other drugs of abuse, emphasize the significant danger of this drug.     

γ-羟基丁酸在急性中毒大鼠体液和组织中的检测及分布

目的建立生物检材中γ-羟基丁酸(GHB)的检测方法,研究GHB急性中毒大鼠体内GHB的分布,为GHB中毒的鉴定提供方法和评价依据。方法用GC/MS法检测生物检材中的GHB;以1000mg/kg剂量给大鼠灌胃使其染毒,分别于1h和3h处死,测定体液和组织中GHB的含量。结果测组织中内源性GHB的线性范围是1～20μg/g,R2=0.9974;测组织中外源性GHB的线性范围为100～1500μg/g,R2=0.9958。相对回收率为98%～103%。体内内源性GHB的含量均≤10μg/mL或10μg/g。尿液中GHB含量为最高,其他依次为:胃、血液、肠、肾、肺、脾、心、肝和脑。结论所建方法准确、便捷,适用于GHB中毒的鉴定;尿液是体内检测GHB的最佳检材。     

Tissue distribution of trichloroethylene in a case of accidental acute intoxication by inhalation

This article describes the toxicological findings in a fatality due to an accidental inhalation of trichloroethylene which took place during wall coating of a poorly ventilated well using trichloroethylene. The man was wearing protective clothing and a mouthmask with adsorbent. He was found dead on the floor of the well 5h after descending. Trichloroethylene was added to the mortar to enhance drying. Identification and quantitation of trichloroethylene in the postmortem samples (blood, lung, liver, kidney, stomach content and bile) and identification of its metabolite trichloroacetic acid in urine was performed using static headspace gas chromatography with mass spectrometric detector. The compounds were separated on a CP-SIL 5CB Low Bleed/MS column using n-butanol as internal standard. The method was linear over the specific range investigated, and showed an accuracy of 104% and an intra-day precision of 11%. Trichloroethylene concentrations of 84mg/l in subclavian blood, 40mg/l in femoral blood, 72mg/kg in liver, 12mg/kg in kidney, 78mg/kg in stomach content, 104mg/l in bile and 21mg/kg in lung were found. Trichloroacetic acid was identified in the urine.     

Fatal intoxication involving 2-methyl AP-237

Novel synthetic opioids contribute considerably to the opioid epidemic, especially with the frequent emergence of structurally similar compounds. This case report describes a fatal intoxication involving 2-methyl AP-237. A 54-year-old Caucasian male was found deceased from an apparent drug overdose. A plastic container labeled “2MAP” and a cut straw were found in the decedent's backpack at the scene. A white substance found in the container tested positive for fentanyl by field testing. According to his medical history, the decedent was treated for a drug overdose 3 years prior to his death. With no diagnostic findings at autopsy, the case was submitted for toxicological analysis. An unknown substance was detected in peripheral blood and urine using gas chromatography with nitrogen phosphorous detection (GC-NPD). Further testing was conducted using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) which confirmed the presence of 2-methyl AP-237 and potential metabolites in blood and urine. Quantitation by GC-NPD revealed concentrations of 2-methyl AP-237 in blood and urine at 480 ng/mL and 4200 ng/mL, respectively. The toxicological analysis also identified and quantitated alprazolam in the blood at 55 ng/mL. Additionally, the metabolism of 2-methyl AP-237 was investigated and three hydroxylated metabolites were identified in peripheral blood and urine. Limited literature is available for the detection and quantitation of 2-methyl AP-237 in postmortem specimens. Given the toxicological findings with unremarkable autopsy findings, this case is an example of a fatal intoxication involving 2-methyl AP-237.     

Urinary excretion of benzoylecgonine following ingestion of Health Inca Tea

Four males ingested one cup of Health Inca Tea which contained 1.87 mg of cocaine. Urine specimens collected for 36 h post-ingestion were analyzed for benzoylecgonine (BE) by EMIT-d.a.u., TDx and gas chromatography/mass spectrometry (GC/MS). Positive immunoassay results were obtained for 21-26 h post tea ingestion. Discrepant immunoassay results occurred with only one specimen: EMIT positive; TDx negative, 0.25 mg/l; GC/MS, 0.273 mg/l. Quantitative TDx results were well correlated with GC/MS results, r2 = 0.963, n = 45. Maximum urinary BE concentrations ranged from 1.4-2.8 mg/l, occurring from 4-11 h, post ingestion. Total BE excretion in 36 h ranged from 1.05 to 1.45 mg, 59-90% of the ingested cocaine dose. Urinary excretion rate constant (Km) ranged from -0.073 to 0.111/h. Health Inca Tea ingestion should be considered when interpreting urinary BE concentrations.