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

In this work, the interconversion of GHB and GBL in a variety of aqueous media was studied. The effects of solution pH and time were determined by spiking GHB or GBL into pure water and buffered aqueous solutions, and determining the GHB and GBL contents at various time intervals. The degree of GBL hydrolysis to GHB was determined for several commercial aqueous-based GBL products, and further studied as a function of time. The effects of temperature and time were also determined for five commercial beverages spiked with GHB or GBL. GHB and GBL contents were determined using high performance liquid chromatography (HPLC). GHB and/or GBL confirmations were made using gas chromatography-mass spectrometry (GC-MS) and/or infrared spectroscopy (IR). Solution pH, time, and storage temperature were determined to be important factors affecting the rate and extent of GBL hydrolysis to GHB. Under strongly alkaline conditions (pH 12.0), GBL was completely converted to GHB within minutes. In pure water, GBL reacted to form an equilibrium mixture comprising ca. 2:1 GBL:GHB over a period of months. This same equilibrium mixture was established from either GHB or GBL in strongly acidic solution (pH 2.0) within days. A substantial portion of GBL (ca. 1/3) was hydrolyzed to GHB in aqueous-based GBL products, and in spiked commercial beverages, after ambient storage for a period ranging from several weeks to several months. Heat increased and refrigeration decreased the rate of GBL hydrolysis relative to ambient conditions. These studies show that hydrolysis of GBL to GHB does occur in aqueous-based solutions, with samples and time frames that are relevant to forensic testing. Implications for forensic testing and recommendations are discussed.     

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.     

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。     

Determination of GHB in urine and serum by LC/MS using a simple one-step derivative

A sensitive and specific method for the determination of gamma-hydroxybutyric acid (GHB) in urine and serum is described. Prior to quantification by LC/MS in a Pauli-type ion trap, the molecule is converted by a fast and simple one-step procedure into its n-butyl ester derivative. Hexa-deutero GHB has been used as internal standard.     

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.     

阿片类成瘾者血清、尿中吗啡TLCS分析

建立阿片类成瘾者血清、尿液中吗啡的薄层色谱扫描 (TCLS)定量检测方法。样品经酸、碱性水解后调至pH9,氯仿 /异丙醇 ( 9∶1)萃取及GDX 40 3柱固相萃取 ,在紫外区可见光区薄层扫描。测得 3种萃取方法吗啡回收率分别为 75 3 %± 4 9% ,80 9%± 3 2 %和 79 4%± 3 5 % ,血清、尿中吗啡最低检出浓度分别为 0 1μg·ml-1,0 0 5 μg·ml-1(信噪比≥ 3 )。本法可用于阿片类药物成瘾者或中毒者血、尿中吗啡的检测。     

Pathologic findings in malignant hyperthermia: a case report and review of literature

Malignant hyperthermia (MH) is a rare, potentially lethal disorder of skeletal muscle calcium homeostasis characterized by muscle contracture and life-threatening hypermetabolic crisis following exposure to halogenated anesthetics and depolarizing muscle relaxants. Susceptibility to MH results from mutations in calcium channel proteins that mediate excitation-contraction coupling, with the ryanodine receptor calcium release channel (RyR1) representing the major locus. The mode of inheritance appears to be autosomal dominant with variable penetrance. The authors report the death of a 60-year-old white male with a history of low back pain. He had undergone 2 back surgeries previously, the first occurring 10 years prior to his current presentation. Both previous procedures were done under generalized anesthetic with no complications. Recently, he developed stenosis and presented for fusion of vertebrae L3 and L4. The procedure was performed under general anesthetic including sevoflurane, with no intraoperative complications. The anesthesiologist noted that, near the end of the 2-hour procedure, the decedent's CO2 levels were slightly elevated. After the procedure, the decedent was extubated, the temperature probe which had been recording normal values was removed, and he was rolled from ventral to dorsal position. He immediately became hypotensive and bradycardic. Lifesaving interventions were begun. Subsequently, he went into cardiac arrest, at which time the temperature probe was reinserted into the trachea, where it read a body temperature of 109 degrees F. Malignant hyperthermia protocol was initiated, and interventions continued for over 2 hours, at which time they failed. At autopsy, the abdomen contained 1800 mL of blood, and bilateral hematomas were present in the psoas muscles. The authors present this case of clinically apparent malignant hyperthermia, discuss how to approach such a case, the gross and microscopic findings, ancillary studies, and a review of the literature.     

Incidence of bite marks in a selected juvenile population: a preliminary report

A study of the frequency of bite marks among sheltered children was conducted for a period of three months in the juvenile care facilities in Las Vegas, NV. The study demonstrated an incidence of 1 545 bite marks per 100 000 population. Analysis of the age, sex, and location of bite marks is presented. The study demonstrated an incidence comparable to diseases such as gonorrhea.     

Identification and quantitation of xenobiotics by 1H NMR spectroscopy in poisoning cases

In order to analyse a wide range of xenobiotics and their metabolites present in biological fluids, NMR spectroscopy can be used. A large variety of xenobiotics (therapeutic agents, pesticides, solvents, alcohols) can be characterized and quantitated directly, without sample preparation. NMR investigations were applied to acute poisoning cases, involving drugs such as salicylates and valproic acid (VPA). In a salicylate poisoning case, the three major metabolites of acetylsalicylic acid have been detected in crude urine, and rapid identification of lysine revealed the origin of the intoxication, namely lysine acetylsalicylate (Aspegic). Valproic acid as its glucuronide was identified in urine samples from two poisoned patients. 1H NMR was also used to identify and quantitate paraquat (Gramoxone) in urine owing to its two aromatic signals at 8.49 and 9.02 ppm, in two acutely poisoned patients (183 and 93 mg/l). An intentional poisoning case with tetrahydrofuran (THF) was also investigated. Serum and urine samples were collected. THF was characterized by its resonances at 1.90 and 3.76 ppm, and quantified at 813 and 850 mg/l in the two biological fluids, respectively. Moreover, two other compounds were detected: lactate and gamma-hydroxybutyric acid (GHB). 1H NMR spectroscopic analysis of serum samples from three poisoned patients revealed methanol in one case and ethylene glycol in the two others. Moreover, in the same spectrum, the corresponding metabolites formate and glycolate were found. Compared with the reference chromatographic or spectrophotometric methods, requiring time-consuming extraction and/or derivatization steps, NMR spectroscopy allows the determination of many exogenous and endogenous compounds, without any pre-selection of the analytes.     

A preliminary report: proximal facet analysis and the recovery of trace restorative materials from unrestored teeth

A small sample of unrestored teeth with wear facets on the proximal surfaces was examined using a scanning electron microscope and energy-dispersive X-ray fluorescence spectrometry (EDS) analysis. Initial results indicate the potential of this technique for detection of residual restorative materials in these facet areas, and its resulting capability to determine the existence and composition of unrecovered adjacent restorations. The potential value of this technique in the identification of incomplete dental remains is discussed.     

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

γ-Hydroxybutyrate (GHB) is an increasingly popular drug of abuse that causes stimulation, euphoria, anxiolysis or hypnosis, depending on the dose used. Low doses of the drug are used recreationally, and also implicated in drug-facilitated sexual assaults. Because of the unusually steep dose–response curves, accidental GHB overdosing, leading to coma, seizures or death can occur. Being a controlled substance, GHB is often substituted with its non-scheduled precursors γ-butyrolactone (GBL) and 1,4-butanediol (BD), which are rapidly metabolized into GHB in the body. Here we describe an assay for GHB, GBL and BD in blood and/or urine samples. GHB and BD were extracted from diluted 200 μL aliquots of samples with t-butylmethylether (plus internal standard benzyl alcohol) in test tubes preloaded with NaCl. After acidification and centrifugation the solvent phase was transferred to a test tube preloaded with Na₂SO₄, incubated for 30 min, centrifuged again, and evaporated in vacuum. The residue was mixed with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) in acetonitrile, and injected into a GC–MS. When analyzing GBL, the salting-out step was omitted, and analysis was performed with a GC–FID apparatus. As revealed by the validation data this procedure is suitable for quantitative determination of GHB and its precursors in blood and/or urine samples.     

Identification and quantitation of 3,4-methylenedioxy-N-methylamphetamine (MDMA,ecstasy) in human urine by 1H NMR spectroscopy. Application to five cases of intoxication

Identification of 3,4-methylenedioxy-N-methylamphetamine (MDMA, ecstasy) in five cases of intoxication using nuclear magnetic resonance (NMR) spectroscopy of human urine is reported. A new water suppression technique PURGE (Presaturation Utilizing Relaxation Gradients and Echoes) was used. A calibration curve was obtained using spiked samples. The method gave a linear response (correlation coefficient of 0.992) over the range 0.01–1 mg/mL. Subsequently, quantitation of the amount of MDMA present in the samples was performed. The benefit and reliability of NMR investigations of human urine for cases of intoxication with MDMA are discussed.     

Is urine a suitable material for the preliminary screening of drugs in autopsy cases?

This paper reports a survey of drug screening in the urine specimens of 45 autopsy cases whose livers contained medicinal substances. The extractions were carried out by a solid phase/liquid technique and the analyses by thin-layer chromatography. Nine compounds out of 43 actually present in the liver were not detectable in the urine; eight cases with high drug concentrations in the liver and also in the blood would have evaded the intoxication suspicion had the urine been used as the only material for the chemical survey. On the basis of these data we advocate that the preliminary drug screening of medical-examiner cases not be carried out on urine alone.     

Age estimation of bloodstains: a preliminary report based on aspartic acid racemization rate

This study describes an innovative application of a well-established method of age determination. The conventional method of aspartic acid racemization (AAR) is based on estimation of the d-l-aspartic acid ratio in slow turnover tissues, such as tooth tissue, to reflect the age of an individual. This method has been recently applied to age estimation in forensic investigations, and is also widely used for archeological dating of fossils. We suggest that the aspartic acid racemization method could be applied to a significant, although unresolved, forensic issue: that of bloodstain dating. Standard kinetic experiments were used to describe the characteristics of the racemization reaction in bloodstains, which were then employed to estimate the age of various samples. The soluble protein fraction of a bloodstain produced a stronger correlation between elapsed time and d-aspartic acid content than total amino acid fractions. According to our preliminary results, the time lapse after the creation of a bloodstain can be determined ex vivo by measuring the extent of aspartic acid racemization. Our analysis highlights the need for further study into the preservation and composition of bloodstains to assist in further development of this pioneering application.     

Determination of glyphosate in biological fluids by 1H and 31P NMR spectroscopy

Identification of glyphosate in four cases of poisoning, using nuclear magnetic resonance spectroscopy of biological fluids is reported. It has been performed by using a combination of 1H and 31P NMR analyses. Characterization of the N-(phosphonomethyl) glycine herbicide was achieved by chemical shift considerations and coupling constant patterns: CH2-(P) presents specific resonance at 3.12 ppm and appears as a doublet with a H-P characteristic coupling constant of 12.3 Hz. Moreover, resonances due to isopropylamine were present, confirming the ingestion of the considered trade formulation. After a calibration step, quantitation was performed by 1H and 31P NMR spectroscopy. The benefit and reliability of NMR investigations of biological fluids are discussed, particularly when the clinical picture is quite confusing.     

Subtyping phosphoglucomutase-1 in semen stains and bloodstains: a report on the method

A method is described for obtaining nondistorted, reproducible phosphoglucomutase-1 subtyping patterns from semen stains and bloodstains. Isoelectric focusing of phosphoglucomutase-1 was accomplished in 80 min in a 0.2-mm-thick polyacrylamide gel with an interelectrode wick distance of 8.0 cm. The gel contained 1.2% (w/v) N-(2-hydroxyethyl) piperazine-N-3-propanesulfonic acid (EPPS) and pH 5 to 7 ampholytes (4% w/v). When maintained at room temperature, laboratory-prepared bloodstains and semen stains could be typed for phosphoglucomutase-1 up to four months and three weeks, respectively. An evaluation of phosphoglucomutase-1 typing by isoelectric focusing and the Group I system was performed on casework samples submitted to the FBI Laboratory. In addition to the increased discriminating probability of phosphoglucomutase-1 when subtyped, isoelectric focusing yielded an increase in positive calls on questioned bloodstains (65.6 versus 36.2%) and dried seminal stains (16.4 versus 13.1%) compared with the Group I system.     

Building bridges between the parole officer and the families of serious juvenile offenders: a preliminary report on a family-based parole program

Responding to a model of juvenile offender case supervision that called for a new intervention paradigm to guide the work of probation and parole officers, the present article reports on the use of a family-based parole initiative known as the Growing Up FAST program. Developed in part as a tool for use within this new intervention paradigm, the Growing Up FAST parole program targets serious youthful offenders who have been released from juvenile correctional facilities and their families. Based on elements contained within the "what works" literature and the Balanced and Restorative Justice model, this program recognizes the central role that field staff can play in rehabilitation efforts. Demographic information and formative data regarding the first set of families to participate in this program are presented, then program limitations and lessons learned as part of the initial offering of this parole initiative are discussed.     

Morphologic patterns of lip prints in a portuguese population: a preliminary analysis

Lip prints are thought to have the ability to distinguish individuals and, hence, have a potential use in human identification purposes. However, questions remain regarding their utility for sex determination. This study aimed to classify lip prints for different individuals in a Portuguese population and to determine whether sex differences exist. Lip prints of 25 females and 25 males were obtained using dark-colored lipstick and cellophane tape. Lip prints were analyzed using a magnifying lens and classified according to the Suzuki and Tsuchihashi classification. A Type II pattern was found to be most common. A comparison of lip-print patterns between males and females showed results with a statistically significant difference: Type III pattern was most common in males, and a Type II pattern in females. This study corroborates the hypothesis that lip prints are able to distinguish individuals and may be useful in sex determination.     

A preliminary study on findings of psychopathy and affective disorders in adult sex offenders

The construct of psychopathy has often been considered mutually exclusive to the presence of genuine depressive or anxiety symptomotology. This article addresses the hypothesized reasons for this dichotomous relationship. In this study, 68 civilly committed adult male sex offenders were evaluated using a variety of psychological measures to determine if psychopathic individuals in this group would demonstrate clinically significant affective symptoms. Results indicate that the men in this sample endorsed high rates of psychopathy on the PCL-R, with 42% of these expressing concurrent symptoms of depression and 26% manifesting symptoms of an anxiety disorder. Relationships between affective symptoms and PCL-R factor scores and qualitative differences between these constructs in child molesters and rapists are also discussed.