Blood cyanide determination in two cases of fatal intoxication: comparison between headspace gas chromatography and a spectrophotometric method

Blood samples of two cases were analyzed preliminarily by a classical spectrophotometric method (VIS) and by an automated headspace gas chromatographic method with nitrogen-phosphorus detector (HS-GC/NPD). In the former, hydrogen cyanide (HCN) was quantitatively determined by measuring the absorbance of chromophores forming as a result of interaction with chloramine T. In the automated HS-GC/NPD method, blood was placed in a headspace vial, internal standard (acetonitrile) and acetic acid were then added. This resulted in cyanide being liberated as HCN. The spectrophotometric (VIS) and HS-GC/NPD methods were validated on postmortem blood samples fortified with potassium cyanide in the ranges 0.5-10 and 0.05-5 mug/mL, respectively. Detection limits were 0.2 mug/mL for VIS and 0.05 mug/mL for HS-GC/NPD. This work shows that results obtained by means of the two procedures were insignificantly different and that they compared favorably. They are suitable for rapid diagnosis of cyanide in postmortem cases.     

Toxicological analysis after asphyxial suicide with helium and a plastic bag

A 23-year-old man was found on a raised hide in lying position, the head wrapped in a plastic bag connected with a helium gas cylinder by a polypropylene tube. The autopsy did not show any specific findings nor did the routine toxicological analysis reveal significant information regarding the cause of death (BAC 0.9 mg/g, diphenhydramine 0.81 μg/ml in heart serum). For the detection of helium in the lungs, gas samples from both lungs were collected by a method ensuring minimal dilution. Gas analyses were performed using a GC–MS with a split–splitless injector and a headspace syringe. As carrier gas the commonly used helium was replaced by nitrogen. Helium was found in clearly elevated concentrations in gas samples from both lungs. Therefore, suffocation by breathing helium enriched, and thus oxygen deficient atmosphere, can strongly be assumed as the cause of death.     

Death due to inhalation of ethyl chloride

A 30-year-old white male was found dead in a locked apartment with a rag held loosely in his mouth. Four cans (3 empty, 1 partially empty) containing ethyl chloride and labeled as VCR head cleaner were found next to the body. Phenylpropanolamine and low therapeutic levels of diazepam (64 microg/L) and nordiazepam (126 microg/L) were detected during toxicological analysis. An unidentified peak was observed when performing ethanol analysis by headspace gas chromatography. The peak was identified as ethyl chloride and the concentrations in the blood, urine, vitreous, brain, and lungs of the deceased were 423 mg/L, 35 mg/L, 12 mg/L, 858 mg/kg, and 86 mg/kg, respectively. The results were compared with previously reported levels of ethyl chloride in blood and vitreous and, based on a literature search, we believe that this is the first report of ethyl chloride levels in tissue.     

直接进样气相色谱法测定全血中乙醇的含量

目的建立一种直接进样气相色谱法检测全血中的乙醇。方法全血经硫酸铝钾沉淀蛋白,加入内标异丙醇后,用直接进样气相色谱法进行检测,FID为检测器,用保留时间定性,内标标准曲线法定量。结果该方法线性范围为0.2-1.4mg/m L,相关系数R=0.999 3,总分析时间不超过5min,最低检测限为0.01mg/m L,相对标准偏差（RSD）〈5%,平均回收率为92.3%;所建立的方法与顶空气相色谱（HS-GC）法比较相对偏差（RD）〈10%。结论该方法可用于全血中乙醇的检测。     

Computer-aided headspace gas chromatography applied to blood-alcohol analysis: importance of online process control

This paper describes the analysis of ethanol in blood specimens from suspect drunk drivers and the associated quality assurance procedures currently used in Sweden for legal purposes. Aliquots of whole blood from two separate Vacutainer tubes are diluted with 1-propanol as internal standard before analysis by headspace gas chromatography (HS-GC) with three different stationary phases: Carbopak B, Carbopak C, and 15% Carbowax 20 M. The actual HS-GC analysis, the integration of chromatographic peaks, the collection and processing of results, as well as the quality control tests involve the use of computer-aided techniques. The standard deviation of analysis(y) increased with concentration of ethanol in the blood specimen(x), and above 0.50 mg/g the regression equation was y = 0.0033 + 0.0153x. The prosecution blood-alcohol concentration (BAC) is the mean of three separate determinations made by different laboratory technicians working independently with different sets of equipment. A deduction is made from the mean analytical result to compensate for random and systematic errors inherent in the method. At BACs of 0.5 and 1.5 mg/g, which are the statutory limits in Sweden, the allowances currently made are 0.06 and 0.09 mg/g, respectively. Accordingly, the reduced prosecution BAC is less than the actual BAC with a statistical confidence of 99.9%.     

血液样品中乙醇稳定性的实验研究

目的考察在不同存放条件下含乙醇的血液样品中乙醇浓度的变化情况。方法采用顶空-气相色谱法,以异丙醇为内标,对存放条件不同的血液样品中乙醇进行检测。结果冷冻(-10℃)条件存放1至30天,血液样品中乙醇含量无显著变化;冷藏(4℃)条件存放1至30天,血液样品中乙醇含量变化不显著;室温(28℃~33℃)条件存放1至30天,血液样品中乙醇含量显著改变。结论含有乙醇的血液样品,在冷冻、冷藏条件下可较稳定的存放30天;在室温条件下存放30天血液样品中乙醇浓度发生显著变化,不可在此条件下存放。     

Tissue distribution of olanzapine in a postmortem case

Olanzapine is a relatively new antipsychotic drug used in the United States for the treatment of schizophrenia. Since its release in the United States market in 1996, few cases of fatal acute intoxication have been reported in the literature. This article describes the case of a 25-year-old man found dead at home who had been prescribed olanzapine for schizophrenia. This case is unique because of the measurement of olanzapine in brain tissue obtained from seven regions in addition to the commonly collected biologic matrices. Olanzapine was detected and quantitated by basic liquid-liquid extraction followed by dual-column gas chromatographic analysis with nitrogen phosphorus detection. The assay had a limit of detection of 0.05 mg/L and an upper limit of linearity of 2 mg/L. The presence of olanzapine was confirmed by gas chromatography-mass spectrometry by use of electron impact ionization. The concentrations of olanzapine measured in this case were as follows (mg/L or mg/kg): 0.40 (heart blood), 0.27 (carotid blood), 0.35 (urine), 0.61 (liver), negative (cerebrospinal fluid), 0.33 mg in 50 ml (gastric contents). In the brain, the following distribution of olanzapine was determined (mg/kg): negative (cerebellum), 0.22 (hippocampus), 0.86 (midbrain), 0.16 (amygdala), 0.39 (caudate/putamen), 0.17 (left frontal cortex), and 0.37 (right frontal cortex). The cause of death was determined to be acute intoxication by olanzapine, and the manner of death was accidental.     

Stability of blood carbon monoxide and hemoglobins during heating

The effects of heating on hemoglobin (Hb) and carbon monoxide (CO) levels in human blood were investigated by in vitro experiments. Head-space gas chromatography (HS-GC) using a molecular sieve 5A stationary phase and thermal conductivity detection was adopted for the measurement of CO gas, and spectrophotometric methods were used for the measurement of various Hb forms, protein and heme contents. Deteriorated absorbance spectra were observed for heat-treated blood samples, and double wavelength spectrophotometry was proven to give wrong percent saturation of carboxyhemoglobin content (% CO-Hb). The blood sample taken from one fatal fire casualty gave significantly higher % CO-Hb measured spectrophotometrically, compared to that by HS-GC. Control blood or purified Hb solution, which was saturated with CO in designated extent, was heated in a sealed vial. Under the incubation below 54 degrees C, all Hb forms were stable, except for oxyhemoglobin (Hb-O(2)), which was partially oxidized to met-hemoglobin (Met-Hb). In contrast, under the incubation at 65 degrees C, Met-Hb was denatured completely to be insoluble, and Hb-O(2) was partially denatured via Met-Hb formation. CO-Hb was resistant against heating. The difference of heat susceptibility and precipitability among Hb forms resulted in artificial increase of % CO-Hb. During heating, spontaneous CO was produced from blood.     

The analysis of an intracerebral hematoma for drugs of abuse

Toxicological investigations were performed on an intracerebral hematoma, antemortem blood, and postmortem blood of an individual who was found unresponsive in his home. The hematoma was found to have ethanol at a concentration of 0.05% (w/v), and benzoylecgonine (a cocaine metabolite) was also confirmed at a concentration of 0.43 mg/L by specific analysis using gas chromatography/mass spectrometry (GC/MS). These results enabled the pathologist to record the cause of death as intracerebral hemorrhage due to acute cocaine intoxication.     

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.     

Concentrations of cocaine and its major metabolite benzoylecgonine in blood samples from apprehended drivers in Sweden

Cocaine and its major metabolite benzoylecgonine (BZE) were determined in blood samples from people arrested in Sweden for driving under the influence of drugs (DUID) over a 5-year period (2000-2004). Venous blood or urine if available, was subjected to a broad toxicological screening analysis for cannabis, cocaine metabolite, amphetamines, opiates and the major benzodiazepines. Verification and quantitative analysis of cocaine and BZE in blood was done by gas chromatography-mass spectrometry (GC-MS) at limits of quantitation (LOQ) of 0.02mg/L for both substances. Over the study period 26,567 blood samples were analyzed and cocaine and/or BZE were verified in 795 cases (3%). The motorists using cocaine were predominantly men (>96%) with an average age of 28.3+/-7.1 years (+/-standard deviation, S.D.). The concentration of cocaine was below LOQ in 574 cases although BZE was determined at mean, median and highest concentrations of 0.19mg/L, 0.12mg/L and 1.3mg/L, respectively. In 221 cases, cocaine and BZE were together in the blood samples at mean and (median) concentrations of 0.076mg/L (0.05mg/L) and 0.859mg/L (0.70mg/L), respectively. The concentrations of BZE were always higher than the parent drug; mean BZE/cocaine ratio 14.2 (median 10.9) range 1-55. Cocaine and BZE were the only psychoactive substances reported in N=61 cases at mean (median) and highest concentrations of 0.095 (0.07) and 0.5mg/L for cocaine and 1.01 (0.70) and 3.1mg/L for BZE. Typical signs of drug influence noted by the arresting police officers included bloodshot and glossy eyes, agitation, difficulty in sitting still and incoherent speech.     

Concentrations of zolpidem and zopiclone in venous blood samples from impaired drivers compared with femoral blood from forensic autopsies

The concentrations of zolpidem and zopiclone were determined in peripheral blood samples in two forensic materials collected over a 10-year period (2001-2010). The z-hypnotics were determined in venous blood from living subjects (impaired drivers) and in femoral blood from deceased persons (forensic autopsies), with the latter classified as intoxication or other causes of death. The z-hypnotics were determined in blood by capillary column gas chromatography (GC) with a nitrogen-phosphorous (N-P) detector after solvent extraction with n-butyl acetate. The analytical limit of quantitation (LOQ) was 0.02mg/L for zopiclone and 0.05mg/L for zolpidem and these have remained unchanged throughout the study. When death was attributed to drug intoxication (N=918), the median concentration of zopiclone in blood was 0.20mg/L compared with 0.06mg/L for other causes of death (N=1215) and 0.07mg/L in traffic offenders (N=691) (p<0.001). Likewise, a higher median concentration (0.30mg/L) was found in intoxication deaths involving zolpidem (N=357) compared with 0.13mg/L for other causes of death (N=397) or 0.19mg/L in impaired drivers (N=837) (p<0.001). Median concentration in blood of both z-hypnotics were appreciably higher in intoxication deaths when no other substances were identified; 0 70mg/L (N=12) for zopiclone and 1.35mg/L (N=12) for zolpidem. The median concentrations of z-hypnotics in blood decreased as the number of co-ingested substances increased for intoxication deaths but not other causes of death. The most prevalent co-ingested substances were ethanol in autopsy cases and diazepam in the motorists. This large compilation of forensic cases should prove useful when toxicologists are required to interpret concentrations of z-hypnotics in blood samples in relation to cause of death.     

A fatal case of mercuric cyanide poisoning

A 57-year-old pharmacist was found dead 11 days after his disappearance. At the autopsy, samples of blood, urine, gastric content were obtained. Presence of ethanol, cyanide and mercury were detected in some samples. Cyanide and mercury were identified and quantified using high-performance liquid chromatography with diode array detector (HPLC) in fluorescence mode and ICP with mass selective detector (ICP-MS) respectively. Whole blood concentrations of ethanol was 1.72 g/L. Cyanide and mercury concentrations in whole blood were respectively 0.16 and 3.8 mg/L. Concentrations of cyanide (27 mg/L) and mercury (150 mg/L) in gastric contents prove a massive oral ingestion of mercuric cyanide or mercuric oxycyanide occurred. In this case report, the death was attributed to the combined toxicity of cyanide and mercury.     

Fatal and nonfatal poisoning by hydrogen sulfide at an industrial waste site

An adult man (A) entered a pit to collect seepage at an industrial waste site in Japan. As he suddenly lost consciousness, three colleagues (B, C, D) entered the pit to rescue him. All of these men lost consciousness in the pit. Two workers (A and B) died soon after the accident, one worker (C) died 22 days after the accident, and one worker (D) survived. Since hydrogen sulfide gas was detected in the atmosphere of the pit, gas poisoning was suspected. Toxicological analyses of sulfide and thiosulfate, a metabolite of sulfide, in blood and urine of the victims were made using the extractive alkylation technique combined with gas chromatography/mass spectrometry (GC/MS). Sulfide was detected in the blood of A and B at levels of 0.13 and 0.11 mg/L, respectively, somewhat higher than in healthy persons. Thiosulfate was detected in whole blood of deceased victims A and B, in the plasma of deceased victim C, at concentrations of 10.53, 4.59, and 4.14 mg/L, respectively. These values were similar to those found in fatal cases of hydrogen sulfide poisoning. Thiosulfate was not detected in the plasma of survivor D. With respect to urine samples, thiosulfate was the highest in the non-acute death victim C (137.20 mg/L), followed by that in the survivor D (29.34 mg/L), and low (0.90 mg/L) and not detected in the acute death victims, A and B, respectively. Based on these results, all four patients were victims of hydrogen sulfide poisoning. The concentrations of thiosulfate in blood and urine were more useful than that for sulfide for determining hydrogen sulfide poisoning. Thiosulfate in urine was the only indicator of hydrogen sulfide poisoning in the non-fatal victim.     

Fatality from olanzapine induced hyperglycemia

A case history of a 31-year-old male schizophrenic patient is presented. The man was treated with olanzapine for three weeks before he died. After one week on a 10 mg daily dose of olanzapine, his fasting blood glucose was elevated to 11.3 mmol/L (203 mg/dL). In order to treat more aggressively his psychosis, the olanzapine dose was raised to 20 mg daily resulting in his fasting blood glucose climbing to 15.8 mmol/l (284 mg/dL). On the days preceding his death, he became progressively weaker, and developed polydipsia with polyuria. He had no personal or family history of diabetes mellitus and he was on no other medication at the time of his death. Postmortem blood, vitreous humor, and urine glucose concentrations were 53 mmol/L (954 mg/dL), 49 mmol/L (882 mg/dL), and 329 mmol/L (5922 mg/dL), respectively. Drug screen on urine and blood indicated only a small amount or olanzapine and no alcohols. Peripheral blood olanzapine concentration was within therapeutic limits, 45 ng/mL. Analysis of vitreous humor and urine revealed severe dehydration with small amounts of ketones. Death was attributed to hyperosmolar nonketotic diabetic coma, and olanzapine was felt most likely to be the cause. Another atypical neuroleptic, clozapine, has also been associated with the development and exacerbation of diabetes mellitus or diabetic ketoacidosis. We recommend including vitreous glucose and beta-hydroxybutyrate analysis as part of postmortem toxicology work up when the drug screen reveals the presence of either olanzapine or clozapine.     

A fatality involving U4Euh, a cyclic derivative of phenylpropanolamine

A fatality following ingestion of diazepam and 4,5-dihydro-4-methyl-5-phenyl-2-oxazolamine, a cyclic derivative of phenylpropanolamine known as U4EuH or 4-methyl aminorex, is described. Solid dosage samples of U4EuH were analyzed using gas chromatography, ultraviolet and infrared spectroscopy, nuclear magnetic resonance, and mass spectrometry. Physiological fluids were analyzed quantitatively by gas chromatography and qualitatively by gas chromatography-mass spectrometry. Concentrations of 4,5-dihydro-4-methyl-5-phenyl-2-oxazolamine were: in blood 21.3 mg/L; in urine 12.3 mg/L. Diazepam concentration in blood was 0.8 mg/L.     

HS-GC内标曲线法测定血中乙醇含量的不确定度评估

目的建立自动顶空-气相色谱(HS-GC)内标曲线法测定血中乙醇含量的不确定评估方法。方法从分析测定程序着手,依据不确定度评定的指导性文件,分析不确定度来源,量化不确定度分量,计算检测结果的合成标准不确定度和扩展不确定度。结果各相对不确定度来自于检材重复性检测为3.4%,乙醇标准溶液为0.71%,检材为0.61%,叔丁醇内标溶液为0.41%,标准曲线为1.1%,气相色谱仪为1.3%,血液中乙醇的相对扩展不确定度为3.9%。结论血液中乙醇含量的不确定度主要来源于检材重复性检测、气相色谱仪、乙醇标准曲线。     

Fatal outcome of poisoning with the benzodiazepines flunitrazepam and diazepam

On a wintry day a 29-year-old woman was found dead beside her car showing head injuries and signs of hypothermia. Several empty packets of sedative and hypnotic drugs were lying inside the car. Toxicological analysis revealed the presence of flunitrazepam (heart blood of the left and right chamber 0.033 mg/L each), norflunitrazepam (left heart blood 0.029 mg/L, right heart blood 0.027 mg/L), 7-amino-flunitrazepam (left heart blood 0.090 mg/L, right heart blood 0.104 mg/L), diazepam (left heart blood 0.395 mg/L, right heart blood 0.386 mg/L), nordazepam (left heart blood 0.112 mg/L, right heart blood 0.115 mg/L) and temazepam (left heart blood 0.034 mg/L, right heart blood 0.033 mg/L). Neither alcohol nor other drugs were found. It was concluded that benzodiazepine intake led to a disturbance of consciousness. Whether the woman died in this situation due to the icy temperature as a result of hypothermia or whether she died or would have died solely due to benzodiazepine overdosage could not be clarified.     

Analytical aspects of volatile substance abuse (VSA)

Through a case report, the authors illustrate the volatile substance abuse (VSA) toxicological investigation difficulties mainly due to evaporation of the compounds from postmortem samples and to the lack of reference data for interpretation. A 17-year-old man, student in a chemistry institute, was found dead with a plastic bag placed over his head. Several chemical substances were found in his belongings. Autopsy findings included serious pulmonary lesions and hemorrhagic digestive ulcerations. A large screening of drugs and toxic compounds and selective analyses for several classes of drugs of abuse were carried out in the autopsy samples. In particular, a headspace (HS), -gas chromatography/-mass spectrometry (GC/MS) technique was used to screen for volatile substances and metabolites in the biological samples and for residues of volatile substances on the surface of the plastic bag and in the chemicals found on the scene. The main analytical finding was the presence of alkanes (heptane, methyl-2-pentane, methyl-3-hexane, methylcyclohexane) in the gastric content. The literature data, VSA practices, long time-delay between death and autopsy, preservation conditions of the biological samples before analysis, and in-lab experiments on evaporation of volatile substances were considered to interpret this result. The present fatality was attributed to VSA with a gasoline-based stain remover like "eau écarlate," associated with a hypoxic recreation practice using a plastic bag.