Comparative studies on tissue distributions of organophosphorus, carbamate and organochlorine pesticides in decedents intoxicated with these chemicals

This paper describes the tissue distributions of dichlorvos, an organophosphate, chlorpyrifos-methyl, an organophosphorothioate, methomyl, a carbamate, and endrin, an organochlorine, in three individuals (Cases 1-3) who died after ingesting insecticidal preparations containing these chemicals. In Case 1 involving dichlorvos and chlorpyrifos-methyl, no dichlorvos was detected in most of the blood and tissue samples. Tiny amounts of dichlorvos (0.067 mg/L and 0.027 mg/L) were detected in the vitreous humor and cerebrospinal fluid, respectively. The chlorpyrifos-methyl concentrations in the blood samples were very site-dependent with a range of 0.615-2.24 mg/L. The tissue concentrations of chlorpyrifos-methyl were within the range 0.379-8.60 mg/kg. The total amounts of dichlorvos and chlorpyrifos-methyl in the stomach were 879 and 612 mg, respectively. The serum cholinesterase activity was 3 IU/L/37 degrees C. In Case 2 involving methomyl, the methomyl concentrations in the blood samples were very site-dependent with a range of 0.56-4.75 mg/L. The tissue concentrations of methomyl were 2.61 mg/kg or less, no methomyl being detected in the spleen, liver and kidney. The methomyl concentrations in the cerebrospinal fluid and vitreous humor were 5.37 and 4.75 mg/L, respectively. The stomach contained 85 mg methomyl. The serum cholinesterase activity was 73 IU/L/37 degrees C. In Case 3 involving endrin, the victim underwent medical treatment for 7 h after ingesting an endrin preparation. The differences in the endrin concentrations among the blood samples were small, with a range of 0.353-0.615 mg/L. The tissue concentrations of endrin were within the range 0.467-13.3 mg/kg. The endrin in the stomach (66 mg) was adsorbed almost completely on the activated charcoal that was administered for medical treatment.     

Acute intoxication caused by overdose of flunitrazepam and triazolam: high concentration of metabolites detected at autopsy examination

ABSTRACT: A 52-year-old woman was found dead on the floor of the living room on the first floor of a house, which belonged to the man with whom she shared the house. On visiting the site, her clothes were found to be undisturbed. Packages of flunitrazepam (Silece, 2 mg/tablet) and triazolam (Halcion, 0.25 mg/tablet) were found strewn around the victim. Toxicological analysis was performed, and the concentrations of flunitrazepam, triazolam, and their metabolites in the victim's blood and urine were measured by high-performance liquid chromatography coupled with photodiode array and mass spectrometry. A high blood concentration of 7-aminoflunitrazepam was detected (1,270 ng/g), and further metabolites such as 7-acetamidoflunitrazepam, 7-acetamidodesmethylflunitrazepam, and 7-aminodesmethylflunitrazepam were detected in the blood and urine samples. In addition, 4-hydroxytriazolam and α-hydroxytriazolam were detected in her urine at a concentration of 950 and 12,100 ng/mL, respectively.On the basis of the autopsy findings and toxicology results of high concentrations of both flunitrazepam and triazolam derivatives, the cause of death was determined to be acute intoxication from flunitrazepam and triazolam.     

用气相色谱法/电子捕获检测器测定尿液中的三唑仑

建立了用气相色谱／电子捕获检测器测定尿液中三唑仑含量的方法。2ml尿样在破性条件下用2ml×2氯仿提取净化后,60℃水浴下用空气吹干,残留物用环己烷定容溶解后,进气相色谱仪分析,三唑仑的保留时间为10．74min。最低检测限为0．5ng／ml,回收率为95．98％,变异系数为7．85％（n＝5）。在2～50ng／ml浓度范围内有良好的线性关系：A＝－67．9＋570．IC,r＝0．9939。     

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.     

Evaluation of nicotine and cotinine in human hair.

To validate data on tobacco use, the authors investigated the use of hair samples for quantifying nicotine and cotinine by gas chromatography/mass spectrometry. Hair was taken from 22 nonsmokers and 42 smokers, cut close to the scalp at the back of the head. The hair (about 100 mg from each subject) was incubated in 3 mL of 1N NaOH at 100 degrees C for 1 h. After this, the samples were extracted by diethyl ether. The drugs were separated on a 12-m BP-5 capillary column and detected using selected ion monitoring (nicotine, m/z 84; cotinine, m/z 98). Hair from nonsmokers and smokers contains nicotine and cotinine. Although it is difficult to determine an absolute cutoff level, an amount greater than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from nonsmokers. In the population of nonsmokers, the influence of environmental smoke exposure was noted.     

Thiodicarb and methomyl tissue distribution in a fatal multiple compounds poisoning

Abstract:  Thiodicarb is a nonsystemic carbamate insecticide whose acetylcholinesterase activity is related to its main methomyl degradation product. A 40-year-old woman was found dead in her car. Empty packages of medicines and an open bottle of Larvin^® containing thiodicarb were found near her body. No signs of violence nor traumatic injuries were noticed upon autopsy, and police investigations strongly suggested a suicide. Systematic toxicological analysis performed on postmortem specimens revealed the presence of various sedatives, hypnotics, and antipsychotic drugs in blood, urine, and gastric content. Some of the compounds identified were determined at blood concentrations well above the known therapeutic concentrations: zolpidem (2.87 mg/L), bromazepam (2.39 mg/L), nordazepam (4.21 mg/L), and levopremazine (0.64 mg/L). Specific analysis of thiodicarb and of its methomyl metabolite was then performed on all fluids and tissues collected during autopsy by liquid chromatography ion trap tandem mass spectrometry (LC-MS-MS). The anticholinesterase capacity of blood, urine, and gastric content collected at autopsy was 83%, 82%, and 32%, respectively (normal value: 0%). The presence of thiodicarb in the bottle found near the body corroborates the hypothesis of an intake of that compound. Although thiodicarb was only detected in gastric content (24.3 mg/L), its methomyl metabolite was quantified in most postmortem tissues and fluids: gastric content (19.9 mg/L), peripheral blood (0.7 mg/L), urine (8.5 mg/L), bile (2.7 mg/L), liver (0.7 mg/kg), kidney (1.7 mg/kg), lung (1.5 mg/kg), brain (9.3 mg/kg), and heart (3.6 mg/kg).     

Nicotine and cotinine levels in body fluids of smokers who committed suicide

Cigarette smoking is associated with a higher risk for suicide. The present study was conducted on the hypothesis that suicide smokers show higher nicotine and cotinine levels in blood and urine than non-suicide smokers. We determined nicotine and cotinine levels in blood and urine of 87 deceased individuals (18 suicides and 69 non-suicides) by gas chromatography. The smoking rate was 77.8% for individuals who committed suicide and 42.0% for those who did not commit suicide. Average nicotine and cotinine levels in blood were significantly higher in the suicide smokers than in the non-suicide smokers (nicotine: 93.2+/-46.6 ng/ml versus 25.8+/-14.4 ng/ml, p<0.0001 and cotinine: 378+/-235 ng/ml versus 201+/-137 ng/ml, p<0.005). Average levels of urinary nicotine and cotinine were also significantly higher in the suicide smokers than in the non-suicide smokers (nicotine: 1980+/-2210 ng/ml versus 394+/-376 ng/ml, p<0.005 and cotinine: 1170+/-1330 ng/ml versus 414+/-290 ng/ml, p<0.05). Twenty-six decedents were intoxicated with alcohol, and they included 7 suicides (7 smokers) and 19 non-suicides (15 smokers). Our data suggest that cigarette smokers who commit suicide smoke more heavily than other cigarette smokers.     

Accidental fatal poisoning by Nicotiana glauca: identification of anabasine by high performance liquid chromatography/photodiode array/mass spectrometry

A method, based on reversed phase high performance liquid chromatography (HPLC) was developed for the detection and quantification of anabasine, the toxic alkaloid of Nicotiana glauca, in forensic applications. A standard solid phase extraction (SPE) method was used for the extraction of anabasine from viscera, but was optimized for the extraction of this alkaloid from plant material. The careful selection of mobile phase components allowed the direct coupling of electron impact (EI) and Z spray mass selective detector (ZMD) of the HPLC. Under these conditions, anabasine was well separated from nicotine and could be detected on the PDA (limit of detection, LOD = 250 ng/ml), TMD (LOD = 10 microg/ml) and ZMD (LOD =1 ng/ml) detectors. Three geographically isolated N. glauca trees were analyzed for alkaloid content and it was found that both the leaves and the flowers contain anabasine. The optimized HPLC method was used to analyze two viscera samples (the stomach and contents of a mother and child who putatively died from food poisoning) and a flower exhibit. Anabasine was detected in both the viscera samples, supporting the finding that these fatalities were due to the ingestion of N. glauca accidentally collected with traditional spinach (marog). The alkaloid profile of the flower exhibit submitted with the viscera samples was similar to those obtained from flowers collected from three different N. glauca trees. The results show that anabasine and/or N. glauca poisoning can easily be confirmed using the forensic methodology described.     

A multi-chemical death involving caffeine, nicotine and malathion

Death of a 21-year-old man who was found in a shower stall in his residence is described in the study. At the scene, a 3/4 filled blue glass bottle labeled "Black Leaf 40" (an insecticide containing nicotine), a white plastic pitcher 1/3 full of thick white fluid, a beer mug 1/4 full of thick white fluid, and an empty carton of milk were found. In addition, a can of malathion and an empty bottle labeled caffeine also were found in the vicinity. Autopsy was performed, and the gross examinations of organs revealed no specific findings to account for the death. However, marked congestion in lung, liver, spleen and kidney were noted at microscopic level. Autolytic degenerative changes were also observed in stomach, small bowel and colon. Toxicological analyses of the autopsy samples (blood, urine, liver and gastric contents) revealed the presence of caffeine and nicotine in each sample. Malathion was found to be present only in gastric content. Caffeine and nicotine were analyzed by utilizing gas liquid chromatography-nitrogen phosphorus detector, while malathion was by gas liquid chromatography-flame photometric detector. Analyses of the fluids from the bottle, pitcher and mug disclosed the presence of nicotine in the concentrations of 17.8%, 3.7% and 5.7% (w/w), respectively. The fluids from the pitcher and mug also contained 2.7-2.9% malathion. Results conclude the death was associated with caffeine, nicotine and malathion.     

Last performance with VIAGRA: post-mortem identification of sildenafil and its metabolites in biological specimens including hair sample

A 43-year-old man was found dead in a hotel room during a sexual relation with a colleague.He was treated both for cardiovascular disease and for erectile dysfunction with VIAGRA. A pillbox was found in the room with several tablets of verapamil (Isoptine), trimetazidine (Vastarel), yohimbine and bromazepam (Lexomil). A box of VIAGRA 25mg was found in his raincoat and two tablets were missing. His wife declared during the investigation that he was also treated by trinitrine. Autopsy revealed severe coronary artery sclerosis as well as signs of previous myocardial infarctions. Blood, urine, bile, gastric content and hair and representative tissues for histology were collected for toxicological analysis.Sildenafil and yohimbine were screened with liquid chromatography/mass spectrometry (LC/MS) and trinitrine with headspace injection (HS)/GC/MS. Verapamil and trimetazidine were identified and quantified with LC/diode array detection (DAD).Sildenafil was identified in blood, urine, bile and gastric content at 105, 246, 1206 and 754ng/ml, respectively. Hair concentration was 177pg/mg. The desmethyl metabolite was quantified in urine at 143ng/ml. Blood concentrations of verapamil and trimetazidine were measured at 659 and 2133ng/ml, respectively and were above therapeutic ranges. Trinitrine and yohimbine were not identified.These results confirm the absorption of sildenafil, verapamil and trimetazidine before the death and hair analysis indicates the chronic use of sildenafil.To the author's knowledge, this is the first report of a fatal sildenafil-verapamil association, probably by hypotension and cardiac dysrhythmia.     

A fentanyl fatality involving midazolam

A case is presented of a 35-year-old black African male anesthesiology resident, found dead in his apartment. At the scene a syringe, butterfly intravenous line and a bottle of Versed (Midazolam) were recovered. A comprehensive screen for common drugs of abuse and therapeutic agents failed to detect any drugs in blood and urine. The blood ethanol concentration was 0.06 g/dl. A GC/MS SIM assay for midazolam was developed. A sub-therapeutic midazolam blood concentration of 7.5 ng/ml was detected and concentrations (ng/ml or ng/g) in bile, urine, and liver were 3.3, 7.5, and 96, respectively. The syringe fluid was then analyzed and found to contain only fentanyl, midazolam was absent. The blood fentanyl concentration was 4.9 ng/ml which is consistent with those reported in fentanyl fatalities. Fentanyl concentrations (ng/ml or ng/g) in bile, urine, and liver were 8.8, 5.0, and 5.9, respectively. The cause of death was ruled to be fentanyl intoxication and the manner of death undetermined.     

Tissue distribution of DDVP after fatal ingestion

Tissue distribution of dichlorvos (DDVP) was determined in a case of fatal ingestion using a rapid and simple gas chromatographic (GC) assay. Remarkable autopsy findings were congestion of the lung and kidneys and bleeding ulcer extending from the dorsum of the tongue to the upper pharynx. The serum cholinesterase activity was 2 IU/l, however, miosis was not observed. In the stomach, 250 ml of volatile fluid was found. Tissue distribution of DDVP was determined using a newly developed simple and rapid GC method. DDVP was found in the spleen and heart at higher concentrations (3340 and 815 μg/g, respectively), and also detected in the urine at the lowest level (4.5 μg/ml). The DDVP concentrations in blood, brain, lung, kidney and liver were 29, 9.7, 81, 80 and 20 μg/ml or g, respectively.     

Fatal acute alcohol intoxication in an ALDH2 heterozygote: a case report

On an evening in November, a 25-year-old man was found dead in his bedroom. There were many empty snap-out sheets for flunitrazepam tablets in the trash at his bedside. He had been beaten by a gang of young people earlier in the morning of the same day. At the medico-legal autopsy, although there were many bruises and/or abrasions on the whole body, only slight subdural hemorrhage was observed, and none of them was thought to be the cause of death. Flunitrazepam and its metabolites were not detected in his body fluid by gas chromatography-mass spectrometry (GC-MS). Marked lung edema and a severe congestion of organs were observed. His blood alcohol concentration from the femoral vein was 2.00 mg/ml. Fatal cases of acute alcohol intoxication usually have shown higher alcohol concentration (2.25-6.23 mg/ml). Although the genotype of aldehyde dehydrogenase 2 (ALDH2) has not previously been mentioned as a contributing factor in determining the cause of death, in this case the genotype of ALDH2 was ALDH2*1/2 and thus is important. Those who possess the ALDH2*2 gene show high concentrations of acetaldehyde (AcH) at even comparatively lower alcohol levels. Consequently, the cause of death was considered to be acute alcohol intoxication including AcH poisoning.     

Plasma,oral fluid and sweat wipe ecstasy concentrations in controlled and real life conditions

In a double-blind placebo controlled study on psychomotor skills important for car driving (Study 1), a 75 mg dose of +/- 3,4-methylenedioxymethamphetamine (MDMA) was administered orally to 12 healthy volunteers who were known to be recreational MDMA-users. Toxicokinetic data were gathered by analysis of blood, urine, oral fluid and sweat wipes collected during the first 5h after administration. Resultant plasma concentrations varied from 21 to 295 ng/ml, with an average peak concentration of 178 ng/ml observed between 2 and 4h after administration. MDA concentrations never exceeded 20 ng/ml. Corresponding MDMA concentrations in oral fluid, as measured with a specific LC-MS/MS method (which required only 50 microl of oral fluid), generally exceeded those in plasma and peaked at an average concentration of 1215 ng/ml. A substantial intra- and inter-subject variability was observed with this matrix, and values ranged from 50 to 6982 ng/ml MDMA. Somewhat surprisingly, even 4-5h after ingestion, the MDMA levels in sweat only averaged 25 ng/wipe. In addition to this controlled study, data were collected from 19 MDMA-users who participated in a driving simulator study (Study 2), comparing sober non-drug conditions with MDMA-only and multiple drug use conditions. In this particular study, urine samples were used for general drug screening and oral fluid was collected as an alternative to blood sampling. Analysis of oral fluid samples by LC-MS/MS revealed an average MDMA/MDEA concentration of 1121 ng/ml in the MDMA-only condition, with large inter-subject variability. This was also the case in the multiple drug condition, where generally, significantly higher concentrations of MDMA, MDEA and/or amphetamine were detected in the oral fluid samples. Urine screening revealed the presence of combinations such as MDMA, MDEA, amph, cannabis, cocaine, LSD and psilocine in the multiple-drug condition.     

The study of metabolite-to-parent drug ratios of methamphetamine and methylenedioxymethamphetamine in hair

The metabolite-to-parent drug ratios were determined in the hair of 2444 methamphetamine (MA) abusers who had produced MA-positive hair results from 2001 to May 2005 and in the hair of 53 ecstasy abusers who had produced positive methylenedioxymethamphetamine (MDMA) hair results from 2002 to May 2005. For the hair analyses, hair strands were washed, cut into small pieces and extracted for 20 h in 1 mL methanol containing 1% HCl. Drugs in the extract were determined by gas chromatography-mass spectrometry (GC-MS) using selective ion monitoring after derivatization with trifluoroacetic anhydride. The six range groups were divided as follows on the basis of MA concentrations in hair (n = 2389): 0.5-5 ng/mg (n = 950), 5-10 ng/mg (n = 582), 10-20 ng/mg (n = 503), 20-30 ng/mg (n = 160), 30-40 ng/mg (n = 80), more than 40 ng/mg (n = 114) to assess the correlations between MA concentrations and metabolite-to-parent drug ratios. In groups of higher MA concentrations, lower ratios of AP/MA were found, and there was a statistically significant difference among six range groups. Comparisons of age groups (tens, twenties, thirties, forties, fifties, and sixties) and male and female subjects for the ratios of AP/MA showed a statistically significant difference. The detection of metabolites and the parent drug with reasonable ratios was found to be a useful indicator for distinguishing internal drug incorporation from external contamination. In our study, MA users can produce 0.4-116% (mean = 9%) of amphetamine (AP) concentrations in hair, and ecstasy users 1-110% (mean = 12%) of methylenedioxyamphetamine (MDA) in appropriately washed hair samples.     

Nails of newborns in monitoring drug exposure during pregnancy

This project was developed to investigate the usefulness of newborn nails for monitoring in utero drug exposure. Cocaine, benzoylecgonine, morphine, methadone, caffeine, nicotine, and cotinine were determined in nail samples from the first 3 months of life of 25 newborns abandoned immediately after birth (group 1) and of 33 babies born at the local maternity hospital whose families were recruited on a voluntary basis (group 2). All substances were measured by gas chromatography-mass spectrometry (detection limit: 0.025 ng/mg). Moreover, analytical results were compared with mothers' self-reported habits when the information was available. In group 1, 12 nails were found positive for caffeine and 13 for both nicotine and cotinine. Six samples tested cocaine- (range, median: 0.14-0.25, 0.175 ng/mg) and benzoylecgonine-positive (range, median: 0.12-0.20, 0.165 ng/mg). Both nicotine and cocaine were always retrieved together with their main metabolite. Morphine was found in four samples (range, median: 0.10-0.15, 0.125 ng/mg), methadone in five samples (range, median: 0.12-0.26, 0.170 ng/mg) that were found negative for all other compounds. In group 2, two samples tested positive for methadone (0.16, 0.17 ng/mg). The mothers self-report of the use of coffee always corresponded to caffeine positivity in the newborn nails (n=6), whereas six samples tested positive for nicotine and/or cotinine with a non-smoking mother. Sixteen out of the 33 samples of group 2 tested negative for all compounds. In conclusion, for the first time, results showed that, once that sample collection problems are solved, nails of the first period of life can be a very interesting indicator of in utero drug exposure.     

A fatal case of pure ethanol ingestion

An adult male was found dead in a car with two empty bottles (500 ml x 2) labeled dehydrated ethanol (>99.5%, v/v). At autopsy, extensive pancreatic necrosis with severe hemorrhage was observed. High concentrations of ethanol were detected in blood (8.14 mg/ml), urine (8.12 mg/ml) and tissue specimens. The cause of death was determined to be an acute alcohol intoxication caused by ingesting approximately 1l dehydrated ethanol.     

A fatal case of oleandrin poisoning

The study presents a case of fatal poisoning with oleander leaves in an adult diabetic male. After repeated vomiting, and gastrointestinal distress the patient was admitted at the hospital with cardiac symptoms 1h after the ingestion. Urine samples were assayed immunochemically and by GC-MS for drugs of abuse and for general toxicological screen. Blood was analyzed for alcohol and volatiles by static head space GC-MS. Blood and oleander leaves were analyzed by LC-MS/MS for oleandrin and related compounds, the main cardiac glycosides of Nerium oleander. Oleandrin was detected by LC-MS/MS in the blood sample at a concentration of approximately 10 ng/ml. Another cardiac glycoside with pseudo-molecular ion of m/z 577, a likely structural isomer of oleandrin, was also detected in the blood and oleander leaves. However, by using the response as a function of concentration for oleandrin, this cardiac glycoside was roughly estimated at a concentration of approximately 10 ng/ml in the deceased blood. This would give a total fatal blood concentration of cardiac glycosides of about approximately 20 ng/ml in the deceased blood.     

UPLC-MS/MS法检测血痕中常见烟草、毒品和药物成分

目的建立UPLC-MS/MS测定血痕中尼古丁、可替宁、甲基苯丙胺、氯胺酮、吗啡、O6-单乙酰吗啡、地西泮、三唑仑、艾司唑仑、佐匹克隆和利血平的方法。方法以甲醇为提取液,采用基质提取溶液配制标准溶液制作定量曲线。采用超高效液相色谱柱对待测样品进行分离;以电喷雾离子源正离子（ESI＋）模式和多反应监测（MRM）模式进行质谱分析。优化实验条件,并进行方法学评价。结果选择UPLC HSS T3色谱柱,乙腈-5mmol/L甲酸铵＋0.1%（v/v）甲酸作为流动相,甲醇作为提取溶剂。11种检测物检出限（S/N=3）和定量限（S/N=10）分别为0.04~2.82ng和0.13~7.64ng,1~500ng/mL范围内的线性关系良好。除利血平、吗啡外的检测物回收率为（53.8±5.3）%~（107.2±12.6）%。结论采用本文UPLC-MS/MS方法对血痕中11种常见烟草、毒品和药物成分进行检测,能够满足实际检案的要求,可在相关检验中选用。