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“Unusual Causes of Death: from Analysis to Interpretation” Society of Forensic Toxicologist Annual Meeting Tuesday, October 29, 2013, 8:00Am-12:00Pm Orlando, FL

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“Unusual Causes of Death: from Analysis to Interpretation” Society of Forensic Toxicologist Annual Meeting Tuesday, October 29, 2013, 8:00Am-12:00Pm Orlando, FL “Unusual Causes of Death: From Analysis to Interpretation” Society of Forensic Toxicologist Annual Meeting Tuesday, October 29, 2013, 8:00am-12:00pm Orlando, FL Workshop Chairs: Pascal J. Kintz and Jean-Pierre Goullé Schedule: 8:00am-8:05am Welcome & Introduction Dr. Pascal Kintz 8:05am-8:30am Deaths Involving Cyanide Dr. Pascal Kintz 8:30am-9:00am Deaths Involving Metals and Elements Prof Jean-Pierre Goullé 9:00am-9:30am Deaths Involving Plants Dr. Marc Deveaux 9:30am-10:00am Deaths Involving Gas and Volatiles Dr. Jean-Michel Gaulier 10:00am-10:30am Morning Break 10:30am-11:00am Deaths Involving Pesticides Prof Jean-Claude Alvarez 11:00am-11:30am Death Involving Glycemia Control Drugs Dr. Patrick Mura 11:30am-12:00pm Recent Trends in Postmortem Redistribution Prof Anne-Laure Pélissier Pascal Kintz Dr. Pascal Kintz has a degree in Pharmacy (1985), a Diplôme d'Etudes Approfondies in Molecular Pharmacology and a PhD in Toxicology (1989) of the Université Louis Pasteur in Strasbourg. He was Associate Director of the Institute of Legal Medicine of Strasbourg and Associate Professor of Legal Medicine until the end of 2004. Then, he was Head of the Scientific Affairs at ChemTox Laboratory, a private structure in Strasbourg, France (2005-2010). Currently, he is consultant in Toxicology, President of his own company, X-Pertise Consulting. His main topics of interest include: alternative specimens with a special focus on hair and oral fluid, pharmacology of drugs of abuse, postmortem toxicology, drug-facilitated crimes and doping control. He is active in several national and international scientific societies, such as Société Française de Toxicologie Analytique, SFTA (President 1997-2003), The International Association of Forensic Toxicologists, TIAFT (President 2005-2008) and the Society of Hair Testing, SoHT (Founding Member in 1995, President 2008-2012). He received the TIAFT Award for Excellence in 2001, the SFTA Grand Prix (2003) and the IATDMCT Irving Sunshine Award in 2011. He is an Expert for Justice, appointed by the Court of Cassation (French Supreme Court) since 2007, for Pharmacology / Toxicology and blood alcohol determination and an Expert certified by the Gesellschaft für Toxicologische und Forensische Chemie (Germany) and Eurotox. Dr Kintz has published more than 300 papers in peer-reviewed journals and 6 books, including 3 in English. He was associate editor of Journal of Analytical Toxicology and regular reviewer for Journal of Chromatography, Forensic Science International, Clinical Chemistry, Journal of Pharmaceutical Sciences, and Annales de Toxicologie Analytique. 10/4/2013 The cyanide nightmare Pascal KINTZ XX--PertisePertise Consulting Oberhausbergen, France http://www.xhttp://www.x--pertise.compertise.com A criminal case … A 4343--yearyear old woman has been accused of murdering her lover, back in 2004 Only solid body remains (skull, bones) were discovered in 2008 in an open sand quarry in Brazil For toxicological analyses, we received 6 teeth and 1 femur (thighbone) Beside a full screening for illegal drugs and pharmaceuticals, the Judge asked us to test for cyanide Cyanide was analysed by headspace GC/MS after pulverization in a ball mill and sulfuric acid liberation (HS vialvial)) Blood calibrators and controls were used during the analysis A criminal case … Cyanide tested positive in the 2 submitted specimens: - tooth: 5308 ng/gng/g --ffemuremur bone: 4353 ng/gng/g These analyses were interpreted as positive for cyanide but could be hardly interpreted due to the lack of suitable literature reference Test of control material: - 6 teeth (old teeth stored at room temp., dentist): 80 to 200 ng/gng/g - 4 femur bones (stored at room temp.): not detected to 100 ng/gng/g 1 10/4/2013 A criminal case … However, these results were challenged because of - the lack of reference data - the strong deny of the accused woman --tthehe possibility for the victimvictim to have ingested cyanide-cyanide-containingcontaining food, such as bitter almonds,,() fruits or manioc (Brazil) --tthehe existence of post mortem cyanide formation A second expert was requested to rere--analyseanalyse the remains (LC(LC--fluofluo)) Teeth (197 and 202 ng/g)ng/g) and bones (2200 and 2500 ngng/g)/g) tested positive for cyanide The woman was sentenced 17 years of jail (October 2012) Cyanide --11 Cyanide: rapidly acting and lethal poison (death generally occurring within min after ingestion but can be delayed) HCN (hydrogen cyanide = prussic acid): weak acid, colourless volatile liquid, boiling point of 26 .5 °C, same density as air (easy diffusion) Characteristic odour of bitter almonds at 0.2 – 5 ppm (missed by 30 %) Salts: potassium and sodium, strong bases that cause skin ulceration and congestion / corrosion of the gastric mucosa Produced as byby--productproduct of incomplete comcombustionbustion (coke ovens, blast furnaces, cigarettes and house fires) but also from the pyrolysis of nitrogennitrogen--containingcontaining materials, such as wool, silk, acrylonitriles … Cyanide --22 Use: fumigant, execution in ““gasgas chamberchamber””,, production of resin monomers such as acrylates and methacrylates (HCN) – processes of metal hardening, metal cleaning, electoplating, gold recovery and refining (cyanide salts) OfOther forms and uses: calcium cyanamide (melamine resins ), cyanuric chlorides (herbicides), acetonitrile (analysis, pharmaceuticals), cyanoacetic acid (phenylbutazone, barbiturates), nitroprussides (hypotensive agents), potassium ferricyanide (photography, blueprints, pigments) Several plants have been found to contain cyanogenic glycosides: amygdalin (bitter almonds), linamarin (manioc, cassava and lima beans) – present in apricot, pear and applapplee seeds, in peach, cherry and plum pits 2 10/4/2013 Natural cyanide Liberation of hydrocyanic acid in case of hydrolysis: Apricot kernels (Algeria): 1200 ppm Peach pits (Australia): 700 ppm Apple pits (Australia): 700 ppm Flax seeds (Australia): 360 – 390 ppm Manioc (Cameroon): 90 – 1500 ppm Almond syrup (Tunisia): 11--33 ppm Bitter almonds (Tunisia) : 900 – 1200 ppm Case Nader et al (Clin Toxicol, 2010) Child (2.5 yearyear--old)old) cyanide poisoning after ingestion of 5 bitter almonds CN- in blood: 2330 ng/ml Cyanide --33 Cyanide stops cellular respiration by inhibiting electron transport at the cytochrom c oxydase step This will stop the production of highhigh--energyenergy ATP molecules and therefore the oxydative phosphorylation and, as a consequence, the KrebKreb’’ss cycle. Pyruvic acid will accumulate, and will be transformed to lactic acid (metabolic acidosis) Alteration of the gradient for release of oxygen from Hb (hypoxia) Cyanide --44 The primary target organ of cyanide is the brain, followed by the heart Lethal doses: 100 mg of HCN and 200 mg of NaCN (1(1--22 mg/kg) Detoxification: enzymatic conversion (by rhodanase and betabeta--mercaptomercapto-- pyruvatepyruvate--cyanidecyanide sulfurtransferasesulfurtransferase)) to thiocyanates(SCN) and subsequent excretion by the kidney 98 % of cyanide is found in red blood cells: analysis of whole blood The toxic effects of cyanide on fire victims should not be evaluated based solely on the concentration in blood, but also taking into consideration methemoglobinemia (combination (combination of cyanide), CO-CO-HbHb and post mortem interval (Moriya, 2001 and 2003) 3 10/4/2013 Analysis --11 Colour test Conversion of cyanide into cyanogen chloride using chloraminechloramine--TT Addition of pyridine + barbituric acid to produce a red colour Spectrophotometric reading at 580 nm Time: about 2 hours, LOD: about 1 μg/ml MicroMicro--diffusiondiffusion in a Conway cell Conversion of blood cyanide to the more volatile HCN by acidification Inner well: dilute base (trapping agent) Outer well: blood + realising agent (acid) Covered for several hours, room temp. Detection of CN in the inner well Time: about 2 hours, LOD: about 1 μg/ml Analysis --22 Liquid chromatography MicroMicro--diffusiondiffusion or onon--lineline extraction, followed by complexation of CN by NDA + taurine to develop a 11--cyanocyano [f] benzoisoindole derivative Detection by LCLC--fluorimetryfluorimetry or LCLC--MSMS or MS/MS (with 13C15N) Time: 30 to 60 min, LOD: 5 ng/ml with 25 μl of blood Headspace gas chromatography – mass spectrometry 1 ml or 1 g of tissue + IS (acetonitrile) in a headspace vial Sealed + 1 ml orthophosphoric acid Incubation for 20 min at 80 °C Injection into an HP1 15 m capillary column Detection by mass spectrometry Time: 60 min, LOD: 10 ng/ml Concentrations There is no consensus about the physiological and toxic concentrations Levine (1999) < 250 ngng/ml,/ml, normal - 250 - 2000 ngng/ml,/ml, toxic -->> 22--30003000 ngng/ml,/ml, fatal Clarke (()2011) < 26 ngng/ml,/ml, normal - > 500 ngng/ml,/ml, toxic --44--50005000 ngng/ml,/ml, fatal Baselt (2002) 16 ngng/ml,/ml, nonnon--smokers,smokers, 41 ngng/ml/ml smokers - > 1100 ngng/ml,/ml, fatal Kintz NonNon--smokers:smokers: up to 50 ngng/ml,/ml, smokers: up to 80 ng/mlng/ml Fatal concentrations: from 500 ngng/ml/ml 4 10/4/2013 Post mortem findings Autopsy Autopsy findings after cyanide poisoning are generally nonspecific Findings often include visceral congestion and oedema, petechial haemorrhages in the brain, pleura, lungs and heart Sometimes, signs of corrosion of the gastric tract Bright red colour of the blood (hypoxia): not consistent Odour of the blood and tissues: bitter almonds ?? Toxicology Analysis of blood, spleen (+++) and liver Low concentrations in urine Heart / peripheral blood ratio: 0.3
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