“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
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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
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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 sulfurtransferase)sulfurtransferase) 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)
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Analysis -1- 1
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 -2- 2
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 ng/ml,ng/ml, normal - > 500 ng/ml,ng/ml, toxic -4- 4--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
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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 - 10.6 (mean 3.4), Rhee et al, 2011 Analysis for cyanide is impaired by formaldehyde ((WinekWinek,, 19901990)) Major issue: storage, delay and stability
Storage, delay and stability
In vitro changes in blood cyanide concentration have been documented From cyanide poisonings, authors have published rapid decreases in cyanide concentrations after death. Others have indicated that cyanide can be produced in blood specimens
Storage Storing blood specimens in glass tubes with as little airspace as possible and maintaining them frozen until analysis can minimize cyanide loss It is recommended that 2 % NaF be added to blood (McAllister, 2011)
Delay It is suggested that specimens be analysed as soon as possible to prevent or minimize any changes in cyanide concentrations
Unusual findings…
Curry, 1963: in a case of cyanide inhalation, a blood specimen taken immediately after death contained 3.5 mg/L of cyanide, while specimens taken at autopsy, the next day, were 0.5 (carotid) and 1 mg/L(femoral)
Ballantyne, 1974: in rabbits sacrificed by injection of cyanide, the concentrations declined to undetectable levels after only 2 weeks in most tissues
Bright, 1990: blood containing the highest concentrations of cyanide depreciated more quickly under all storage conditions (4 °°CC and --2020 °°C)C) than those samples at lower concentrations
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General rules …
Within minutes of death a number of processes occur that can significantly affect concentrations of drugs
Lividity and partitioning of fluids
Redistribution
MtbliMetabolism
StabilityStability--inducedinduced changes
These changes must be understood in order to interpret post mortem toxicology
The cyanide paradox …
Authentic cases of cyanidecyanide--relatedrelated poisoning and cyanidecyanide--freefree Comparison between immimmediateediate test and re-re-testtest after 2 months Bloods were stored with no preservative, NaF or Li heparin
Disappearance of cyanide 15 to 70 % of decrease in concentration in most cases In some cases: no change
Formation of cyanide In several cases, increase of levels, from physiological to lethal
General: if at T0, the concentration is low, it will be stable at T22monthsmonths If the concentration is high at T0, a decrease will be observed at T22monthsmonths
Post mortem cyanide alterations
To account for falsefalse--positives,positives, 3 alteratialterationsons should be considered
- Metabolic cyanide production
- Cyanide production during storage
- Cyanide production during analysis
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Metabolic cyanide production
Some compounds, when ingested, produce actions and symptoms that are consistent with cyanide poisoning
Nitroprusside (hypotensive medication):medication): partly degraded to produce NO and cyanide
Hypothiocyanate (antibacterial agent): degrades to cyanide
Aliphatic nitriles (solvents,(solvents, acetonitracetonitrile):ile): can be oxidized by cytochrome PP--450450 to cyanhydrine,cyanhydrine, which easily decomposes to cyanide
Production from nitrite
Cyanide can be produced from reactions of nitrite with organic compounds (Hb, serum protein) under acidic conditions
The Okinawa case (Seto et al)
isobutyl nitrile + H2O : isobutyl alcohol + nitrite
- nitrite (NO2) + O2 : nitrate (NO3 ) nitrite (NO2) + organic compounds : cyanide
Cyanide production during storage
Microorganisms can produce cyanide through putrefaction and many bacteria are involved in cyanogenesis (Knowles, 1976)
Bacteria producing cyanide:cyanide: Pseudomonas aeruginosaaeruginosa,, Pseudomonas fluorescens, Chromobacterium violaceum
Bacteria using cyanide for their metabolism:metabolism: Bacillus cereus, Chromobacterium violaceum
Detox bacteria:bacteria: Bacillus, Thiobacillus,Thiobacillus, E. coli
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Cyanide production during analysis
Cyanide may be produced as an artefact during the assayassay’’ss extraction step (Seto, 1993; Tsuge, 2000)
One notable phenomenon is the formation of cyanide from thiocyanate in the presence of erythrocytes, particularly in smokers To avoid this, it is recommended to add ascorbic acid (Seto, 1995)
Under acidic conditions, cyanide is produced from thiocyanate by nitrite and hydrogen peroxide (Wilson, 1961)
Cyanide is also produced nonnon--specificallyspecifically from blood during heating at 50 °°CC (Seto, 1996)
The issue in fire fatalities
In forensic science, HbCO and cyanide concentrations are used to document whether the victim was alive at the beginning of the fire
CaplanCaplan,, 1976: cyanide ranged from 0.010.01 to 4.36 mg/L (mean 1.12) AthiltifidillfiAs a consequence, there is no elevation of cyanide in all fire cases
There is an endogenous production of cyanide in fire fatalities, dependent of the time to exposure to high temperature and the temperature ititselfself (Krabowska(Krabowska et al, Arch Med Sado Kryminol, Kryminol, 2011)
There is a strong potential for cyanide levels to increase by the absorption of atmospheric cyanide (Thoren(Thoren et al, JAT, 2013)
Passive uptake of HCN at 100 and 200 ppm atmospheric concentrations in undisturbed pooled whole blood samples.
Thoren T M et al. J Anal Toxicol 2013;37:203-207
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Identification of cyanide by IRIR--MSMS
In some cases, the identification of the source of cyanide is very important for the forensic investigation (affaire de la Josacine, 1994)
Stable isotope analysis can be used to identify cyanide source (Tea, 2012) The isotopic signature of the commercial samples varies: 13 15 from -51.96-51.96 to -25.77-25.77 °/°° for δ C and -4.51-4.51 to +3.81 °/°° for δ N
Future interest in cyanide discrimination ?
LL’’affaireaffaire de la Josacine
A young girl (9(9--yearyear old) was killed by sodium cyanide contained in an antibiotic medicine ((JosacineJosacine).). On 11 June 1994, at 8 pm Emilie took a spoonful of Josacine,Josacine, as prescribed. She collapsed at 8.15 and despyppite the best efforts of doctors was dead by 10.30 pm A suspect was arrested and it was supposed that he mixed the cyanide with the medicine in order to kill the owner of the house (he had an affair with his wife). It was necessary to determine if the cyanide found in the residue of the medicine and that possessed by a suspect for professional use were of the same origin. The analysis was based on the determination and comparison of impurities, but the results were challenged due to chemical instability. The suspect was sentenced to 20 years of imprisonment.
Conclusion
Cyanide is inherently unstable in cadavers and in stored tissue samples, including blood
The rate of transformation of cyanide in blood is dependent on:
- the initial cyanide concentration in the sample at time of death --tthehe length of time thatthat a sample remains in the cadaver - the length of time that a sample remains in storage before analysis --tthehe preservation conditions (addition of NaF)NaF) --tthehe storage conditionsconditions (temperature)
9 Jean-Pierre Goullé
Pr. Jean-Pierre Goullé, 66 years old, PhD, Professor of Pharmacy in Toxicology at the University of Rouen, Head of Department of Pharmacology–Toxicology at the General Hospital of Le Havre. He is an Expert for Justice in blood alcohol determination, pharmacology and toxicology, appointed by the Court of Appeal of Rouen and the Court of Cassation (French Supreme Court); 97 Medline referenced publications in biochemistry, pharmacology and toxicology ; participation in 11 books; member of international scientific societies (TIAFT, IATDMCT, SoHT); and French societies (past member of the board of the French Society of Clinical Biology SFBC, past-President of the French Society of Analytical Toxicology SFTA , past-President of a French organization of legal experts CNBAE). Death involving metals and elements
E. Saussereau1,L. Mahieu1, MGM. Guer btbet2, J. P. Gou llé121,2 1- Laboratoire de Toxicologie, Groupe Hospitalier du Havre, France 2 - Laboratoire de Toxicologie, EA 4651, UFR de Médecine et de Pharmacie, Rouen 2013 SOFT Annual Meeting Orlando, Florida – October 28 to November 1, 2013
INTRODUCTION
. Metals = one of the oldest toxicants . Abundant in the environment: water, food, insecticides … . Human exposure inevitable . Growing concern over their presence in air, water and soil
INTRODUCTION
. Metals loosing electrons cations . Cations not destroyed in human body . Cations react in biological systems
Toxicity ++
1 INTRODUCTION
Essential to identify/quantify metal species vs total element determination in clinical toxicology . Toxicity of an element depends on: . Chemical form . Oxidation state . Chemical form more valuable information
Total element concentration may be of little value
INTRODUCTION Chemical specie*: specific form of an element defined as the isotopic composition, electronic or oxidation state, and/or complex or molecular structure.
SitiSpeciation analilysis*:analtillytical activ ity to identif y and/or to measure the quantities of one or more individual chemical species in a sample.
Speciationofanelement*: distribution of an element among defined chemical species in a system.
*IUPAC : Templeton et alal..PurePure ApplAppl..ChemChem..2000 ;;7272::14531453--7070
INTRODUCTION
MAJOR TOXICESSENTIAL Arsenic Chromium III Cadmium Cobalt Chromium VI Copper Lead Iron Mercury Magnesium Nickel Selenium Platinum Zinc Thallium
2 ANALYTICAL CHALLENGE
Yesterday: Marsh apparatus
ANALYTICAL CHALLENGE
. Today: analytical progress . ICP-MS . Simultaneous analysis of 30 metals: . 0,3 mL whole blood . 20 mg hair, nails . 40 mg tissues
ARSENIC (As)
Among metals and elements:
As has been known and used since ancient times: « Poison of Kings and the King of Poisons »
Mithridate ingested every day small quantities of As to escape poisoning.
As is still used for volunteer poisonings or murders.
3 As DERIVATIVES
. Inorganic derivativesderivatives::highhightoxicity
. Arsenite AsIII (lethal dose ingested = 120 - 200 mg) . Arsenate AsV . Organic derivatives: innocuous . Arsenobetaine AB . Arsenocholine AC . Methylated metabolites: metabolites: intermediate toxicity . Monomethylarsonate MMAV . Dimethylarsenate DMA V
As SOURCES OF EXPOSURE
. Inorganic derivatives . Environmental
. BangladeshBangladesh::drinkingdrinkingwater > 300 µg/l (WHO ≤ 10 µg/l) cancer death ++ (lung, bladder, skin) . Industrial workers
III . As2O3 ((AsAs ): medical (APL) or criminal . ToxicokineticsToxicokinetics::slowslowelimination (10 (10 days) Lethal dose = 200 mg (2 – 3 mg/kg) . Organic derivatives (AB, AC)AC)::alimentary
As TOXICITY
Acute toxicity Chronic toxicity
Digestive Skin lesions (hypo(hypo-- hyperpigmentation, blackfoot Cardiovascular disease, cancer …) Neurologic Bladder cancer Cutaneous Ischemic heart disease Renal Hypertension Diabetes mellitus
4 Millions of people are suffering from arsenic poisoning
5 As NORMAL CONCENTRATIONS (µg/L or µg/kg1)
Whole Urine Hair Nail Liver Muscle Kidney blood
N 54 54 45 130 21 19 19
Median 2.3 10.0* 50 30 20 13 27
95e P 12.9 293* 80 86 45 21 45
1. Goullé et al . Forensic Sci Int, J Anal Toxicol, Ann Toxicol Anal, 2005-2009
*Total As
As CONCENTRATIONS WITH VARIOUS EXPOSURES
As in whole blood:
▪ Chronic exposure: As > 200 µg/L ▪ Acu te in tox icati on: A s > 500 µg /L ▪ Fatal As: > 1000 µg/L
As in hair: ▪ Chronic inorganic exposure: > 0.1 ng/mg
6 As CONCENTRATIONS IN FATAL CASES (µg/L or µg/kg)
Whole Liver Kidney blood
Rehling, 1967 N = 49 N = 49 N = 49
Median 3,300 29,000 15,000
5e-95e P 600-9,300 2,000-120,000 200-70,000
Whole blood (fatal): 1,200 to 9,000 µg/L (personnal data)
As SPECIATION: HPLC-ICP-MS
. HPLC::AsAs derivatives chromatographic separation (ion(ion--exchange)exchange) . ICPICP--MSMS:: As derivatives highly sensitive detection ( 75As)
Its distribution in tissues
Its speciesspecies--specificspecific toxicity to target organs
7 As SPECIATION: HPLC-ICP-MS
Arsenic derivatives (urine) : 20 µg/l
24 h after seafood consumption
48 h after seafood consumption
As SPECIATION: HPLC-ICP-MS
Arsenic derivatives : 20 µg/l
Suspicious powder
Thallium (Tl) SOURCES OF EXPOSURE
. Rat poisons and insecticides (now banned)
. Industrial: jewelry, semiconductors, dyes
. It has been used medicinally as a depilatory
. A common source of human poisoning:
the use of Tl as rodenticides/insecticides
8 Tl TOXICITY
. One of the most toxic metals . Interfere with biological fonctions of K . Toxicokinetics: very slow elimination (30 days) . Classic syndrome of Tl poisoning: . Gastroenteritis . Polyneuropathy . Alopecia (10 30 days) . Death: renal, CNS and cardiac failure Lethal dose 1 g (10 – 15 mg/kg)
Tl NORMAL CONCENTRATIONS (µg/L or µg/kg1)
Whole Urine Hair Nail Liver Muscle Kidney blood
N 54 54 45 130 21 19 19
Median 0.09 0.14 0.2 0.4 0.8 0.7 1.4
95e P 0.22 0.76 0.4 1.2 1.0 1.0 1.8
1. Goullé et al . Forensic Sci Int, J Anal Toxicol, Ann Toxicol Anal, 2005-2009
TL CONCENTRATIONS IN FATAL CASES (µg/L or µg/kg1)
Whole blood Tl > 800 µg/L in fatal cases
Whole Urine Liver Kidney blood
N 5 555
Median 4,000 5,200 15,000 11,000
95e P 11,000 11,000 29,000 20,000
1. Baselt, 2011
9 LEAD (Pb) SOURCES OF EXPOSURES
. Industrial: batteries, bearing alloys, paints, pigments, ceramic . Pb is now banned in gasoline (USA, Europe) . Regulations in many countries: . Air . Water . Home
Pb TOXICITY
TOXICOKINETICS IS PARTICULAR IN KIDS
• When lead is ingested:
. Adult s ab sorb 5 -15% (<5% re ta ined)
(+++ Kids absorb 42% (32% retained . TOXICOKINETICS OF Pb IN INHALED AIR
. 90% of Pb partially absorbed by lungs
. 50% of Pb retained through alveoli +++
Pb TOXICITY
. Wide range of effects (doses, duration) . Toxic effects range: . Inhibition of enzymes . Severe pathology . Death . Children most senstitive to effects in CNS . Adults develop peripheral neuropathy . Other target tissues: gastrointestinal, skeletal Lethal dose > 10 g
10 Pb NORMAL CONCENTRATIONS (µg/L or µg/kg1)
Whole Urine Hair Nail Liver Muscle Kidney blood
N 54 54 45 130 21 19 19
Median 49 1.5 410 520 86 0.3 17
95e P 184 6.5 4,570 3,710 870 4.9 62
1. Goullé et al . Forensic Sci Int, J Anal Toxicol, Ann Toxicol Anal, 2005-2009
FATAL CASES INVOLVING Pb (µg/L or µg/kg1)
Whole Age Exposure blood Brain Liver Kidney Bone
Contaminated 17 year 950 ------flour 1,110 3 young Pica to ------children 3,500 1 female Shot in the leg 5,300 ------Apple juice 400 2 subjects glazed --- and 13,000 18,000 --- earthenware 2,200 268,000 Pb 1 child --- 5,800 40,000 8,800 to poisoning 132,000
1 - Baselt, 2011
Pb intoxication that could have been fatal due to an earthenware dish
11 Pb ISOTOPIC ANALYSIS
Most of the elements have many isotopes
Pb ISOTOPIC ANALYSIS Pb ISOTOPIC RATIOS
according
Dis h Father Mother Son Others
Chemical Target form Exposure Toxicity organs Biomarker
Vapors Brain, High kidney, lung Urine, plasma Elemental Hg0
Dental Kidney ? amalgams Low Urine, plasma
Skin- Inorganic Kidney lightening Intermediate Urine, plasma Hg+ Hg2+ cream, med. Digestive tract treatment (ingestion ++)
Organic Fish, paints, Very high Brain, Fetal W. blood, hair, + fungicides brain umbilical cord CH3Hg (100 mg lethal)
12 Hg SOURCES OF EXPOSURES
. Dietary = methylmercury (MeHg)
. Occupational = inhalation of Hg vapor
. Medicinal = Thimerosal, dental amalgam
. Fatal Hg poisonings mainly accidental:
. Inhalation of Hg vapor
. Oral ingestion of HgCl2 . Consumption of food contaminated with MeHg
Hg TOXICITY
. Two controversals:
. Thimerosal (ethylmercury)
used in chilhood vaccines is safe at the doses used in vaccines (< 1 mg) (Clarkson et al., 2003)
. Mercury amalgam
exposure is too low to cause significant toxicological effects (DeRouen et al., 2006)
. Lethal dose: MeHg 100 mg - Hg mineral salts 1g
Hg NORMAL CONCENTRATIONS (µg/L or µg/kg1)
Whole Urine Hair Nail Liver Muscle Kidney Brain blood
N 54 54 45 130 21 19 19 20
Median 3.6 1.7 660 290 45 5.0 77 9.2
95e P 13.9 8.9 1,660 830 170 11 440 20
1. Goullé et al . Forensic Sci Int, J Anal Toxicol, Ann Toxicol Anal, 2005-2009
13 Hg CONCENTRATIONS AND TOXICITY
Hg in whole blood:
. Neurological disorders > 200 µg/L
. Acute intoxication > 500 µg/L
. Fatal > 1 000 µg/L
Hg in hair according to WHO:
Hair Hg (µg/g) = whole blood Hg (µg/L) x 250
Hg CONCENTRATIONS IN FATAL CASES µg/L or µg/kg
Whole Liver Kidney Brain blood MeHg1 18,000 accidental 600-6,000 4,200 - 78,000 2,400 - 4,000 - 35,000 (n=8) MeHg2 Irak (n=51) --- 1400 - 76,000 ------Minamata --- 22,000 - 71,000 ------
HgCl -HgBr 3 33,000 47,000 2 2 800-22,000 200-3,000 (n=3) - 56,000 136,000
Hg° vapors4 400-900 ------(n=3, fatal=2)
1. Al-Saleem, 1976 - 2. Magos et al. 1976 - 3. Baselt, 2011 - 4. Jaeger et al. 1979
CHROMIUM (Cr) SOURCES OF EXPOSURES
. Chromium plating and alloys . Manufacture of dyes and pigments . Leather tanning . Photographic processing . Wood preserving and coloring . Traditional remedy in some countries . Artificial limbs
14 Cr TOXICITY
. Cr is a powerfull toxic (CrVI +++)
. CrVI is rapidly and highly absorbed
. Causes severe skin , pulmonary or gastrointestinal injury (corrosive)
. Hepatic and renal failure
. Genotoxic / carcinogenic (IARC. Group 1) Cr use requires substitution
Cr TOXICITY
. Severe intoxication = 0.5 g Cr VI
. Lethal dose = 2 – 3 g Cr VI
. AtAcute po ison ing i s not common
. Extremely poor prognosis
HIGH FATALITY RATE
Cr NORMAL CONCENTRATIONS (µg/L or µg/kg1) 52Cr (CRC*) 53Cr
Whole Urine* Hair Nails Liver Muscle Kidney blood*
N 106 30 45 130 21 19 19
Median 0.55 0.33 200 380 60 <40 43
95e P 0.87 1.04 520 1,890 100 <40 110
1. Goullé et al . Forensic Sci Int, J Anal Toxicol, Ann Toxicol Anal, 2005-2009 CRC* = collision reaction cell
15 Cr CONCENTRATIONS IN FATAL CASES (µg/L or µg/kg)
Age Whole Liver Kidney Issue Ingestion blood
2 year old1 4,200 62,000 33,000 Fatal 48 H 1 g CrVI
Adult2 68,000 187,000 135,000 Fatal 12 H 50 mL H2Cr04
Adult3 Fatal 1,000 ------x mL H2Cr04 1month 15 days 1 month ♂ 58 year old4 Pl : 2,088 Pl : 62 Pl : 5 Alive 3 g CrVI RBC: 631 RBC: 271 RBC : 129
1. Meert et al. 1994 – 2. Saryan et al. 1988 – 3. Loubieres et al. 1999 4. Goullé et al. 2011
TO CONCLUDE
Here is your take-home message : ICP-MS
. Is sensitive, specific, rather fast
. This technique explores many elements
. The sophisticated ICP-MS isotope study and HPLC-MS speciation technique are useful tools
. Is a of major interest for death involving metals and elements
. Is complementary to the widely used chromatographic techniques
Thank you for listening !
With the financial support of the FhSitfFFhSitfAltilrench Society of AltilAnalytical Toxicology.
16 Marc Deveaux
Marc Deveaux, Pharmacist (1980), PharmD (1982), PhD in Forensic Toxicology (1990), 60 year-old, Chief Executive Officer of Laboratoire Toxlab, Paris, since 2005 Formerly (1980-2004) assistant-professor in forensic toxicology (University of Lille, France) Judicial expert at the Court of Appea, Paris Judicial expert at the High Court of France Editor-in-Chief of the Annales de Toxicologie Analytique 138 publications in peer-reviewed journals 98 papers in national journals 280 scientific communications and conferences 51 chapters in books and various publications Member of the SOFT, TIAFT & SFTA.
Unusual Causes of Death:
Death Involving Plants
Marc Deveaux, Marjorie Chèze, Guillaume Hoizey & Gilbert Pépin Laboratoire TOXLAB, Paris, France
Orlando 2013 SOFT Annual Meeting -Workshop -
Incidence of plant poisoning during the last years:
France 1.5 %
Belgium 5 %
Italy 6.5 %
Switzerland 7%
Turkey 6 %
United-States 4th rank of intoxications behind drugs, household cleaning products and cosmetics.
Difficulty in assessing the number of fatalities
The mains species involved in poisoning, are:
Europe Genus Aconitum Monkshood aconitine Atractylis glummifera GlueThistle atractyloside Atropa belladonna Deadly Nightshade atropine Colchicum automnale Meadow Saffron colchicine Datura stramonium Jimson Weed scopolamine Digitalis purpurea Foxglove digitaline LbliLobelia IfltInflata LbliLobelia lblilobeline Prunus laurocerasus Cherry Laurel oleandrine Veratrum album White Hellebore veratrine Genus Taxus Common Yew paclitaxel Glycyrrhiza glabra Licorice glycyrrhizine
Oversea Pausinystalia yohimbe Yohimbe yohimbine Ipomea tricolor Morning Glory LSA Cerbera manghas Sea Mango cerberine
1 Pump LC LC -MS/MS -TQ
AB
Spray Column LC Q1 Q2 Q3
Nebulizer Parent ion Fragmentation Daughter ion selection selection
Creamer
LC-MS/MS triple stage quadrupole with an electrospray source
Interest of LC-MS/MS
1. Very usefull for: - thermolabile - Mol Weight > 400 uma 2. Allows to shorten the extraction
3. Specific
4. Very sensitive
5. No need of derivatization
6. Screening easier than with GC
TSQ Quantum Thermo
An example where LC-MS/MS helped to enhance the LoD:
Colchicum automnale
2 Meadow saffron Colchicum sp.
. Colchicine : highly toxic alcaloid from genus Colchicum (Liliaceae). . The plant may be mixed up with Wild Garlic. (before flowering) Ingestion of 40 flowers may led to death. . All parts of the plant are toxic . Mainly used as medication against gout . Therapeutic concentrations are 0,3-2,4 ng/mL . Toxic concentrations: up to 5 ng/mL . Very short half-life (1h), low blood concentration : LC-ESI-MS/MS
Colchicine determination in blood
• 1 mL blood • pH 9,6 • Extraction with dichlorométhane + 5% isopropanol • i.s.: 10 ng clonazepam-d4
• evaporate and dilute with 50 µL methanol.
• column: C18-XTerra (3,5 µm -150 x 2 mm) Waters. • mobile phase : 2 mM formate buffer pH3 / acetonitrile, gradient mode , flow rate 200 µL/min • Total analysis time : 15 min.
• Positive mode
• pseudo-molécular ion (MH+; m/z = 400,4) is fragmented
• 4 daughter ions • MRM mode :
m/z = 295.1 – E. Coll. = 35 m/z = 310.1 – E. Coll. = 24 m/z = 326.1 – E. Coll. = 25 m/z = 358.2 – E. Coll. = 20
Colchicine in blood: 5,2 ng/mL
3 Case Cardiac Femoral Gastric Vitreous ng/mL Urine Bile blood blood content humor
1 5.2 17.4 19.4 42.8 348 3 2 22.8 21.9 148.5 1818.5 219.8 0.5
CONCLUSION: enhancement of the LoD
Previous LoD & LoQ
Extraction yield: 50-70 % -
Limit of Detection 0.1 ng/mL 0.6; 2.0 (LC-MS-IT; LC-DAD)
Limit of Quantification 0.5 ng/mL 0.5; 4.0 (LC-MS-IT; LC-DAD)
An example where LC-MS/MS helps to confirm the intoxication by a plant:
Taxus baccata L.
4 • Many parts of the tree are toxic, except the bright red aril surrounding the seed
• The foliage remains toxic even when wilted or dried. Horses, cattle, pigs and other livestock are vulnerable, with a lethal dose of 200– 400 mg/kg
• Fatal poisoning in humans is very rare, only occurring after eating a lot of foliage. The lethal dose is reported to be between 50 and 100 g of leaves (a handful) The leaves are lanceolate, flat, dark green, 1-4 cm long and 2-3 mm broad
Toxic substances in Yew: taxanes • Introduced by Avicenne in 1021 as a cardiac remedy;
• Traditional treatment for brest and ovary cancer in Central Himalayas;
• Extract of leaves and leaves used for homicides and suicides;
• Taxicatines: cyanogenic glycosides + aglycone (3,5-dimethoxyphenol)
• Taxine fraction: mixture of at least 20 diterpene pseudo-alcaloids including paclitaxel, taxine B and isotaxine B;
• Taxol® (paclitaxel): used to treat patients with lung, ovarian, breast cancer,
Toxic action: Based on cardiac effects (inhibits Ca and Na transport accross the cell membranes): heart failure; staggering gait, muscle tremors, convulsions, collapse, difficulty breathing, coldness
.
5 Paclitaxel determination in blood • in-house screening method for plant toxin • 2 mL blood or 2g organ pH 9.0 • Extraction with dichlorométhane + 5% isopropanol i.s. clonazepam-d4
• evaporate and dilute with 50 µL methanol.
• column: C18-XTerra (5 µm -150 x 2 mm) Waters. • mobile phase : 2 mM formate buffer pH3 / acetonitrile, gradient mode , flow rate 200 µL/min.
Detection: SRM - positive mode Parent ion: 854.5 daughter ions: 240.2, 286.2, 509.3
Quantification with calibration curve LoQ: 10 ng/mL LoD: 1 ng/mL
193 ng/mL paclitaxel in periph. blood
QUANTUM C:\Xcalibur\methods\toxiques vegetaux\alkB.meth D:1 IML716 Alk B foie / AL C:\Xcalibur\...\C - Mars 09\310309-18 3/31/2009 10:26:59 PM 10.000000 RT: 0.00 - 15.08 SM: 5G RT: 10.39 NL: 1.32E6 MA: 18975595 TIC F: + c SRM ms2 [email protected] 1000000 [ 217.62-274.66] MS 310309-18 Clonazepam d4 (STDI) 0 RT: 12.01 NL: 6.44E3 MA: 145091 TIC F: + c SRM ms2 [email protected] 5000 [ 239.37-509.88] MS 310309-18 Taxol 0 RT: 12.01 NL: 9.99E2 MA: 17017 m/z= 239.70-240.70 F: + c SRM ms2 [email protected] [ 239.37-509.88] MS 310309-18 0 Intensity RT: 11.91 NL: 4.85E3 MA: 109631 m/z= 285.70-286.70 F: + c SRM ms2 [email protected] [ 239.37-509.88] MS 310309-18 0 RT: 12.11 NL: 1.15E3 MA: 18253 m/z= 508.80-509.80 F: + c SRM 1000 ms2 [email protected] [ 239.37-509.88] 0 MS 310309-18 0 2 4 6 8 10 12 14 Time (min)
310309-18 #1988-2038 RT: 12.01-12.21 AV: 3 NL: 2.78E3 F: + c SRM ms2 [email protected] [ 239.37-509.88] 286.08 100 90 25 ng/g paclitaxel in 80 liver 70
60
50
40 509.18 240.05 Relative Abundance 30
20
10
0 239.5 240.0 285.5 286.0 509.0 509.5 m/z m/z m/z
6 Conclusion: why to determine Paclitaxel concentrations ? • Peripheral blood: 193 ng/mL • Liver: 25 ng/mg • Bile: 230 ng/mL • Gastric content: 252 ng/mL
• Pure standard of taxin B and isotaxin B are not commercially available, unless extract yourselves from Yew leaves
• Determination of 3,5-DMP by GC-MS could be an other way to confirm a poisoning Yew, but the litterature on this matter is rare
• Knowing the case history, and using a screening method for plant toxins, we prefered to determine Paclitaxel directly by LC-MS/MS, considering that it could allowed to confirm the cause of death as the ingestion of Yew leaves
An example where LC-MS/MS allows to determine the concentration without the standard substance:
Ipomoea tricolor
Ipomoea tricolor Morning Glory (Convolvulaceae)
Seeds
Flowers Plant
7 Ipomoea tricolor seeds were used as psychedelic by Aztec shamans (tlitlitzin)
Today, available without any control on the Internet and used for the same purpose (not by chamans !)
The main active component is Lysergamide (=( ergine, = LSA)
About 10 times less potent than LSD, slightly different action (more sedative, very bad taste, emetic)
Dosage: a handfull
Lysergamide
LSA = Lysergamide = Ergine . “Morning Glory”
. C16H17N3O PM : 267.3 . Metabolites: not well known . Not controlled
LSD, Lysergide . “Acid”
. C20H25N3O PM : 323.4 . metabolites: iso-LSD, 2-oxo-LSD . Controlled
Analysis
• When finalizing the method for LSA determination, no LSA standard was commercially available
• We decided to use LSD-d3 as internal standard
• Results were to be given in LSD-equivalent :
– seeds – urine in one Morning Glory seeds user
8 Morning Glory seeds analysis
LC-MS/MS identification and quantification
- Ten seeds (m=322 g) + 2 mL ethanol, 30 min sonication, then overnight at 20 C - 5µL sol. + 5µL LSD-d3 (1 µg/mL)+ 90 µL MeOH
LSA: MRM spectrum LSA: 98 ng/g seed
LSA: Identification and quantification in urine
RT: 0.00 - 10.01 SM: 9G RT: 6.35 NL: 2.46E4 MA: 149192 TIC F: + c sid=-10.00 SRM 20000 ms2 -1 mL urine + Toxitube A [email protected] [ 15000 LSD-d 207.57-226.62] 3 MS 171104-24 10000 -Evaporation In ten sity 5000 9.13 LSD-d3 9.70 0 RT: 6.06 NL: 4.70E4 + 50 µL methanol MA: 275464 TIC F: + c sid=-10.00 SRM 40000 ms2 [email protected] [ 30000 179.93-223.52] LSA MS 171104-24 20000 Intensity
10000 LSA 0 Estimated LoD = 0,5 ng/mL 0 1 2 3 4 5 6 7 8 9 10 Time (m in)
171104-24 #1356 RT: 6.04 AV: 1 NL: 3.46E4 F: + c sid=-10.00 SRM ms2 [email protected] [ 179.93-223.52] 223.298 100
Estimated LoQ = 1 ng/mL 90
80
70 60 LSA: MRM spectrum 50
40 207.284
LSA in urine: 5 ng/mL Relative Abundance 30 180.216 20
10
0 180.0 180.1 180.2 180.3 180.4 207.1 207.2 207.3 207.4 207.5 223.1 223.2 223.3 223.4 223.5 m/z m/z m/z
Conclusion: LC-MS/MS allows to identify and quantify without the target i.s.
An example where LC-MS/MS helped to answer the question who is the real guilty party ?
Cerbera manghas vs. Birgus latro
9 The Protagonists
1) a Crab 2) a Tree Birgus latro Cerbera manghas
Loyalty Islands
New Caledonia
3) eight Victims TIAFT 2010 – Bonn, Germany from Wallis Island
Case history
• 8 victims, native from Wallis Islands (2000 km from Loyalty Islands and New Caledonia) • They had eaten flesh and hepatopancreas of Coconut Crabs (Birgus latro) • Death occurred in less than 12 hours • With digitalic-like s ymptoms of into xication : – digestive .vomits, diarrhea – cardiovascular .severe bradycardia +++, .low blood pressure .conduction disorders .asystolia – major hyperkaliemia +++
• It is well known in New Caledonia and Loyalty Islands that the Coconut Crab (Birgus latro L.) is edible: the oily abdominal sack is called “foie gras” or “caviar”:
How could be the Crab so highly toxic ?
• Melanesian native from New Caledonia and Loyalty Islands known also perfectly that the fruits of Red-Eye-Sea Mangoo Tree (Cerbera manghas L.), are extremely toxic;
The question was: Is there a link between the animal and the fruit ?
Who has to be blamed: Cerbera manghas or Birgus latro ?
One hypothesis is that the Crab feeds on fleshy fruits of Cerbera manghas (among others) and so its flesh and gut become toxic.
10 Birgus latro L. (Coenobitiadeae)
The largest land-crab
TIAFT 2010 – Bonn, ever known ! Germany
Toxicity of Cerbera manghas L.
Cerbera manghas L. . (Apocynaceae)
= False mangoo tree = Native frangipani
The fruit (drupe) has a kernel containing active cardiotoxic glycosides, notably:
- Cerberine - Neriifoline (= desacetylcerberine) - Cerberigenine (= digitoxigenine)
Development of a LC-MS/MS method to determine the cardenolides:
- cerberine: no standard commercially available
- cerberigenine: no standard commercially available
- neeoeriifoline: was the ooynly ssatanda dadrd comme rc ia lly available (but is no more sold ! )
- we had to develop a method to determine these 3 cardenolides, using the similarity of their structures
- determination had to be performed in different matrices: . Fruit . Crab . Biological samples
11 Cardenolides neriifoline, cerberine and cerberigenine have similar structures
cerberine M = 576.6 O O O O neriifoline M = 534,7
O O OH O HO OH OH O OH O O O O
O O cerberigenine M = 374.1 digitoxine M = 764.9 O O
OH OH HO O O OH OH HO O O O O OH
Method: summary
• Pure solutions of neriifoline : results will be given in neriifoline equiv. • Internal standard: digitoxine • Column: column UP5 Bio P2 150 x 1 mm ; 5 µm
• Apparatus: ion-trap LC-MS/MS
• Solvent (gradient mode): formate buffer 2mM pH3, ACN / MeOH • Linearity: linear response to neriifoline 0.1 to 100 g/mL
neriifoline
Detection of cerberigenine:
Using the similarity of its molecular structure to neriifoline (ESI +)
neriifoline M = O O . ion-trap LC-MS/MS (LCQ-Duo) 534.7 O . Using an option of the source (MS3): HO OH OH O O - neriifoline is fragmented into the source by Collision Induce Dissociation (CID),
375 - daughter-ion of neriifoline = parent-ion
O O of cerberigenine cerberigenine M = 374.1 - the daughter-ion is then re-fragmented into the ion-trap MS m/z = 375 OH HO
12 Daughter-ions of cerberigenine
375.03 NL : 1 .24E8 85 150509-25#408 RT: O 11.02 AV: 1 F: + c O 80 neriifoline M = ESI Full ms2 75 [email protected] [145.00-450.00] 534.7 70 65
O 60 fragmentation HO OH 55 50 spectrum of OH 45 40 neriifoline O O 35 Relative Abundance 30
25 339.05 20 357.00 375 15
10
5 376.17 320.55 337.45 339.96 354.88 358.10 373.38 387.77 394.69 O 0 O 339.09 NL : 2 .94E7 85 150509-25#368 RT: cerberigenine M = 10.14 AV: 1 F: + c 80 ESI Full ms2 75 [email protected] 374.1 [100.00-500.00] 70
65 m/z = 375 60 OH 55 MS/MS spectrum 50 HO 45 of cerberigenine 40
35
30 356.90 Daughter-ions of 25 20 340.11 cerberigenine = 15 10 358.01 5 355.91 338.07 341.04 375.13 321.09 329.07 354.03 358.85 388.72 Granddaughter-ion of 0 320 330 340 350 360 370 380 390 neriifoline m/z
Detection of cerberigenine:
Using the similarity of its molecular structure to neriifoline (ESI +)
O O neriifoline M = O O cerberine M = 576.6 534.7 O O O OH OH HO OH O OH O O O O 202 375
We thought that the fragmentation 375 will take place according this diagram;
But which fragment will be charged + ?
This has to be tested in analysing kernel, which contains cerberine
Confirmation by analysing dryed kernel
C:\LCQ Duo\LCQ2009\2009\150509-25 5/15/2009 10:41:51 PM extrait noyau 150 mg /mc-cl A:36 C:\Xcalibur\methods\toxiques vegetaux\cerberine-1mm.meth 10.000000 RT: 0.00 - 20.19 SM: 7G RT: 12.79 NL: 2.61E5 MA: 7689646 m/z= 100 374.50-375.50+504.50-505.50 F: + c ESI Full ms2 50 [email protected] [170.00-550.00] MS 150509-25 1.83 Digitoxine = Standard interne 14.71 17.35 0 RT: 11.18 NL: 2.55E8 MA: 8600802644 m/z= 374.50-375.50 F: + c ESI 100 Full ms2 [email protected] [145.00-450.00] MS 150509-25 Neriifoline 375 50 Neriifoline 0 RT: 10.14 NL: 2.00E7 Relative Abundance MA: 773610681 m/z= 338.50-339.50 F: + c ESI 100 Full ms2 [email protected] [100.00-500.00] MS 150509-25 50 Cerberigenine 339 11.22 Cerberigenine 0 RT: 12.26 NL: 1.47E7 MA: 470625521 m/z= 202.35-203.35 F: + c ESI 100 Full ms2 [email protected] [155.00-550.00] MS 150509-25 Cerberine 202 50
Cerberine 0 0 2 4 6 8 10 12 14 16 18 20 Time (min)
374.98 NL: 2.30E8 100 150509-25#404-434 RT: 11.02-11.48 AV: 4 F: + c 80 ESI Full ms2 ion 202 is the [email protected] 60 [145.00-450.00] positive charged Neriifoline 375 40 339.08 20 160.94 230.94 279.16 337.18 415.13 449.11 fragment, 0 339.10 NL: 1.58E7 100 150509-25#363-394 RT: 10.14-10.61 AV: 4 F: + c 80 ESI Full ms2 and is typical of [email protected] Cerberigenine 339 60 [100.00-500.00] 40 356.94 cerberine 20 358.01 Relative Abundance 135.03173.11 219.01 293.14 337.09 397.82 0 202.79 NL: 1.27E7 100 150509-25#447-479 RT: 11.97-12.41 AV: 4 F: + c 80 ESI Full ms2 [email protected] 60 Cerberine 202 [155.00-550.00] 40 375.01 20 170.82 203.81 243.15 312.98 339.06376.02 415.64 506.85 541.04 0 100 150 200 250 300 350 400 450 500 550 m/z
13 Results: Coconut Crab C:\Xcalibur\methods\toxiques vegetaux\cerberine 1mm.meth 10.000000 RT: 0.00 - 20.14 SM: 7G RT: 12.63 NL: 7.12E6 MA: 272380666 m/z= 100 374.50-375.50+504.50-505.50 F: + c ESI Full ms2 50 [email protected] Crab’s gut (N=2): [170.00-550.00] MS 080110-10 Digitoxine = Standard interne 0 RT: 10.98 NL: 7.79E6 MA: 299208546 m/z= 374.50-375.50 F: + c ESI 100 Full ms2 [email protected] [145.00-450.00] MS 080110-10 Concentration 50 Neriifol ine 0 NL: 1.68E6
Relative Abundance Relative RT: 9.99 (µg/g) 12.65 m/z= 338.50-339.50 F: + c ESI 100 MA: 78043264 Full ms2 [email protected] [100.00-500.00] MS 080110-10 Neriifoline 2 and 56.6 50 Cerberigenine 0 RT: 11.90 NL: 1.07E4 MA: 418590 m/z= 374.50-375.50 F: + c ESI 6.3 and 9.8 100 Cerberigenine Full ms2 [email protected] (Neriifoline equiv.) [155.00-550.00] MS 080110-10 50
Cerberine 0.01 and 111.1 0 Cerberine 0 2 4 6 8 10 12 14 16 18 20 (Neriifoline equiv.) Time (min)
375.01 NL: 8.14E6 100 080110-10#334-355 RT: 10.78-11.17 AV: 3 F: + c 80 ESI Full ms2 [email protected] 60 [145.00-450.00] Crab’s flesh : 40 339.06 20 162.81238.84 295.14 337.11 415.56 448.86 0 Concentration 339.08 NL: 2.43E6 100 080110-10#308-322 RT: 9.99-10.20 AV: 2 F: + c ESI (µg/g) 80 Full ms2 [email protected] [100.00-500.00] 60 7.7 40 356.93 Neriifoline 20 292.97 293.97 357.99 Relative Abundance 134.82 184.22 231.02 274.91 407.46 439.26 0 202.77 NL: 5.77E4 100 Cerberigenine 1.9 (Neriifoline equiv.) 080110-10#367-384 RT: 11.68-12.11 AV: 3 F: + c 80 ESI Full ms2 [email protected] 60 <0.1 (Neriifoline [155.00-550.00] Cerberine 40 equiv.) 374.89 20 527.19 170.70 203.68 297.12 369.90 402.26 454.89 0 100 150 200 250 300 350 400 450 500 550 m/z
Results: deceased patient # 3
C:\Xcalibur\methods\toxiques vegetaux\cerberine-1mm.meth 10.000000 RT: 0.00 - 20.15 SM: 7G RT: 12.61 NL: 9.25E6 MA: 332503577 m/z= 100 374.50-375.50+504.50-505.50 Serum (1 mL): F: + c ESI Full ms2 50 [email protected] [170.00-550.00] MS 080110-15 Digitoxine = Standard interne 0 RT: 11.08 NL: 4.35E4 MA: 1666761 m/z= 374.50-375.50 F: + c ESI 100 Full ms2 [email protected] Concentration [145.00-450.00] MS 080110-15 50 13.45 (µg/mL) Neriifoline 0 NL: 2.17E6 Relative Abundance 12.63 m/z= 338.50-339.50 F: + c ESI 100 Full ms2 [email protected] Neriifoline 16.6 [100.00-500.00] MS 080110-15 50
Cerberigenine Cerberigenin 0 NL: 1.67E3 N.D. 14.57 m/z= 202.35-203.35 F: + c ESI 100 e Full ms2 [email protected] [155.00-550.00] MS 080110-15 50
Cerberine N.D Cerberine 0 0 2 4 6 8 10 12 14 16 18 20 Time (min)
375.14 NL: 9.53E4 100 080110-15#352 RT: 11.08 AV: 1 F: + c ESI 80 Blood (1 mL): Full ms2 [email protected] 60 [145.00-450.00] 40
20 262.87 339.01 226.53 408.91 Concentration 164.14 305.49 444.19 0 368.58 NL: 1.37E5 100 080110-15#312 RT: 9.96 (µg/mL) AV: 1 F: + c ESI Full 80 364.17 ms2 [email protected] 372.02 [100.00-500.00] 60 375.33 Neriifoline 24.3 40 377.25 20 357.06 263.82 286.09 426.39
Relative Abundance 146.70193.97 237.34 449.51 486.90 0 Cerberigenin 540.91 NL: 7.40E3 100 N.D. 080110-15#383 RT: 340.92 11.92 AV: 1 F: + c ESI e 80 491.89 Full ms2 [email protected] 60 404.59 [155.00-550.00] 40 280.34 Cerberine N.D. 332.89 450.66 20 300.61 0 100 150 200 250 300 350 400 450 500 550 m/z
Conclusion These observations and the determination of 3 cardenolides in the tree C. mangha, the crab B. latro and biological samples confirm our hypothesis of an indirect intoxication:
This allows us also to establish a scientific base for the Melanesian customs which prescribe taboos and very strict rules about eating some special food
14 Conclusion
• Most of the toxins from plants involved in intoxications in Man can be identified by LC-MS/MS
• The main advantage is its sensitivity
• Allows to shorten the extraction
• No need of derivatization
• Using Collision Induce Dissociation (CID) may help if the reference substance does not exist.,
Some others plants and mushrooms cases where LC‐MS/MS may lead the inquiries
Glycyrrhiza glabra L. Liquorice
15 Hallucinogenic mushrooms
Genus Psilocybe • Psilocybine • Psilocine
Amanita muscaria • Muscarine • Muscimol • Ibotenic acid
Ricinus communis Castor bean : ricin
But one may also work without LC-MS/MS :
16 Catha edulis: cathine and cathinone GC-MS
Prunus amygdalis amara: Cyanides LC‐Fluorimetry
Amygdaline
17 Jean-michel Gaulier
Dr. Jean-michel Gaulier, 48 years old, PharmD (Institute of Pharmaceutical and Biological Sciences, University of Lyon), Ph D in Analytical Chemistry (University of Lyon), Head of the Biological and ForensicToxicological Unit, University Hospital of Limoges, Expert in pharmacology and toxicology to the Court of Appeal of Limoges ; 70 publications in Journals indexed in the “Science Citation Index” - 5 book chapters ; Member of The International Association of ForensicToxicologists and of the Society of Hair Testing. Deaths involving gas and volatiles
Jeanean--mmihlGAULIERichel GAULIER
Biological and Forensic Toxicology Lab, Pharmacology and Toxicology Unit ACCREDITATION N° 8-2512 University Hospital Dupuytren, PORTEE DISPONIBLE SUR Limoges, France WWW.COFRAC.FR ACCREDITATION N° 8-2607 PORTEE DISPONIBLE SUR WWW.COFRAC.FR Unusual causes of death: from analysis to interpretation 2013 SOFT Annual Meeting, Orlando - October 2013
Introduction
gas and volatiles in post-mortem toxicology:
involuntary expositions professional accidental
voluntary expositions suicides VSA crimes
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
Objective
to scan gas and volatile substances analyses in post-mortem situations, in a nonexhaustive way, using some examples
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
1 Plan
pre-analytical (samples) rules which samples?
analytical
post-analytical
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
pre-analytical
5 rules … early biological samplings air-tight containers full containers (minimum headspace) conservation under cold conditions (4ºC or less) analysis occurring in a short delay
Gill R et al. Sample handling and storage for the quantitative analysis of volatile compounds in blood: the determination of toluene by headspace gas chromatography. J Anal Toxicol 1988;12:141-6. Gaulier JM et al. Analytical aspects of Volatile Substance Abuse (VSA): about a case report. J Forensic Sci 2003;48(4):880-882
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
pre-analytical
evident rules for samples … illustration: butane intoxication cases
case # 3 # 4 # 5 # 6 # 7 # 8 # 9 post-mortem 80 blood 200 168 94 1,173 1,876 1,800 610 concentrations 267 391 246 (µg/L) + + +++++ + + + + high time-delay (+) elapsed between death and sampling (and/or analysis) is linked to low observed post-mortem blood concentrations
Gaulier JM et al.Interpretation of n-butane findings in post-mortem samples. XX SFTA Congres, Chambéry, France, Septembre 2012
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
2 pre-analytical
evident rules for samples … illustration: butane intoxication cases
case # 3 # 4 # 5 # 6 # 7 # 8 # 9 post-mortem 80 blood 200 168 94 1,173 1,876 1,800 610 concentrations 267 391 246 (µg/L) cases with several blood samples
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
pre-analytical
evident rules for samples … illustration: butane intoxication cases
case # 3 # 4 # 5 # 6 # 7 # 8 # 9 post-mortem 80 blood 200 168 94 1,173 1,876 1,800 610 concentrations 267 391 246 (µg/L) cases with several blood samples
200 µg/L
267 µg/L
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
pre-analytical
evident rules for samples … illustration: butane intoxication cases
case # 3 # 4 # 5 # 6 # 7 # 8 # 9 post-mortem 80 blood 200 168 94 1,173 1,876 1,800 610 concentrations 267 391 246 (µg/L) cases with several blood samples
168 µg/L
391 µg/L
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
3 pre-analytical
evident rules for samples … illustration: butane intoxication cases
case # 3 # 4 # 5 # 6 # 7 # 8 # 9 post-mortem 80 blood 200 168 94 1,173 1,876 1,800 610 concentrations 267 391 246 (µg/L) cases with several blood samples
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
pre-analytical which samples?
blood (concentrations interpretation?) urine (metabolites) gastric content (hydrocarbons) lungs (absorption/elimination) liver heart (+) brain (+) kidney
others: i.e. non biological samples : liquids, gas cylinders, textiles (!!!), …
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
Plan
pre-analytical (samples)
analytical Volatiles = HS-GC-MS
post-analytical
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
4 analytical Volatiles = HS-GC-MS direct injection using « transfer line » or injection using gas syringe
IS choice . Deutered IS, but costly and some problems, i.e.
. benzene-D6 in methanol !!! . acetonitrile-D3 (but chromatographic picks which are not satisfactory …) . in situ generation of IS? . Do not use IS?!
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
analytical Volatiles = HS-GC-MS generate an internal labeled standard in situ
Varlet V et al. Validation of methane measurement using headspace-gas chromatography-mass spectrometry and quantification by a stable isotope-labeled internal standard generated in situ. J Sep Sci. 2013 ; doi: 10.1002/ jssc.201300080 « generate an internal labeled standard gas in vial on the basis of the formation of methane by the reaction of Grignard reagent methyl-magnesium chloride with deuterated water »
CH3MgCl + H2O → CH4 + MgClOH CH3MgCl + D2O → CDH3+ MgClOD
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
analytical Volatiles = HS-GC-MS : our choice… HS Turbomatrix 40 system (Perkin-Elmer, Norwalk, CT, U.S.A.) QP2010 detector (Shimadzu, Japan)
direct injection –transfer line platinum needle columns directly implemented on the head-space system columns: 30 m x 0.25 mm i.d. 1,4 µm Rxi-624SilMS (Restek, France) « ”all terrains” solution, screening » 30 m x 0.32 mm i.d. 30 µm RT-Msieve-5A (Restek, France) CO, organs 30 m x 0.32 mm i.d. 10 µm RT-Q-Bond (Restek, France) alkanes 1 mL ou 1 g organ 22 mL HS-vial
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
5 Plan
pre-analytical (samples)
analytical Volatiles = HS-GC-MS metabolites specific methods
post-analytical
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
analytical
H2S (hydrogen sulphide)
+ concentrated orthoppphosphoric acid hydrolysis Supel-Q plot column HS-CG-SM m/z 32 ; 33 et 34
external calibration using Na2S (sodium sulphide)
Cirimele V et al. Dosage de l’hydrogène sulfuré : bilan de 10 ans d’expérience et contribution du dosage analytique dans l’élucidation de cas d’intoxication chez l’Homme et l’animal. 21ème Congrès de la Société Française de Toxicologie Analytique, St Malo, France, Juin 2013
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
analytical CO
2 g of organ (finely cute < 2 mm) + H2SO4 22°mL HS-vial HS Turbomatrix 40 system (Perkin-Elmer, Norwalk, CT, U.S.A.) 30 m x 0.32 mm i.d. RT-Msieve 5A column (Restek, France) QP2010 detector (Shimadzu, Japan) / SIM mode (m/z = 28) external formic acid calibration LOD: 0,1 µL/g – LOQ : 1 µL/g
Kintz P. et al. Dosage du monoxyde de carbone par HS-GC-MS. Journal de Médecine Légale 2000;43:145-8
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
6 Plan
pre-analytical (samples)
analytical
post-analytical (interpretation) false positive results fermentation of substrates present in a decaying corpse (carbohydrates, amino acids, fatty acids, …) can generate post- mortem volatile compounds: ethanol, acetaldehyde, acetone, 2- propanol, 1-propanol, 1-butanol, isobutanol, isoamyl alcohol, d- amyl alcohol, acetate, propionate, butyrate, isobutyrate, …
Boumba A et al. Biochemical pathways generating post-mortem volatile compounds co-detected during forensic ethanol analyses. Forensic Sci Int 2008;174:133-151
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
Plan
pre-analytical (samples)
analytical
post-analytical (interpretation) false positive results false negative results volatiles are volatile (in the body, in the sample) intensive metabolization, i.e. isobutyl nitrite inhalant
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
Plan
pre-analytical (samples)
analytical
post-analytical (interpretation) false positive results false negative results qualitative results
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
7 post-analytical (interpretation)
Case story 17 yearyear--oldold boy student in a chemistry institute found dead in his bedroom at daybreak corpse in sitting position on the bed aablackblack plastic bag placed on his head without any ties around his neck Gaulier JM et al. Analytical aspects of Volatile Substance Abuse (VSA): about a case report. J Forensic Sci 2003;48 (4):880-882.
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation)
Police search for clues
Several chemical substances
Oxidants: ferric chloride, ethyl methyl ketone peroxide Acids: phosphoric acid, acetic acid Petroleum derivatives: acetone, ethoxyethyl, developers for photography (« White Spirit », xylene…), stain remover called « eau écarlate »
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation)
Corpse examination
No mark of traumatic origin, nor external lesion
Hemorrhagic regurgitation marks around the mouth and nose; a labial wound due to self-biting
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
8 post-analytical (interpretation)
Autopsy findings (≈ 48 hours after death)
Oedematous and inflammatory aspect of the oropharyngeal tract
Bilateral pulmonary oedema
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Methods
Large screening for drugs and toxic compounds (HPLC(HPLC--DADDAD and GCGC--MS)MS)in blood, urine, gastric content, lungs, internal and external methanol rinsing of the plastic bag, chemical substances
More selective assays, ii..ee.. Ethanol and other alcohols in blood, urine, gastric content and VH (GC(GC--FID)FID) DOA in urine (GC- (GC-MSMSand LCLC--ESES--MS)MS) Anticoagulants and rodenticides (HPLC(HPLC--DAD)DAD) …
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Results
large screening and selective assays revealed no drugs, nor toxic compounds in biological samples
only one positive result, in gastric content, using the GCGC--MSMSscreening technique for volatilesvolatiles::
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
9 post-analytical (interpretation)
alkanes in gastric content
9000000 Abundance 8000000
7000000 25 5025 75 50 100 75 2525 50 50m/z 75 75m/z 100 100 6000000 m/zm/z MethMethylcyclohexaHeptaneMethyl-2-pentaneyl-3-hexane 5000000 Rt 5.035 min RtneRt 8.084 8.687 min min 4000000 Rt 9.374 min
3000000
2000000
1000000
0 5.00 10.00 15.00 Time (minutes)
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) MethylMethyl--33--hexanehexane
8.13
Heptane MethylMethyl--22--pentanepentane Volatile 8.72 substances in «eau écarlate » Methyyylcyclohexane
9.41 5.10 Volatile substances in gastric content
5.00 10.00 15.00
Unusual causes of death: from analysis to interpretationTime - deaths (minutes) involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Discussion alkanes in gastric content
Origin: due to sniffing practice and/or psychiatric deviance sniffing drinking
Fukunaga T et al. Liquefied petroleum gas (LPG) poisoning: report of two cases and review of the literature. Forensic Sci Int 1996;82(3):193-200.
Gastric content and « eau écarlate »: same, slightly volatile alkanes « eau écarlate » VSA compatible
Barrière A et al.Poisoning caused by Eau Ecarlate: apropos of a fatal case in a child. Cah Anesthesiol 1987;35(2):125-7
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
10 post-analytical (interpretation) Discussion Hypotheses for the negative results in biological samples
Long time delay between death and sampling (autopsy at T 48 h)
Samples packaged in non airair--tighttight vials
Sent by post to the laboratory at room temperature
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Discussion Plastic bag sniffing practices: use of plastic or paper bags (with or without a support inside) no support (paper, cloth…) found experiments and analyses: similar black ppglastic bag deposit of 2 mL « eau écarlate » 48 h at room temperature in open air (macroscopic evaporation in 2 min without any visible residue) volatile compounds analysis by GCGC--MSMS
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation)
Presence of alkanes,2400000
2200000
2000000
1800000
1600000
contrary to the 1400000
black plastic bag 1200000 found 1000000 on the victim’s head 800000 (blue line) 600000 400000
200000
0 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Time (min) Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
11 post-analytical (interpretation) Discussion Plastic bag
plastic bag probably not used for « eau écarlate » VSA
probably used for a hypoxic recreation practice
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Conclusion
fatality probably due to a VSA with a gasolinegasoline--basedbased cleaner, soso--calledcalled “eau écarlate”, associated with a hypoxic recreation practice using a plastic bag
≠ “simple” « sudden sniffing death syndrome », as seen in teenagers
Barriere A et al. Poisoning caused by Eau Ecarlate: a propos of a fatal case in a child. Cah Anesthesiol 1987;35:125-7
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
Plan
pre-analytical (samples)
analytical
post-analytical (interpretation) false positive results false negative results qualitative results quantitative results
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
12 post-analytical (interpretation) Butane Method 1 mL ou 1 g sample 22 mL HS-vial RT-Q-Bond Column (30 m x 0,32 mm i.d.) (Restek, France) detection: SIM m/z 41 (43/58) external calibration (from 0.078 to 3.9 µg of n-butane) by means of volumetric dilutions from a calibration gas mixture (Scott Specialty Gases, Takkebijsters, Netherlands) using a gas-tight sample lock syringe + specific connecting device (Interchrom, Montlucon, France gas cartridge)
Gaulier JM et al. Interpretation of n-butane findings in post-mortem samples. 20ème Congrès de la Société Française de Toxicologie Analytique, Chambéry, France, Septembre 2012
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Butane Results Case #1-suicide
samples # 1 # 402 yrs #woman, 3 # 4 found # 5 dead # 6 in #the 7 garden, # 8 # 9 after she shut80 herself in the kennel with a 200 168 blood detected detected 94 1,173 1,876 1,800 610 camping267 gas bottle … 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 < 78 liver 285 heart 786 4,090 kidney 457
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Butane Results Case #2-suicide
samples # 1 # 2 # 243 yrs # 4 woman, # 5 dead # 6 after # 7 a # butane8 # 9 explosion80 (+ fire) in her home 200 168 blood detected detected 94 1,173 1,876 1,800 610 267 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 < 78 liver 285 heart 786 4,090 kidney 457
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
13 post-analytical (interpretation) Butane Results Case #3-suicide 64 yrs man, found dead at home in samples # 1 # 2 # 3 # 4the #bedroom 5 # 6 with # 7 a gas # 8 cylinder # 9 of 80 200 168 blood detected detected 94 butane1,173 1,876 1,800 610 267 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 < 78 liver 285 heart 786 4,090 kidney 457
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Butane Case #4-VSA Results 19 yrs boy, ≈ 12 deodorizing sprays around the corpse and samples # 1 # 2 # 3 # 4 # in5 the # refrigerator;6 # 7 # 8 # 9 80 known for chronic use of 200 168 blood detected detected 94 1,173 1,876 1,800 610 267 volatiles 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 < 78 liver 285 heart 786 4,090 kidney 457
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Butane Case #5-VSA Results 19 yrs girl, several deodorizing sprays samples # 1 # 2 # 3 # 4 # 5 #around 6 # 7 # 8 # 9 80 the corpse 200 168 blood detected detected 94 1,173 1,876 1,800 610 267 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 < 78 liver 285 heart 786 4,090 kidney 457
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
14 post-analytical (interpretation)
Butane Case #6-VSA Results 14 yrs girl, at a family party, sniffing a samplesdeodorizing # 1 # 2spray #in 3 a car # 4 # 5 # 6 # 7 # 8 # 9 80 found comatose200 168 blood detected detected 94 1,173 1,876 1,800 610 267 391 deceased following 246 cardiac arrests several urine <78 hours later, in spite of gastric content 347 medica l care lungs 319 820 2,280 134 brain 1,874 <78 liver 285 heart 786 4,090 kidney 457
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Butane Cases #7 and #8 – Results suspected crimes samples # 1Two girls # 2 (sisters): # 3 15 # 4 mo # 5 # 6 # 7 # 8 # 9 80 and 3 ½ yrs found200 dead in a 168 blood detected detected 94 1,173 1,876 1,800 610 267 391 car, together with their246 mother (comatose) homicide urine <78 / suicide tentative attested gastric content 347 by messages open lungs 319 820 2,280 134 butane gas cylinder in the braincar 1,874 < 78 liver 285 heart 786 4,090 kidney 457
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Butane Case #9 – suspected crime 54 yrs women, found dead in a car / gas Results cylinder found in the car and several samples # 1others #in 2 the garage # 3 # 4/ her # husband5 # 6 was # 7 # 8 # 9 also in the car, alive80 - suspected of having 200 168 blood detectedmurdered detected his wife 94 1,173 1,876 1,800 610 267 391 Dumestre V et al. Quantitative determination246 of n-butane in post-mortem tissues: a case report . urine TIAFT annual Congress, Hamamatsu, Japon, Juin 2012 <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 <78 liver 285 heart 786 4,090 kidney 457
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
15 post-analytical (interpretation) Butane Discussion
samples # 1 # 2 # 3 # 4 # 5 # 6 # 7 # 8 # 9 80 200 168 blood detected detected 94 1,173 1,876 1,800 610 267 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 < 78 liver 285 heart 786 4,090 kidney 457
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Butane Discussion
samples # 1 # 2 # 3 # 4 # 5 # 6 # 7 # 8 # 9 80 200 168 blood detected detected 94 1,173 1,876 1,800 610 267 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 <78 liver 285 heart 786 4,090 kidney 457 interest of n-butane determinations in post-mortem tissues or organs?
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Butane Discussion
samples # 1 # 2 # 3 # 4 # 5 # 6 # 7 # 8 # 9 80 200 168 blood detected detected 94 1,173 1,876 1,800 610 267 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 < 78 liver 285 heart 786 4,090 kidney 457 interest of n-butane determinations in post-mortem tissues or organs? only few data, but, it seems no correlation between [lungs] and [blood]
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
16 post-analytical (interpretation) Butane Discussion
samples # 1 # 2 # 3 # 4 # 5 # 6 # 7 # 8 # 9 80 200 168 blood detected detected 94 1,173 1,876 1,800 610 267 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 < 78 liver 285 heart 786 4,090 kidney 457 interest of n-butane determinations in post-mortem tissues or organs? only few data, but, it seems no correlation between [heart] and [blood]
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Butane Discussion
samples # 1 # 2 # 3 # 4 # 5 # 6 # 7 # 8 # 9 80 200 168 blood detected detected 94 1,173 1,876 1,800 610 267 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 <78 liver 285 heart 786 4,090 kidney 457 interest of n-butane determinations in post-mortem tissues or organs? only few data, but, it seems “perhaps” a correlation between [brain] and [blood]?
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
post-analytical (interpretation) Butane Discussion
samples # 1 # 2 # 3 # 4 # 5 # 6 # 7 # 8 # 9 80 200 168 blood detected detected 94 1,173 1,876 1,800 610 267 391 246 urine <78 gastric content 347 lungs 319 820 2,280 134 brain 1,874 < 78 liver 285 heart 786 4,090 kidney 457
in accordance with n-butane blood Ago M et coll. Leg Med. 2002; 4: 113113--118118 concentrations reported in the literature Fuke C et coll. Leg Med. 2002; 4: 134134--138138 Jackowski C et coll. Am J For Med Path. 2005; 26: in such fatalities : > 100 µg/L 355355--359359 and? Sugie H et coll. For Sci Int. 2004; 143: 211211--214214 Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
17 post-analytical (interpretation) Conclusion are blood post-mortem n-butane determinations required in n- butane intoxication ? established (proven) suicide NO suspected VSA fatalities, or murder YES
what is the blood lethal concentration? > 1000 µg/L If > 1,000 µg/L no doubt with a “massive” n- butane exposition close to the DC with potential lethal toxic effects: hypoxemia, SSDS < 1000 µg/L other items should be taken into account (especially sampling!) i.e. if no full containers, or air-tight containers, then a “massive” n-butane exposition close to the DC with potential lethal toxic effects can be considered
any interest in n-butane determinations in other post-mortem tissues or organs? lungs, heart: hum! … brain: perhaps…
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
Conclusion
pre-analytical (samples) analytical post-analytical (interpretation) critical importance of the quality of sampling only few pitfalls in analyses but the interpretation is limited and should be performed with caution
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
Thank you for your attention
Unusual causes of death: from analysis to interpretation - deaths involving gas and volatiles JeanJean--michelmichel GAULIER
18 Jean-Claude Alvarez
Pr. Jean-Claude Alvarez, 49 years old, Professor of Medicine in Pharmacology, PhD, Director of the platform of mass spectrometry at the University, Versailles Saint Quentin- en-Yvelines, Head of Department of Pharmacology–Toxicology at the University Hospital of Garches, expert in pharmacology and toxicology to the Court of Appeal of Versailles ; 78 publications in pharmacology and toxicology ; participation in 3 books, President-Elect of the French Society of Analytical Toxicology (SFTA, President in 2015).
10/4/2013
Intoxications and deaths involving pesticides Pr. JC Alvarez
Department of Pharmacology – Toxicology Medical Faculty of « Paris Ile-de-France Ouest » Versailles Saint-Quentin-en-Yvelines University Teaching Hospital of Garches, France
1
What are Pesticides ?
Pesticides or plant protection products are drugs for plants: Kill insects: insecticides Kill rodents: rodenticides (talpicine) Kill weeds: herbicides Against diseases that attack plants: fongicides, antiparasitic (but these compounds are less toxic)
Natural active ingredients (copper, sulfur…) or synthetics +++
2
What are Pesticides ?
Sprayed onto plants or near them
Used in large quantities since the ‘50s in intensive agriculture in the world and in malaria eradication
Presence in many areas: water, soil, air...
Presence in many foods: 50% of fruits and vegetables issued of intensive agriculture may be contaminated 3
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Use of pesticides in World
France is the largest European user
- But median consumption if reported by harvested acres
- United States > Japan > France
- Herbicides are the most widely used
- Glyphosate is the most common
4 From ECPA « European Crop Protection Association », 2009
Chronic toxicity of pesticides
Most of these chemicals are not specific to their target and could contaminate other species including humans
May be the cause of many diseases including cancers (prostate and non-Hodgkin lyypmphoma)
France: 2 studies have found side effects in 20% of manipulators during one year of professional use
Lasso herbicide (Monsanto) prohibited in France in 2007 5
Chronic exposure of pesticides : biomonitoring1
Hair analysis is a promising biomarker for the biomonitoring of human exposure to environmental chemicals
Chemical concentration in hair may be representative of both the level of exposure and the chemical concentration in other compartments of the body
Considerations highlight the need to develop multi-class methods with high sensitivity for the biomonitoring of human exposure to pesticides based on hair analysis
1BMR Appenzeller, AM Tsatsakisb, Toxicology letters, 2012 6
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Acute toxicity of pesticides
Rating of World Health Organization: 1 million of severe poisoning each year
220 000 deaths (India +++)
Few cases describes in literature
Even fewer cases with confirmation by blood measurement
7
Acute toxicity of pesticides
2006 in Poison Control Center in France : 5100 cases out of 130,000 voluntary and accidental exposures to a toxic (= 3.9%, Villa et al. Rev Prat 2008 ; 58 : 825-31)
15 deaths: mortalityyp of 2.9 per thousand
138 total deaths in 2006 from toxic, 10.9% with pesticides
2012 in Paris : 970 cases (mainly accidental) out of 26,000 cases of intoxication (= 3.7%)
8
Penetration and distribution
Penetration Essentially dermal, respiratory and mucous in professional environment But mainly digestive in acute administration Hand hygiene for professional Ingestion in domestic environment, either by food (chronic) consumption or by deliberate ingestion (suicide)
Distribution : practically every organ
Fat storage for some of them (DDT)
9
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Analytical aspects: difference between acute and chronic exposure
When assessing environmental exposure by the quantification of chemicals in a biological matrix, analytical sensitivity is a fundamental parameter since levels of concentration resulting from environmental exposure are relatively low : In cases of acute poisoning with pesticides serum [C] range from 20 g/L (for bifenthrin) up to 1.5 (carbofuran) or 6.5 mg/L (endosulfan, Lacassie et al., 2001) In cases of chronic exposure, levels of pesticide concentration detected in serum collected from raw population is generally about 10 ng/L or lower (Berman et al., 2011), and about 10-100 pg/mg in hair
10
Analytical aspects: Methods
Most of them were based on GC/MS and focused on specific compounds or on a single chemical class, contrary to environmental matrices or food where multi-class methods are more common Cirimele et al (1999) were probably the first to siltimultaneous lly analyze 15 agriltlicultural pestic ides from several chemical classes in hair collected from farm workers (GC/MS) Recently-developed methods based on Solid Phase MicroExtraction (SPME) coupled with GC-MS/MS enabled researchers to reach high sensitivity while analyzing 22 pesticides simultaneously from several classes (Salquebre et al., 2011) 11
1. Insecticides : organochlorines (1)
DDT, aldrine, chlordane, heptachlore, lindane, endosulfan
Highly lipophilic and toxic Very stable, have long metabolic disposition and are stored in fatty tissues Can persist in the environment (water, soil, plants)… but also Human
All prohibited in Europe ; Many now banned in the US. For example: lindane banned in California and elsewhere used for control of lice and scabies
12
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1. Insecticides : organophosphates (2)
Parathion, chlorfenvinphos, diméfox, dichlorvos, methyl-parathion, malathion, trichlorfon
Very liposoluble but rapidly degraded, no accumulation, have replaced organochlorine
Most of OPs are highly toxic, would represent half of all deaths linked to pesticides in the world
3 groups according to their toxicity:
Highly toxic: DL50 < 50 mg/kg : chlorfenvinphos, parathion, diméfox
Moderately toxic: 50 < DL50 < 500 mg/kg : dichlorvos
Low toxicity: DL50 > 500 mg/kg : malathion (metabolite 60 times more toxic), trichlorfon 13
1. Insecticides : organophosphates (2)
Mechanism of action: inhibition of the acetylcholinesterase enzyme, producing accumulation of acetylcholine at the synapses and neuromuscular junction, leading to excessive stimulation
Reaction is generally an irreversible binding, with phosphorylation of the esterase site of the enzyme
Release of the enzyme is very slow, but can be accelerated by enzyme reactivator: oxime (pralidoxime), which is an specific antidote of Ops intoxication
14
1. Insecticides : organophosphates (2)
Accumulation of acetylcholine : First toxicity by muscarinic effects: nausea, vomiting, abdominal pain, hypersecretions, miosis, bradycardia..
Then nicotinic effects: seizures, respiratory depression,coma and respiratory failure.
Laboratory confirmation: red blood cell and plasma cholinesterase levels depressed with acute poisoning; Evolution well correlated with symptomatology
Measurement of OP possible, even if sometimes unstable, usually with GC/MS 15
5 10/4/2013
1. Insecticides : organophosphates (2) : case reports of deceased patients
Method Heart blood Peri blood Viscera Ref (µg/mL) (µg/mL)
Methidathion GC/MS 66 8.3 Yes Takayasu, Legal Med, 2012 1.9 0.4 Yes Arao, Legal Med, 2002 Dimethoate GC/MS 38 -- Yes Tarbah, FSI, 2007
Omethoate GC/MS 208 -- Yes Pavlic, Int J Leg Med, 2002
Chlorfenvinphos GC/MS 8.6 and 4.4 -- Yes Martinez, JAT, 2012
Dichlorvos GC/MS 4.4 1.3 Yes Abe, JFS, 2008 GC/MS 29 -- Yes Shimizu FSI 1996 Fenthion GC/MS 3.8 -- No Meyer, JAT, 1998
Malathion GC/MS 1.8 -- No Thomson, FSI 1998
Dichlorophen GC/MS 9.8 -- No Kintz, Int J Leg Med, 1997
Chlorpyrifos GC/MS 5.3 (alive) -- No Martinez, JAT, 2004
Disulfoton GC/MS 1.1 (alive) -- No Futagami, J Toxicol16 Clin Toxicol, 1995
1. Insecticides : organophosphates (2)
Heart blood = 4.4 µg/mL Peripheral blood = 1.3 µg/mL Heart Concentration = 1400 µg/g
17
1. Insecticides : carbamates (3)
Used for insect control in agricultural and home settings Acting like OP but less toxic because acetylcholinesterase inhibition is reversible
3 groups according to their toxicity:
Highly toxic DL50 < 20 mg/kg : aldicarb, carbofuran, methomyl ® Moderately toxic 20 < DL50 < 100 mg/kg: propoxur (Baygon )
Low toxicity DL50 > 100 mg/kg : carbaryl
Pralidoxime ineffective: interest of the identification of the compound (OP or carbamate) and of the measurement of its biological concentration when plasma cholinesterases are decreased 18
6 10/4/2013
1. Insecticides : carbamates (3)
Case reports
Metho Heart blood Perif blood Viscer Ref d (µg/mL) (µg/mL) a Aldicarb HPLC/fl 6.2 -- Yes Proença, FSI, 2004 uo Methomyl GC/MS 8 3 Yes Moriya, FSI, 2005 0.7 Yes Hoizey, JFS, 2008 Ethiofencarb GC/MS 26.4 -- No Al-Samarraie, JAT 2009 Propanil + GC/MS 21.6 and 8.1 -- No Yamazaki, JFS, 2001 carbaryl Carbofuran GC/MS 0.32-11.6 No Ameno, FSI, 2001 (n=4)
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1. Insecticides : carbamates (3)
Carbofuran: compound with Carbofuran 3-OH cardiac tropism (not OH- carbofuran carbofuran) Peripheral blood 225.5 080.8 (mg/L) Necessity of measurement Heart blood 4.0 1.0 in peripheral blood (mg/L) Gastric content 41230 < 0.2 Liver and kidney elimination (mg/Kg) (and not exclusively urinary) Bile (mg/L) 19.5 10.2 Urine (mg/L) 11.4 17.0
C. Bocca et al. Dosage of carbofuran in blood and viscera by LC/MS/MS, application to two forensic cases. Ann Toxicol Anal, 2011, 23(S1): 28 20
1. Insecticides : pyrethrins - pyrethroids (4)
Used as pediculicide, to control insect and often used in home and garden
Lipophilic compounds, but unstable in soil, non-retentive with no residues
Low toxicity in humans because of their rapid metabolism but risk of seizures if massive ingestion
No death reported with biological concentration in literature
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2. Herbicides
Highly variable toxicity Toxicity is related to the product, but also to the solvent
2004: Atrazine was banned in EU, because of its persistent groundwater contamination (up to one year) In the US, it is one of the most widely used herbicide, with 76 million pounds applied each year
2007: Paraquat forbidden in EU because of its high toxicity (linked to development of Parkinson’s disease) In the US, It is classified as "restricted use," which means that it can be used by licensed applicators only
Most widely important is glyphosate +++ 22
2. Herbicides : glyphosate (1)
Aminophosphonate: non-selectif systemic herbicide
No anticholinesterase activity, should block the synthesis of the aromatic amino acids in the plant
Ingestion > 100 mL : can cause death Digestive disorders: caustic effect by associated excipient Multivisceral disorders: respiratory, renal and cardiovascular ;
Some deaths described with blood concentrations
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2. Herbicides : glyphosate, case report
Gly AMPA Clinical Ingested vol Decease Ref (µg/mL) (µg/mL) or number of patients 22.6 0.18 10 mL Non Hori, 2003 ?? ?? Digestive 300 mL Oui Delaunay, 2011 disorders, solution 30% nécrosis 3050 ?? Congestion, 500 mL Oui Sribanditmongkol Pulmonary solution 41% 2012 edema 0.6 - 150 0.17 – 0.76 Low to n = 5 Non Zouaoui, 2013 moderate 690 - 0.8 – 24.6 Decease n = 6 Oui Zouaoui, 2013 7480
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3. Rodenticides (1)
Anticoagulants like antivitamine K agents Warfarin and derivatives Clinical symptomatology if massive ingestion in human Many intoxications
α-Chloralose : causes convulsive coma with bronchial hypersecretion Treatment: anticonvulsant like diazepam and atropine for secretion; good prognosis Toxic dose 2-4 g, lethal: 10 g
Gerace, 2012 : 50 g Death [C] = 65.1 µg/mL Kintz 1999 : 15 g Death [C] = 175.7 µg/mL 25
3. Rodenticides (2)
Crimidine Lethal dose: 100 mg ? Besnard, 2002 : unknown dose , [C] = 368 ng/mL, not dead
Strychnine : used against foxes, talpicide and only authorized for veterinary in France since 2000 Intoxication : tetanization with convulsion after 20-30 min, patient being conscious, death by respiratory paralysis Treatment: anticonvulsant like diazepam + hydration Lethal dose : 20 to 50 mg Interest of blood concentration: Toxic [C] = 0.075-0.1 mg/L Lethal [C] > 0.2 mg/L
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[C] Bottle = 850 mg/L Blood [C] = 25 mg/L Q ingested about 27 200 mg
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Sulfate of strychnine 10%, expired since many years, 3% of strychnine when measured
STRYCHNINE IS: CHLOROQUINE
Intoxication on D1, sampling of blood and urine on D7, hair (4 cm) collected on D12 Blood and urine: Strychnine not detected Hair: Segment A Segment Segment Segment = 1 cm B = 1 cm C = 1 cm D = 1 cm (20.3 mg) (20.5 mg) (20.2 mg) (19.5 mg) Strychnine 207 pg/mg Absence Absence Absence28
Case 2 :
Patient whose wife is veterinarian. Subcutaneous abdominal injection of an ampule marketed as « Kill dog » (expired since 15 years !!) Painful stifness of the 4 members, trismus At hospital, classical toxicology was negative and alcohol = 0.18 g/dL
Blood and urine were collected on D2, hair (5 cm) one month later Blood: Not detected Urines: Presence at concentration < 1 mg/L Hair:
Segment A = 2,5 cm Segment B = 2,5 cm (20.6 mg) (21.1 mg) Strychnine 1000 pg/mg 98 pg/mg
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Conclusion
• Many cases of deaths from acute poisoning, but few are documented • When documented, most of the time there is no dosage of the compound • Recent development of multi-class method for simultaneous measurement of many pesticides is of great interest, standards for calibration not so easy to find • However, blood concentration correlate well with prognosis for some of them, in particular glyphosate the most widely used • Characteristic smelling when opening stomach at autopsy, especially garlic smelling, must evoke pesticide poisoning • Great interest of hair for retrospectively diagnosis, and probably for chronic exposure monitoring 30
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Thank you for your attention
With financial support of
11 Patrick Mura
Dr. Patrick Mura, 64 years old, PhD, Head of the Toxicological and Pharmacokinetic Unit, University Hospital of Poitiers, France. Expert in pharmacology and toxicology to the Court of Appeal of Poitiers. 75 publications in Journals indexed in the “Science Citation Index”. Editor of 2 books ; participation in 9 books. Past-President of the French Society of Analytical Toxicology (2003-2009). French Regional Representative for TIAFT. Member of the French National Academy of Pharmacy.
04/10/2013
Deaths involving glycemia
Patrick MURA University Hospital Poitiers, France
2013 SOFT Annual Meeting. Orlando, Florida, USA.
Glycemia in forensic toxicology Toxicological investigations
Death Doping
Hyperg ly cemia Hypoglycemia
• Diabetes mellitus (most frequenly) • Excess of insulin • Drugs (olanzapine, duloxetine, • Excess of other amphetamines) hypoglycaemic drugs • Critical illness •Failure to produce glucose
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Hyperglycemia and forensic toxicology
A 54‐year‐old man with history of diabete Found dead in his bed Blood, urine and vitreous humor are sent to our laboratory We had to determine the cause of death
Toxicological screenings (alcohols, drugs, drugs of abuse, …) were performed in blood and urine by chromatographic methods (HPLC, LC/MS/MS, GC/MS) negative
Vitreous humor glucose (enzymology) 62.7 mmoles/L
Glucose in vitreous humor 62.7 mmoles/L
Guillermo Vivero and Eduardo Osuna (spanish group, urcia and Madrid) The Review of Diabetic Studies 2008; 5: 220‐4.
Vitreous humor from 377 cadavers divided in 2 groups, one diabetic and the other non‐diabetic Mean post‐mortem interval of 14.9 h
Diabetic Non‐diabetic Mean S.D. Mean S.D. Post‐mortem 15.4 5.7 14.7 6.1 interval (h) Glucose 5.2 6.5 1.1 1.1 (mmol/L)
1/ Glucose in vitreous humor 62.7 mmoles/L
Antemortem hyperglycemia
2/ Pathologist results revealed an acute pancreatitis
Interpretation
Acute pancreatitis
Intensive liberation of glucagon
Hyperglycemia
Death
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Glucose in vitreous humor 62.7 mmoles/L
With such a value , it was not too difficult to conclude. Some other biochemical markers canbeuseduseful:
Diabetic Non‐diabetic Mean S.D. Mean S.D. Post‐mortem interval (h) 15.4 5.7 14.7 6.1 Glucose (mmol/L)* 5.2 6.5 1.1 1.1 Fructosamine* 0.8 1.2 0.2 0.2 Lactate* 16.7 4.0 13.3 4.2 Glucose + lactate* 22.0 8.2 14.5 4.4 Glucose + fructosamine* 6.1 7.1 1.3 1.1 In all cases p< 0.001
Postmortem vitreous humor ‐hydroxybutyrate: its utility for the postmortem interpretation of diabetes mellitus Osuna E. et al. Forensic Science International 2005; 153: 189‐95
These authors vitreous humor glucose levels may fluctuate and be influenced by the environmental temperature
Taking into account that ketoacidotic coma is one of the most serious complications arising from diabetes mellitus, specially type I, which may terminate in the sudden death of the patient,
On 453 cadavers, determination of : ‐hydroxybutyrate (enzymologic method using ‐OHB dehydrogenase and nicotinamide‐adenine dinucleotide), + Glucose, lactate and fructosamine (Hitachi autoanalyzer Roche)
With comparison of two groups : diabetic and non‐diabetic
Diabetic Non‐diabetic Mean S.D. Mean S.D. Post‐mortem interval < 72 h Glucose (mg/dL) 100.3 116.0 21.7 27.5 Fructosamine (mmol/L) 0.88 1.28 0.16 0.22 Lactate (mg/dL) 153.2 37.4 124.4 40.0 Glucose + lactate 253.5 129.5 146.2 50.6 (mg/dL) ‐OHB (mg/mL) 6.32 16.50 2.02 7.34 In all cases p< 0.001 Also, they classified the study sample into 3 groups according to the glucose values: below 50 mg/dL, from 50 to 200mg/dL and above 200 mg/dL The highest ‐OHB values in vitreous humor were obtained in the group of cases with glucose values above 200 mg/dL
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The authors conclude that ‐OHB levels could be determined in the cases where glucose levels are high and it is necessary to ascertain the possible existence of diabetic ketoacidosis.
These studies confirmed the results of a number of papers, such as for example :
Karlovsek MZ (Ljubljana, Slovenia). Forensic Sci Int 2004; 146: 19‐23 [on the interest of combined values of glucose and lactate in vitreous humor]
Terekhina NA and Akimov PA (Russia) Patol Fiziol Eksp Ter 2005; 2: 24‐5
All these studies underline the major interest of the determination of glucose and lactate in vitreous humor, with the assumption that pre‐ existing glucose is gradually converted to lactate under anaerobic conditions during agonal phase and the early postmortem period.
But
an other study showed that the use of the sum value of vitreous glucose and lactate can lead to erroneous diagnosis of hypoglycemia.
Postmortem identification of hyperglycemia Zilg B, Alkass K, Berg S, Druid H (from Sweden) Forensic Sci Int 2009; 185: 89‐95
3076 cases After an initial drop (as soon as possible after arrival at the morgue) of vitreous humor (0.2 mL aspirated with a 1mL syringe equipped with an 18‐ gauge needle) from the center of each eye and pooled, Followed by a second sample collected at autopsy 1‐3 days later Analyses were performed using a blood gas instrument
Glucose levels stayed stable but lactate levels showed a steady increase The sum glucose + lactate increases with post‐mortem time
They conclude by suggesting that vitreous glucose alone should be used to diagnose hyperglycemia postmortem and that the limit of 10 mmol/L should have a good specificity for diabetic coma, which theoretically would equal an original blood glucose value of about 26 mmol/L
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Investigation of markers to indicate and distinguish death due to alcoholic ketoacidosis, diabetic ketoacidosis and hyperosmolar hyperglycemic state using post‐mortem samples Hockenhull J., Dhillo W., Andrews R., Paterson S. (from United Kingdom) Forensic Sci Int 2012; 214: 142‐7
191 post‐mortem cases
They suggest blood ‐OHB concentration should be measured in all cases where acetone has been detected in post‐mortem blood greater than 2 mg/dL.
They also suggest vitreous humor glucose concentrations should be measured in all cases where ‐OHB is detected in significant concentrations and more generally in all unexplained deaths and especially in cases with risk factors for diabetes including obesity or old age.
Glycemia in forensic toxicology Toxicological investigations
Death Doping
Hyperg ly cemia Hypoglycemia
• Diabetes mellitus (most frequenly) • Excess of insulin • Drugs (olanzapine, duloxetine, • Failure to produce amphetamines) glucose • Critical illness
Fatality from olanzapine induced hyperglycemia Meatherall R, Younes J. (from Canada) J Forensic Sci 2002; 47: 893‐6
A 31‐year‐old male schizophrenic patient, Treated with olanzapine (10 mg one week followed by 20 mg) for three weeks before he died, with no other medication, Without history of diabete mellitus Drug screen on urine and blood only a small amount of olanzapine (45 ng/mL) Analysis of vitreous humor Glucose 49 mmol/L, small amounts of cetones. Death was attributed to hyperosmolar nonketotic diabetic coma, Olanzapine was felt to be the cause. So they 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.
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Hypoglycemia in forensic toxicology Toxicological investigations
Death ? Doping Hypoglycemia Why is hypoglycemia so dangerous ? Answer in Medscape Diabetes and Endocrinology • Excess of insulin by Leszek Czupryniak and Brian M. Frier •Excess of other hypoglycaemic drugs • Failure to produce glucose
Hypoglycemia would not kill everybody Neurologic death, brain death occurring from hypoglycemia is relatively rare The brain is very resistant. We may consider that at least 6 hours of very deep hypoglycemia before getting permanent brain damage and subsequent death.
The main cause of death is cardiac Hypoglycemia causes cardiac arrhythmia, cardiac ischemia or exacerbating cardiac failure
Hypoglycemia in forensic toxicology Toxicological investigations
Death Doping
Hypoglycemia
• Excess of insulin • Excess of other hypoglycaemic drugs • Failure to produce glucose
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INSULIN
Computer‐generated image of six insulin molecules assembled in a hexamer, highlighting the threefold symmetry, the zinc ions holding it together, and the histidine residues involved in zinc binding. Insulin is stored in the body as a hexamer, while the active form is the monomer
Effect of insulin on glucose uptake and metabolism. Insulin binds to its receptor (1), which starts many protein activation cascades (2). These include translocation of Glut‐4 transporter to the plasma membrane and influx of glucose (3), glycogen synthesis (4),glycolysis (5) and triglyceride
Insulin, a Nobel Prizes story
Frederick Banting joined by Charles Best in office, 1924 The Nobblel Prize committee in 1923 credddited the practical extraction of insulin to a team at the University of Toronto and awarded the Nobel Prize to two men: Frederick Banting and J.J.R. Macleod. They were awarded the Nobel Prize in Physiology or Medicine in 1923 for the discovery of insulin. Banting, insulted that Best was not mentioned, shared his prize with him, and Macleod immediately shared his with James Collip. The patent for insulin was sold to the University of Toronto for one half‐dollar.
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The primary structure of insulin was determined by a British molecular biologist, Frederick Sanger. It was the first protein to have its sequence be determined. He was awarded the 1958 Nobel Prize in Chemistry for this work.
In 1969, after decades of work, Dorothy Hodgkin determined the spatial conformation of the molecule. She had been awarded a Nobel Prize in Chemistry in 1964.
Rosalyn Sussman Yalow received the 1977 Nobel Prize in Medicine for the development of the radioimmunoassay for insulin.
INSULIN AND DOPING
After being thrown out of the Tour de France in 2007, professional cyclist Michael Rasmussen have now confessed taking multiple illegal substances, including insulin
Insulin, a doping agent also used in equine sports
Insulin can act as a powerful anabolic agent. It helps to drive glucose and amino acids into muscle cells, thereby helping to increase glycogen synthesis and lean muscle mass. When combined with anabolic steroids, insulin also helps prevent muscle tissue breakdown. The International Olympic Committee banned the use of this hormone by non‐diabetic athletes in 1998, but the problem is that a test to catch insulin abusers does not yet exist because chemical procedures to date have not been able to differentiate sufficientlyreliablybetwwenthe synthetic insulins produced for medical use, and the endogenously produced insulin produced naturally by the body.
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INSULIN and MURDERS OR SUICIDES
«The present account describes the medical and scientific investigations in a case in which a man was convicted of murdering his wife by injecting her with insulin. » It was the first time «in which insulin has been demonstrated in human tissue, other than pancreas, after death. »
6 hours after death, glucose in blood was found to be 210 mg/ 100 mL [it is known that glucose concentration in blood may rise after death, due to glycogenolysis in the liver and subsequent diffusion of glucose into the inferior vena cava and the right heart]
At this time, no chemical or physico‐chemical method was devised for its identification.
So, they realized tissues extracts of the victim and injected these extracts purified to animals :
The results showed that the biologically active substance present in the extracts from the dead woman’s tissues (skin, fat and muscle) had the following properties : 1/ It produced hypoglycaemia on injection into mice and guinea‐pigs 2/ It stimulated glucose consumption by the isolated rat diaphragm 3/ These biological effects were reduced or abolished by (a) cysteine, (b) proteolytic enzymes (pepsin and insulinase), (c) serum of insulin‐ densitized guinea‐pigs, and (d) anaerobic incubation
These properties are characteristic of insulin, and no other known hypoglycaemic agent could have produced the same combination of effects.
The woman’s husband, a trained male nurse, was accused and convicted of his wife’s murder
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Practical considerations
The normal plasma insulin level is said to be 6 –26 mU/mL and some authors say that the post‐mortem insulin level should be at a maximum of 0.07 mU/mL When considering whether the death is related to exogenous insulin administration, one must exclude an islet cell tumour (pancreatic endocrine neoplasia for example insulinoma) Endogenous insulin production (for example from tumours) is accompanied by a corresponding equivalent increase in c‐ peptide (which is split from proinsulin) Exogenous insulin administration is not associated with a rise in the c‐peptide levels.
Practical considerations
Secretogogue drugs, such as tolbutamide and chlorpropramide, cause there to be a rise in endogenous insulin production, with a corresponding rise in c‐peptide. One can’t calculate the dose of insulin administered exogenously from post‐mortem blood analysis. No post‐ mortem correlation has been found between insulin dosages and post‐mortem values, or the time intervals of administration. No defined « fatal » post‐mortem levels of insulin in blood have been identified but some concentrations measured in fatal cases of suspected exogenous insulin administration have been recorded : Some authors report 12 –40 mU/mL (with blood glucose 0.25 mmol/L and c‐peptide < 0.1 –4.4 ng/mL)
Practical considerations Insulin to C‐peptide ratio It is normally 0.1 – 0.47, never >1 Exogenous insulin administration leads to a rise in this ratio.
HbA1c analysis Post‐mortem blood analysis of non‐diabetics has shown HbA1c to be : between 4 and 6% (Valenzuela 1998) Between 3.25 and 6.25% (Goulle et al 2002)
Post‐mortem HbA1c in diabetics were found 6.5 – 10.7% in diabetics
Kernbach‐Wighton and Puschel found post‐mortem HbA1c in exogenous insulin administration cases to be between 8.1 – 10.7% and > 12% in diabetic comas
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Practical considerations
Vincent Marks. Murder by Insulin Med Leg J 1999; 67: 147‐63
The amount of insulin required to kill infants may be quite small and easilyadiitdministered . In the sentient adult, the amount is large, requires injection of a large volume of fluid, 10 to 20 mL, and needs 20 minutes to take effect.
Quantification of human insulin and its synthetic analogues in human specimen : new data
Disorders of glucose metabolism: post mortem analyses in forensic cases‐part II Franck Musshoff, Hess C, Madea B. Int J Legal Med 2011; 125: 171‐80
Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography‐mass spectrometry Hess C, Thomas A, Thevis M, Stratmann B, Quester W, Tschoepe D, Madea B, Musshoff F. Anal Bioanal Chem 2012; 404: 1813‐22
Measuring insulin in human vitreous humour using LC‐MS/MS Thevis M, Thomas A, Schänzer W, Östman P, Ojanperä I. Drug Test Analysis 2012; 4: 53‐6
Determination of hypoglycemia induced by insulin or its synthetic analogues* post mortem Hess C, Madea B, Daldrup T, Musshoff F Drug Test Anal 2013 Jul 11. doi: 10.1002/dta. 1500 *
Measuring insulin in human vitreous humour using LC‐MS/MS Thevis M, Thomas A, Schänzer W, Östman P, Ojanperä I. Drug Test Analysis 2012; 4: 53‐6
A 55‐year‐old non‐diabetic female, died from an insulin overdose. This insulin poisoning occured at the hospital. After five weeks of inpatient health care, a sudden and severe hypoglycemia occured : 2 mg/dL (before: 89 mg/dL).
Immunological method Blood insulin level was 5551 mIU/L (194 ng/mL) = extremely elevated (before: 5.9 mIU/L, 0.2 ng/mL) C‐peptide concentration s remained within normal ranges (1.7 –4.8 ng/mL)
The patient was comatose, survived upon normalization of blood glucose levels without regaining consciousness, and deceased after four days.
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During autopsy, an adenoma of the pancreatic tissue was not observed.
Sample preparation 500 µL of vitreous humor Enriched with 1 pmol of bovine insulin (ISTD) + 1 mL of acetonitrile, vortexed, centrifuged, supernatant evaporated Reconstitution in 500 µL of phosphate‐buffered saline (PBS), + Anti‐insulin antibodies and anti‐mouse IgG coated magnetic beads The mixture was incubated for 1h at 20°C The beads were separated, washed with PBS, Target analytes were eluted with 50 µL of aqueous acetic acid for LC‐MS/MS
Liquid chromatography‐(tandem) mass spectrometry
Two instrumental setups were applied : 1/ a nanoUPLC (WATERS Acquity) and a Thermo LTQ equipped with a nanospray source 2/ An Accela untrahigh performance liquid chromatograph (UHPLC) + an exactive high resolution/high accuracy orbitrap mass spectrometer (Thermo), interfaced by means of an Advion Triversa Nanomate (Ithaca).
«The combined information obtained by both instruments was used to unambiguously identify insulin in vitreous humor. »
Determination of hypoglycemia induced by insulin or its synthetic analogues post mortem Hess C, Madea B, Daldrup T, Musshoff F Drug Test Anal 2013 Jul 11. doi: 10.1002/dta. 1500
Case 1: antemortem material was available Human insulin was detected by immunopurification with magnetic beads and LC‐MS/MS analyses at a concentration of 5180 µU/mL. The molar ratio human insulin / c‐peptide was 111.
Case 2: a suicide by self‐injection of Insulin lispro. The determination of the drug was performed after pre‐ extraction with methanol and immunopurification by LC‐ MS/MS at the injection site, in vitreous humor and organs.
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As a conclusion concerning hypoglycemia induced by insulin
«The determination of human insulin or its synthetic analogues in post‐mortem specimens represents a challenge for forensic toxicologists due to its proven instability in post‐ mortem blood. Apart femoral blood and urine, vitreous humor and the injection site promise the best possibilities for a proof of insulin at autopsy. In addition, c‐peptide, glucose, lactate should be measured in case of suspected fatal hypoglycemia. »
Hess C, Madea B, Daldrup T, Musshoff F
Glycemia in forensic toxicology Toxicological investigations
Death Doping
Hyperg ly cemia Hypoglycemia
• Diabetes mellitus (most frequenly) • Excess of insulin • Drugs (olanzapine, duloxetine, • Excess of other amphetamines) hypoglycaemic drugs • Critical illness •Failure to produce glucose
Metformin intoxication
Metformin, a member of the biguanide class
Metformin promotes the conversion of glucose to lactate in the splanchnic bed of small intestine
Metformin inhibits hepatic gluconeogenesis from lactate, pyruvate, alanine, resulting in additional lactate and substrate for lactate production (mainly by decreased pyruvate carboxylase activity, a rate limiting enzyme in the formation of glucose from lactate)
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Does plasma metformin level predict lactate level / mortality?
Lalau et al. Diabetes Care 1998
Fatal metformin intoxication with markedly elevated blood and liver concentrations F. Lee Cantrell, Craig L Nelson, Ray D Gary and Lain M McIntyre J. Anal. Toxicol 2012; 36: 657‐659
HPLC –DAD
Postmortem metformin concentrations : Peripheral blood 240 mg/L Liver 240 mg/kg a ratio blood/liver = 1 (no postmortem redistribution)
A record : the highest values observed before were 77.3 in blood and 146 mg/kg in liver
Simultaneous identification and validated quantification of 11 oral hypoglycaemic drugs in plasma by electrospray ionisation liquid chromatography‐mass spectrometry Hess C, Musshoff F, Madea B Anal Bioanal Chem 2011; 400: 33‐41
They developped a LC‐MS/MS procedure (single run) for identification and quantification of: Glimepiride Glibenclamide Gliquidone Glibornuride Glisopexide Glipizide and Gliclazide (sulfonylurea type) Nateglinide and repaglinide (glinide type) Rosiglitazone and pioglitazone (thiazolidinedione type) Vildagliptin, sitagliptin and saxagliptin (dipeptidyl peptidase inhibitors)
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General conclusion
THE PROBLEM
The diagnosis of glucose metabolism disorders post mortem is often complicated and imprecise caused by the missing of characteristic morphological findings.
THE STATE OF THE ART
A number of works have allowed to progress in this field : Concerning hyperglycemia with the use of vitreous humor and with the association of several biological markers, Concerning hypoglycemia induced by drugs such as insulin with the fantastic technological progress that occured during the last decades but also owing to the implication of a number of toxicologists such as Frank Musshoff and his team.
AND NOW, WHAT TO DO ?
To develop calibrated and validated methods for vitreous humor, To continue to progress in new tthechnol liogies, more specific, more sensitive And why not, to find postmortem biological markers for other frequent causes of death such as myocardial infarction
15 Bertrand Brunet
Dr. Bertrand Brunet, 36 years old, Toxicologist at the university hospital of Poitiers (FRANCE) in the department of toxicology and pharmacokinetics, in charge of the toxicology unit. Dr. Brunet earned a PhD in pharmaceutical sciences working on distribution and postmortem redistribution of cannabinoids (University of Poitiers). He earned a Pharm Degree at the university of Limoges (FRANCE). Dr. Brunet worked for a year at the National Institute on Drug Abuse, Chemistry and drug metabolism section, Baltimore (USA) on drug disposition in sweat. Dr. Brunet has written or participated in 22 publications and 3 book chapters.
10/4/2013
Redistribution from drug reservoirs • Gastrointestinal tract • Lungs • Myocardium • Liver Agonic and cadaveric changes • Cell lysis • Putrefactive process Physicochemical properties and pharmacokinetics • Kp • pKa • Vd
Pélissier-Alicot A.L. et al. J Anal Toxicol 2003; 27: 533-44
Redistribution from gastric content1‐3 • Transparietal diffusion towards surrounding organs • Blood vessels (aorta, right cardiac chambers, inferior vena cava) • Regurgitation into airways • Agony • Passive relilaxation of the lower oesophlhageal sphincter • Increasing blood concentrations » Left cardiac chambers »Lowerlobe of the left lung » Superior vena cava ‐> right cardiac chambers
1Pounder D.J. et al. Am J Forensic Med Pathol 1996;17: 1-7 2Pounder D.J. & Yonemitsu K. Forensic Sci Int 1991; 51: 189-95 3Pounder D.J. & Smith D.R.W. Am J Forensic Med Pathol 1995; 16: 89-96
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LA LV RL LL
IVC Ao
STO
Frontal section of the thorax and abdomen through the left atrium (according Sobota J., Atlas of Human Anatomy, 2th ed.)
Redistribution from gastric content • Early onset after death • Depending from gastric concentration • Widely described for ethanol1, amitriptyline2 and fluoxetine3 • Recent data on cyanide »21 deaths related to cyanide intoxication by oral ingestion4 »[GC]m = 859 mg/L »[C]m = 38.1 mg/L »[P]m = 17.1 mg/L »Ratio (C:P)m : 3.4 »Ratio (C:P)m obtained from suicide cases by cyanide inhalation5: 1.3
1Hilberg T. et al. J Forensic Sci 1993; 38: 81-90 2Pounder D.J. & Smith D.R.W. Am J Forensic Med Pathol 1995; 16: 89-96 3Pohland R. & Bernhard N.R. J Forensic Sci 1997; 42: 812-6 4Rhee J. et al. J Forensic Sci 2011; 210: e12-15 5Dalpe-Scott M. et al. J forensic Sci 1995; 28: 113-21
Redistribution from lungs • In vivo accumulation of weak lipophilic bases • Redistribution into pulmonary vessels • Increasing blood concentrations in left cardiac chambers » Amphetamines2 » Antidepressants3 » Methadone1 »Lidocaine4 • Intense and early mechanism
1McIntyre A.C. & Cutler D.J. Biopharm Drug Dispos 1998; 9: 513-26 2 Miyazaki T. et al. Am J Forensic Med Pathol 1993; 14: 121-4 3 Hilberg T. et al. Forensic Sci Int 1994; 64: 47-55 4 Pounder D.J. J Forensic Sci 1997; 42: 965-6
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Redistribution from myocardium • In vivo accumulation » Cardiac drugs : digoxin1, quinidine1, metoprolol1, diltiazem2 » Other drugs : amphetamine1, metamphetamine1 • High ratio [myocardium/cardiac blood] • RditibtiRedistribution to riihtght and lftleft cardiac bloo d • Redistribution to subclavian blood ? Redistribution from liver • Inferior vena cava‐> right cardiac chambers • Gastrointestinal tract (stomach, duodenum)
1 Prouty R.W. & Anderson WH. J Forensic Sci 1990; 35:243-73 2Moriya F & Hashimoto Y. J Anal Toxicol 2004; 28: 269-71
Redistribution into body fat1 • Redistribution from blood slowly after death • Low blood flow • Slow equilibrium between blood and tissue concentrations • Decrease in peripheral blood concentrations • Highly lipophilic drugs »Anaesthetics » Volatiles compounds » Antidepressants » Cannabis2
1Brunet B. & Pélissier-Alicot A.L. In Kintz P. ed. Traité de Toxicologie Médico- Judiciaire 2e ed. 2012: 51-73 2Brunet B. et al. Int J Legal Med 2010; 124: 543-9
Cell lysis
Putrefactive process
Blood movements???
Coagulation and hypostasis????
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Starting within minutes of death1,2 Self‐digestion by endogenous enzymes (amylases, proteases, lipases)1,2 • Release of nutrient‐rich fluids (amino‐acids, fatty acids, carbohydrates)2 • Release of basic lipophilic drugs1,2 Accelerated process in • Lysosome‐rich organs/tissues (liver, pancreas, spleen, lungs)2 • High water content organs (brain)3
1Butzbach D.M. Forensic Sci Med Pathol 2010; 6: 35-45 2Zhou C. et al. J Forensic Legal Med 2011; 18: 6-9 3Vass AA. Microbiol Today 2001; 28: 190-2
Bacterial proliferation/fermentation1,2 • Early onset • Highly variable process » Environmental conditions (temperature, humidity, motion of air) » State of the body (integrity of skin, fever, sepsis) • Glucidi c / protidicsubtbstra tes Alcaligenes faecalis Proteus mirabilis • Synthesis Bacillus cereus Providentia species Bacillus species Pseudomonas • Degradation Bacteroides fragilis aeruginosa Clostridium Pseudomonas species perfringens Serratia marcescens Escherichia Coli Shigella flexneri Klebsiella aerogenes Staphylococcus aureus Klebsiella Staphylococcus pneumoniae epidermidis Lactobaccilus Streptococcus faecalis species Streptococcus 1Butzbach D.M. Forensic Sci Med Pathol 2010; 6: 35-45 Micrococcus species pneumoniae 2Zhou C. et al. J Forensic Legal Med 2011; 18: 6-9 Proteus mirabilis
Ethanol postmortem neoformation1‐2 • Within the corpse/ in the stored sample • Alcoholic fermentation by yeasts • Anaerobic breakdown of carbohydrates by bacteria • Additional putrefactive alcohols (n‐propanol, isopropanol, n‐butanol.. ) • Unaffected matrices : vitreous humour ±urines
Ethanol postmortem degradation3 • Rarer than neoformation • Bacteria/yeasts with fermentative and/or oxidative metabolisms (Candida albicans, Serratia marcescens)
1Huckenbeck W. Forensic Patho Rev 2006; 4: 205-59 2Kugelberg F.C. & Jones A.W. Forensic Sci Int; 2007; 165: 10-29 3Laviano C. Ann Biol Clin 1998; 56: 96-9
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Nitrobenzodiazepine degradation1‐3 • Flunitrazepam, nitrazepam, clonazepam • Enterobacteria containing oxygen‐sensitive nitroreductase • Reduction into 7‐amino metabolites • Decrease in parent molecule concentrations • Limitation of the degradation by » NaF adjonction »Preservationof corpses at 4°C
1Robertson M.D. & Drummer O.H. J Forensic Sci 1995; 40: 382-6 2Robertson M.D. & Drummer O.H. J Forensic Sci 1998; 43: 5-8 3Robertson M.D. & Drummer O.H. J Forensic Sci 1998; 43: 9-13
Recent data concerning cyanides • Conflicting observations in the literature1 • Recent study2 » 14 fire victims » Assays at D9/D11 and D25/D30 » 2 groups : with or withou t NFNaF (2%) • Results » Slight increase at D9/D11 in the 2 groups » Significant increase at D25/D30 in the 2 groups »No significant difference between the 2 groups at D9/D11 » Significant increase in the group without NaF at D25/D30
1McAllister J.L. & Roby R.J. J Anal Toxicol 2008; 32: 612-20 2McAllister J.L. et el. Forensic Sci Int 2011; 209:29-33
Issue of subclavian blood • Early study in the 60s1 » Rigor mortis‐induced ventricular contractions »Reflux of cardiac blood into superior vena cava and subclavian vessels • Recent studies » CiComparison of ethano l concenttitrations btbetween sublbclav ian (SC), cardiac (C) and femoral (F) blood (n=50)2 No signifiant difference between [SC] and [F] [C] >> to [SC] and [F] » Comparison of [SC], [C] and [F] (n=26) for 6 therapeutic classes3 [F] < [SC] << [C] excepted for benzodiazepines
1Fallani M. Minerva Medicoleg. 1961; 81:108-15 2Sastre C. et al. Int J Legal Med 2013; 127: 379-84 3Molina D.K. & Hargrove V.M. Am J Forensic Med Pathol 2013; 34: 155-8
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Dissociation constant (pKa) • Molecules with pKa > 7 • In vivo accumulation of weak bases in cells • Postmortem release into the extracellular space1 • Conflicting results for diazepam, clonazepam, oxazepam, temazepam2 Partition coefficient (Kp) • Molecules with Kp > 0.5 • In vivo accumulation of lipophilic molecules in the cells • Postmortem release into the extracellular space3
1Moriya F. & Hashimoto Y. J Forensic Sci 1999; 44: 10-16 2Ferner R.E. Br J Clin Pharamcol 2008; 66: 430-3 3Pélissier-Alicot A.L. et al. Int J Legal Med 2006; 120: 226-32
Plasma proteins and erythrocytes binding1 • Changes in the balance between free and bound fractions »Proteinbreakdown »Hemolysis • Difficulty circumvented by measuring whole blood Distribution to organs and tissues • Molllecular size • Lipophilicity • Ionization degree • Affinity for organs/tissues Tissue uptake of weak basic lipophilic molecules • Perfusion rate • Apparent Vd »Vd 3‐4 L/kg2 • Conflicting results in the literature3
1Pélissier-Alicot A.L. et al. J Anal Toxicol 2003; 27: 533-44 2Hilberg T. et al. J Forensic Sci 1999; 44: 956-6 3Brunet B. & Pelissier-Alicot A.L. In Kintz P. ed. Traité de Toxicologie Médico-Judiciaire 2e ed. 2012: 51-73
Metabolism • Variable kinetic • Persistence of enzymatic activity »UDP‐glucuronyltransférase1 » ‐glucuronidase2 »Hepaticand plasmatic esterases3 • Decrease in parent drug concentration in the benefit of metabolites »Cocaine3 »Dichlorvos4 » Morphine5
1Butzbach D.M. Forensic Sci Med Pathol 2010; 6: 35-45 2Yamazaki M. & Wakasugi C. J Forensic Sci 1994; 67: 155-68 3Isenschmidt D.S. et al. J Anal Toxicol 1992 16: 319-24 4Yamazaki M. & Wakasugi C.Forensic Sci Int 1994; 67: 155-68 5Gerostamoulos J. & Drummer O.H. J Forensic Sci 2000; 45: 843-5
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Central:peripheral blood ratio (C:P)1‐5 • C:P > 3 ‐> PMR • C:P < 1 ‐> no PMR • Variations according to the studies • Absorbed amount • Time of ddtheath/phhkitiarmacokineticstage • Resuscitation attempts • Postmortem delay • Method of sampling • Arterio‐venous differences • Anatomical variability
1Dalpe-Scott M. et al. Can Soc Forensic Sci 1995: 28: 113-21 2Leikin J.B. & Watson W.A. J Toxicol Clin Toxicol 2003; 41: 47-56 3McIntyre I.M. et al. J anal Toxicol 2012; 36: 177-81 4McIntyre I.M. et al. Forensic Sci Int. 2013; 28-33 5Pelissier-Alicot A.L. et al. J Anal Toxicol 2003; 27: 533-44
Liver:peripheral blood ratio (L:P)1‐6 • L:P > 20 ‐> PMR and L:P < 5 ‐> no PMR • Metformin, carisoprodol, meprobamate, sertraline, hydroxyzine, metamphetamine, fentanyl • No corr ela tio nwiththe centra l:pe rip he ra l (()C:P) bl ood raaotio • Influence of the absorbed amount (overdose) • Influence of the route of administration • Influence the postmortem delay
1McIntyre I.M. & Mallett P. Forensic Sci Int 2012 ;223: 349-52 2McIntyre I.M. & Mayer Escott C. J Forensic Res 2012; e108. 3McIntyre I.M. et al. J Anal Toxicol 2012; 36: 177-81 4McIntyre I.M. et al. Forensic Sci Int. 2013; 28-33 5McIntyre I.M. et al. J Anal Toxicol 2013; 37: 386-9 6McIntyre I.M. et al. Int J Legal Med 2013;
Pharmacokinetic modelisation1 • QSAR methodology » Predictive models using quantitative structure‐activity relationship » Multivariate data analysis Application to postmortem redistribution • Molllecular properties » Flexibility, Van der Waals volume, Van der Waals size area, refractivity, polarizability, dipole moment, energy parameters (binding energy, electronic energy, nuclear energy…) • Structural properties » Molecular size, polarity, hydrophobicity • Physicochemical properties »Kpand pKa
1Giaginis et al. Forensic Sci Int 2009;190: 9-15
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Methodology1
• Application of the model to 77 structurally diverse drugs: PMRpred 2 • Comparison to the central:peripheral (C:P) ratio : PMRobs Results
• PMRpred is comparable to PMRobs for 56 (77%) drugs • Predictive contributors »High lipophilicity »High molecular size »High flexibility »High number of halogens
1Giaginis et al. Forensic Sci Int 2009;190: 9-15 2Leikin J.B. & Watson W.A. J Toxicol clin Toxicol 2003; 41: 47-56
1‐6 Situations of interest Samples Molecules • Putrefied, burned or bloodless Vitreous humor Ethanol ,6‐MAM corpses THC‐COOH, MDMA • Suspicion of neoformation Brain Cocaine/cocaethylene (ethanol +++) Cannabinoids, • Scientific purposes metamphetamine Muscle Cocaine/cocaethylene Samples of interest MDMA, HbCO • Vitreous humor Bone Marrow Alcohols, antidepressant, benzodiazepines, paracetamol, • Brain morphine, meprobamate… • Muscle Pericardial fluid Ethanol • Bone marrow Cerebrospinal fluid Opiates • Pericardial fluid 1Kugelberg F.C. & Jones A.W. Forensic Sci Int 2007; 165: 10-29 2Cartiser N. et al. Int J Legal Med 2011; 125: 181-98 3Madea H. et al Forensic Sci Int. 2006; 161: 141-3 4Moriya F. & Hashimoto Y. J Forensic Sci 1996; 41: 612-6 5Wyman J. & Bultman S. J Anal Toxicol 2004 28: 260-3 6Brunet B. & Pelissier-Alicot A.L. In Kintz P. ed. Traité de Toxicologie Médico-Judiciaire 2e ed. 2012: 51-73
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Recent conflicting results • Holland et al.1 »Ratio C:P < 2 for THC, 11‐OH‐THC and THC‐COOH »Ratio C:P increasing with postmortem delay • Lemos & Ingle2
» [THC_ C] m : 8 ng/mL and [THC_ P] m = 15.8 ng/mL
»[THC‐COOH_C] m : 55,2 ng/mL and [THC‐COOH_P] m = 60.6 ng/mL
»[11‐OH‐THC_C] m : 17 ng/mL and [11‐OH‐THC_P] m = 12 ng/mL »Influence of postmortem delay • Brunet et al.3 »Decreasing[THC] in P and increasing in C with postmortem delay » Redistribution into fat tissues?
1Holland M.G. et al. Forensic Sci Int 2011; 212: 247-51 2Lemos N.P. & Ingle E.A. J Anal Toxicol 2011; 35: 394-401 3Brunet B. et al. Int J Legal Med 2010; 124: 543-9
Recent studies • Tolliver et al.1 » Morphine, codeine and 6‐MAM »Peri‐ and postmortem periods »Variables sampling sites »Variables C:P according to the cases » Probable influence of the PMD • Olson et al.2 »Fentanyl » Admission to the morgue : femoral blood sampling [FB1] »Autopsy: heart blood [HB] and femoral blood [FB2] » [HB]/[FB1] >> [HB]/[FB2] » Femoral postmortem redistribution
1Tolliver S.S. et al. J Anal Toxiol 2010; 34: 491-7 2Olson K.N. et al. Am J Clin Pathol 2010; 133: 447-53
Saar et al.1 • 10 antipsychotic drugs • Femoral blood at admission to mortuary (AD) and at autopsy (PM) (n=273) • Significant increase for chlorpromazine, olanzapine, promethazine, zucllthilopenthixol and amiilidsulpride • Significant decrease for risperidone and 9‐OH‐risperidone • No significant modification for haloperidol and quetiapine, • Influence of the postmortem delay • No correlation with Vd or Kp
1Saar E. et al. Forensic Sci Int 2012; 222: 223-7
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To limit PMR phenomena • Sample quickly after admission to mortuary • Take care when sampling femoral blood • Sample alternative specimen if necessary • Add NaF to blood samples • Keep cold • Analyze as soon as possible To avoid misinterpretation • Measure parent drug and metabolites • Know the postmortem delay • Take into account data from the literature »C:P and L:P ratios »Data bank
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