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Toxicology Drugs, poisons, alcohol in forensic medicine 2012 History What is forensic toxicology? Intoxications Mayer Mátyás Forensic toxicology Toxicology Paracelsus (born Philippus Aureolus Theophrastus Bombastus von Hohenheim, 1493 – the father of toxicology, wrote: Alle Ding' sind Gift, und nichts ohn' Gift; allein die Dosis macht, daß ein Ding kein Gift ist.* 1541), *All things are poison, and nothing is without poison; only the dose permits something not to be poisonous What is a Poison? All substances are poisons; there is none that is not a poison. The right dose differentiates a poison and a remedy. Dose-Response Relationship As the dose of a toxicant increases, so does the response. RESPONSE Therapeutic range 5 4 therapeutic effects undesired/unwanted effects 0-1 NOAEL (No Observable Effect Level) 3 2-3 Linear Range 4 Maximum Response 5 Plateau 2 0 1 DOSAGE What is a lethal Dose? • LD50 is the idea that is used by pharmacologist and companies in a statistical sense in animal experiments. • LD50 Over a large number of tests, it provides a toxic level at which half the animals will be expected to die. Comparison of LD50 Chemical Ethyl Alcohol Sodium Chloride Ferrous Sulfate Morphine Sulfate Strychnine Sulfate Nicotine Black Widow Curare Rattle Snake Dioxin (TCDD) Botulinum toxin LD50 (mg/kg) 10,000 4,000 1,500 900 150 1 0.55 0.50 0.24 0.001 0.0001 Toxicology as science Toxicodynamics investigates the effect of xenobiotics to the human body (dose-response relationship, mechanism of action etc.) Toxikokinetics investigates the effect of the organism to the harmful xenobiotics (absorption , distribution, metabolism, routes of excretion) • Xenobiotics is derived from the Greek words xenos (foreigner, stranger) and bios (life) Toxic substance • Medicaments (TCA, neuroleptics, opiates, NSAID, ...) • Pleasure drugs (heroine, PCP, ethanol, mephedrone, ethanol+BZD/barbiturates, …) • Household chemicals (HCL, HOCl, NaOH, ethylene glycol) • Pesticides • Toxins from plants, animals and mushrooms Pathways of intoxication • Routes and Sites of Exposure – Ingestion (Gastrointestinal Tract) – Inhalation (Lungs) – Dermal/Topical (Skin) – Injection • intravenous, intramuscular, intraperitoneal Typical Effectiveness of Route of Exposure iv > inhale > ip > im > ingest > topical Duration of exposure Acute < 24hr usually 1 exposure Subacute 1 month repeated doses Subchronic 1-3 month repeated doses Chronic > 3 month repeated doses Over time, the amount of chemical in the body can build up, it can redistribute, or it can overwhelm repair and removal mechanisms Individual Susceptibility • Genetics-species, strain variation, interindividual variations (yet still can extrapolate between mammalssimilar biological mechanisms) • There can be 10-30 fold difference in response to a toxicant in a population • Gender (gasoline nephrotoxic in male mice only) • Nutritional status • Health conditions Individual Susceptibility II. • Age – young • underdeveloped excretory mechanisms, biotransformation enzymes and blood-brain barrier – old • changes in excretion rates, metabolism rates and body fat • Previous or Concurrent Exposures – additive – synergistic – antagonistic Causes of death • Respiratory failure – RCD (opiates, EtOH) – Airway obstruction (aspiration, swallowing the tongue) • Circulatory failure – Cardial – Vascular – Hypovolaemia • Systemic hypoxaemia – O2 transporrt/use • Seizures • Damage of other organs – Liver, lung • Accidents – Behavioral effects (gasoline+whiskey=car crash) 11-10-2011 Mayer Mátyás Forensic toxicology Forensic toxicology Mathieu Joseph Bonaventure Orfila (1787–1853), the father of forensic toxicology published the first comprehensive work on forensic toxicology in 1813. Marie LaFarge 1840 (arsenic) 11-10-2011 Mayer Mátyás Forensic toxicology • Toxicology –Experimental Toxicology –Applied toxicology • Clinical toxicology • Forensic toxicology • Ecotoxicology • Occupational toxicology 11-10-2011 Mayer Mátyás Forensic toxicology Forensic toxicology • The study of alcohol, drugs and poisons, including their chemical composition, preparations and identification • The science of detecting and identifying the presence of drugs and poisons in body fluids, tissues, and organs. • Forensic toxicology is toxicology with medicolegal applications Multidisciplinar applied science includes toxicology, analytical chemistry, pharmacology and clinical chemistry 11-10-2011 Mayer Mátyás Forensic toxicology Objectives • Postmortem forensic toxicology is used in death investigations to establish whether or not drugs were the cause or contributing factor in death. • Human performance toxicology – Alcohol in the body an its effects – Drugs and driving – Workplace drug testing • Forensic drug testing is the evaluation of illegal drug consumption 11-10-2011 Mayer Mátyás Forensic toxicology Samples (biological matrices) • • • • • • • • • Evidence from the crimescene Urine Femoral blood PM (venous blood)* Blood from the heart PM (arterial blood)* Liquor Saliva Tissues (liver, kidney, etc.) Nails Hair *plasma or serum 11-10-2011 Mayer Mátyás Forensic toxicology Narcotics • • • • • Pains relievers Depressing action on CNS and RC Used legally also Opium: from poppy bulb Contains alkaloids: – – – – – Morphine: heroin is synthetised from it (semi-synthetic opiate) Codeine Thebaine Narcotine Narceine Opioids: non-peptide components which bind to the opiate receptors Abuse potential • Opiates addictions are chronic problems • Compulsive drug seeking • Neurochemical and molecular changes in the brain • Heroin: tolerance and physical dependence Depressants • Depress the CNS, inhibition of neurotransmitter receptors • Side effects: slow reflexes, slow thinking, poor decision making (influenced driving behaviour!) • Alcohol • Barbiturates • TCA Stimulants Caffeine, Theophylline, Nicotin legal, socially accepted drugs Amphetamine, Metamphetamine, MDMA Cathinones Atropine, Scopolamine • Exitement of central nervous system • Create a sense of euphoria • Long term use: stroke, high blood pressure, paranoia, heart attack • Hyperthermia, water poisoning MDMA Amphetamines • In contrast to opiates, these drugs created an excitatory condition characterized by elevated heart rate • Extreme feelings of euphoria • These drugs have some of the highest incidences of abuse Cocaine • Cocaine is a stimulant that resembles amphetamine in it’s abusive potential. • Unlike amphetamine, cocaine is a natural stimulant found in the coca leaf. • Cocaine can be refined to produce crack cocaine: intensifies its euphoric effects and cost effectiveness. • Increase of potency when the drug is smoked, the large surface area of the lungs provides rapid absorption into the body • Cocaine is fully metabolized 11-10-2011 Mayer Mátyás Forensic toxicology Hallucinogens • • • • Changes in thoughts, perception, moods Marijuana Ketamine and phencyclidine (PCP): dissociative anesthetics Ketamine:Fast acting anaesthetic and pain killer used primarily in veterinary surgery, and human medicine • Produces vivid dreams of hallucinations • Makes user feel that the mind is separated from the body “dissociation” • Ecstasy is called “love pill” increases perception of color, sound and sensations Cannabinoids • Marijuana is a name that applies to parts of the Cannabis sativa plant. • Tetrahydrocannabinol (THC) is the major active agent in marijuana. • Typically marijuana has a 2-6 % by volume concentration of THC, Hashish has higher concentration of THC (12 %). • The average marijuana cigarette contains about 75 mg of THC but it is rapidly absorbed into the blood stream reaching peak concentration around 10 – 20 minutes after smoking. • Physical dependency is rare • Long term use: risk of harm (cardiodepression Marijuana sinsemilla Short Term Effects: • Increase in heart rate, lead to anxiety and paranoia • Distorted concept of time and space • Decrease in concentration skills, short-term memory capacity • Feeling tired after the “high” wears off • Increase in appetite, weight gain Long Term Effects: • Breathing problems • Lung cancer • Damage cells and tissues in the body that fight disease • Lack of motivation • Difficulty processing new information Phencyclidine • Commonly known as PCP • Developed by the Park Davis company and was intended for use as a surgical anesthetic. – However it was found to be unsatisfactory because some patients exhibited manic behavior after usage. – PCP is measured in blood or urine levels around 25 ng/ml. Levels of greater than 100 ng/ml have been related to seizure and some deaths. „Designer” Drugs New challenges Cyanide • Highly toxic substance found in various places throughout nature. • Cyanide is dangerous because it binds with ferric ions in cytochrom oxidase, which is an enzyme critical for electron transport. • Because it interrupts electron transport it stops the bodies main mechanism for energy production. • Most labs can test for cyanide in whole blood and its concentration correlates well with severity of poisoning. • Levels higher than 1000 nanograms per mil are associated with stupor and amounts greater than 2500 nanograms per mil are usually fatal. Carbon Monoxide CO • Some studies suggest that CO causes more deaths than any other toxin. • This is because CO is present in all fires and typically is the cause of death in fire. • CO binds to hemoglobin much more tightly than O2 does and decreases our blood’s ability to carry oxygen. • CO also binds with myoglobin which is also important in electron transport. Metal poisoning • Copper – Copper-sulphate most frequent – Ulceration – Vomiting: first greenish, later bloody • Lead – Concentrates in blood, soft tissues, bones – Causes haemolysis, anemia, encephalopathy • Cadmium – Itai-itai disease • Mercury – limate (HgCl2), calomel (Hg2Cl2), merury nitrite – Oral ingestion: metallic taste in the mouth,, pain in the pharynx, bloody vomiting, oral mucosa is grayish-white, coagulation necrosis, anuria – Can accumulate in the hair – the fish and shellfish of Minamata Bay methylmercury atomic absorption spectroscopy Kayser–Fleischer ring 11-10-2011 Mayer Mátyás Forensic toxicology Lead dense metaphyseal bands Dark deposits of lead sulphid on the gum (lead lines) 11-10-2011 Mayer Mátyás Forensic toxicology Mercury Mercury-Amalgam filling 11-10-2011 Mayer Mátyás Forensic toxicology Cadmium Tobacco accumulates Cd 11-10-2011 Mayer Mátyás Forensic toxicology Itai-itai Disease • • • • weak and brittle bones spinal and leg pain bone deformities complications • coughing • anemia • kidney failure Acid poisoning • • • • • • • Coagulative necrosis Acidosis, haemolysis, hypercapnia Hydrochloric gas: laryngeal oedema and spasm Hydrochloric acid: grayish-white scab Sulphuric acid: grayish-black scab Nitric acid: yellow colour (xanthoprotein reaction) Healing occurs with scarring Alkaline poisoning • Ammonium or sodium hydroxide: household substances, mainly children by accident, or with the intention of suicide • They cause colliquative necrosis – Surface becomes softened and slippery – Necrosis is deep, may cause perforation – Healing with serious scarring • Dysphasia, vomitting, gastric pain, salivation, haematemesis Corrosive metal salts • Silver nitrate – Grayish-white area on buccal mucosa – Later becomes grayish-black • Zink chloride – From batteries – Burning pain in the mouth, nausea, vomitting, bloody diarrhoea – Liver and kindney damage • Potassium permanganate – Coagulation necrosis – Irritated mucosa turns into a purplish-red colour Alcohols • Ethanol is beverage alcohol • Low molecular weight alcohols such as methanol and isopropyl may also contribute to human injury. • It takes around 90 minutes for the peak level of ethanol to enter the blood. • Gas Chromatography is the preferred method for testing alcohol in blood and a proper chain of custody must be observed for criminal cases. Toxicology of Alcohol • Alcohol intoxication depends on – Amount of alcohol consumed – Time of consumption – Body weight – Rate of alcohol absorption – Gender Factors that Affect Alcohol Absorption • Time of consumption • Type of alcoholic beverage • Presence of food in stomach Calculation of BAC • • • • Widmark formula: C= A/ W x r C – concentration of alcohol (mg/ 100 ml) A – amount of alcohol in gramms – Multiply volume% with 0.8 • W – body weight in kg • r – Widmark factor: 0.7 males, 0.6 females • In general as average: 1 dl wine or 3 dl beer is eliminated completely in an hour (means: 7-8 gramms of pure alcohol) Effects of alcohol Physical Effects • Co-ordination is impaired, clumsiness, slower reflexes • High blood pressure, damage to the heart • Liver damage • If drinking when pregnant • Life threatening when mixed with other drugs Mental and Emotional • Behave in ways that you normally wouldn’t • Increase in aggressive and violent behaviour • Problems with school and learning Fate of Alcohol I. • Alcohol is absorbed into the bloodstream • Distributed through-out the body’s water • And finally eliminated by oxidation and excretion Note: A. Oxidation is the combination of oxygen and alcohol to produce new products by the liver B. Elimination is removing alcohol from the body in an unchanged state; normally excreted in breath and urine Alcohol and the Law • 1939-1964: intoxicated = 0.15% BAC • 1965: intoxicated = 0.10% BAC • 2003: intoxicated = 0.08% BAC In Hungary: zero tolerance From 0.081 % - crime Assay Procedure • Studying the anamnesis • Screening test for substance group (immunological tests) • Confirmational analysis qualitative or Fourier IR spectroscopy, mass quantitative (NMR, spectrometry, UV-VIS spectroscopy) Mayer Mátyás Forensic toxicology Rapid Drug Screen (RDS) OPI TCA COC THC MTD mAMP AMP BZO PCP BAR 11-10-2011 Mayer Mátyás Forensic toxicology Fluorescence Polarization Immunoassay (FPIA) I. • Fully automated immunoassay analyzer • Fit for medium and high volume clinical laboratories • Up to 81 tests analyzed per hour • FPIA is a patented technology used for measurement of serum and urine drug concentration Axsym Abott 11-10-2011 Mayer Mátyás Forensic toxicology Fluorescence Polarization Immunoassay (FPIA) II. SAMPLE PREPARATION • Protein precipitation • Separation (filtration, centrifugation, dialysis, etc.) Protein precipitation • • • • • „salting out” ((NH4)2SO4) Organic solvent Acid (TCA, HClO4) Heavy metal salt Heat denaturation HClO4 + KHCO3 KClO4 + H2O + CO2 Control: MALDI-TOF EXTRACTION METHODS • Dialysis • Supercritical Fluid Extraction • Liquid-liquid extraction (LLE) • Solid-phase extraction (SPE) Mayer Mátyás 2012.10.18. MICROEXTRACTION METHODS • Microdialysis (in vivo) • Micro-diffusion • Liquid-liquid microextraction - DLLME, Direct-SDME, LLLME, HS-SDME, HF-LPME • Solid-phase microextraction - DI-SPME, HS-SPME, MEPS Liquid-liquid extraction (LLE) Dispersive liquid-liquid microextraction (DLLME) Extraction and derivatisation in one step Extracting and dispersing solvents Δ-9-THC, cannabinol, cannabidiol urine Moradi M, Yamini Y, Baheri T. J Sep Sci. 2011 Jul;34(14):1722-9. MA, MDMA, ketamine, heroine (59-81%) Liang Meng, Bin Wang, Feng Luo, Guijun Shen, Zhengqiao Wang, Ming Guo Forensic Science International 209 (2011) 42–47 THC-COOH, A, MA, MDA (65-76%) Blood, urine, seum Abdulmumin A.Nuhu, ChanbashaBasheer, BahruddinSaadb Journal ofChromatographyB (2011) M. Rezaee, Y. Assadi, M.R. Milani Hosseini, E. Aghaee, F. Ahmadi, S. Berijani, J. Chromatogr. A 1116, 2006 Mayer Mátyás 2012.10.18. Single-drop microextraction (SDME) • Cheap • Not labor intensive • Slow (A) direct SDME (B) liquid-liquid-liquid microextraction (LLLME) (C) headspace SDME (HS-SDME). A, MA (99-107%) urine LLLME 500X Yi He, Youn-Jung Kang Journal of Chromatography A, 1133 (2006) 35–40 Francisco Pena-Pereira, Isela Lavilla, Carlos Bendicho Trends in Analytical Chemistry, Vol. 29, No. 7, 2010 Hollow fibre liquid phase microextraction (HF-LPME) • 1990’s • Very clear extract relatively fast A, MA (60-70%) Blood, serum, saliva Knut Einar Rasmussen, Stig Pedersen-Bjergaard Trends in Analytical Chemistry, Vol. 23, No. 1, 2004 A, MA, MDMA, MDA (56-77%) hair Lorena do Nascimento Pantaleão, Beatriz Aparecida Passos Bismara Paranhos, Mauricio Yonamine Journal of Chromatography A, 1254 (2012) 1– 7 Mayer Mátyás 2012.10.18. Solid-phase extraction (SPE) Normal Phase SPE procedure Reversed phase SPE Ion exchange SPE – Anion exchange – Cation Exchange Vacuummanifold Mayer Mátyás Forensic toxicology Solid Phase Microextraction (SPME) • DI-SPME • HS-SPME Synthetic cannabinoids saliva Mayer Mátyás 2012.10.18. Melissa Toms Northern Kentucky University, poster SPME II. divinylbenzene/carboxene/ polydimethylsiloxane Coating: polypyrrole, immuneaffinity, monolithic, C18… GHB Hiroyuki KATAOKA ANALYTICAL SCIENCES SEPTEMBER 2011, VOL. 27 Kenneth G. Furton, JingWang, Ya-Li Hsu, JohnWalton, José R. Almirall Journal of Chromatographic Science, Vol. 38, July 2000 GHB On column derivatization Jodi E. Meyers, Jos´e R. Almirall J Forensic Sci, Jan. 2005, Vol. 50, No. 1 A, MA, MDMA, MDA, MDE urine Benkő András; Artemis Dona; Kovács Anikó; Maravelias, Costas; Mikóné Hideg Zsuzsanna; Kerner Ágnes Acta Pharmaceutica Hungarica - 1998. 68. évf. 5. sz. A, MA, EPH (89-98%) urine Mayer Mátyás 2012.10.18. Sunanta Wangkarn, Wisitsak Wutiadirek Mj. Int. J. Sci. Tech. 2007, 01(2), 145-156 MEPS microextraction by packed sorbent 45µm silica baesed particles with 60Å pore size C2, C4, C8, C18, Sil, M1 ... A, MA (98-100%) haj Hajime Miyaguchi, Yuko T. Iwata, Tatsuyuki Kanamori, Kenji Tsujikawa, Kenji Kuwayama, Hiroyuki Inoue Journal of Chromatography A, 1216 (2009) 4063–4070 Molecular imprinting A. Prieto, S. Schrader, C. Bauer, M. Möder Analytica Chimica Acta 685 (2011) 146–152 Mayer Mátyás 2012.10.18. CAPILLARY ELECTROPHORESIS • Fast • Low sample needs MASS SPECTROMETRY • M/Z (mass per charge) • Molecular ion • Fragmentation Chromatography I. • • • • • Stationary phase Mobile phase (eluent) Elution Analyte Retention time Chromatography II. • Mobile phase – Gas chromatography – Liquid chromatography • Bed shape – Column chromatography – Planar chromatography • Separation mechanism – Ion exchange chromatography – Size-exclusion chromatography – Normal-/Reversed-phase chromatography – Chiral chromatography 11-10-2011 Mayer Mátyás Forensic toxicology Thin Layer Chromatography TLC/HP-TLC Densitogram • Normal phase • Multiple samples • Detection with Densitometer Mayer Mátyás Forensic toxicology Thin Layer Chromatography II. Gas chromatography (GC) Analysing compounds that can be vaporized without decomposition Mobile phase is an inert unreactive carrier gas (helium, nitrogen) Similar to fractional distillation High-performance liquid chromatography (HPLC) Pressure: 100-150 bar -> 1000-1200 bar Time of analysis: 30-60 min. -> 0,5-5 min Solvent consumption: 40-50 ml -> 0,5-5 ml (green chemistry) Flow rate: 1-1,5 ml/min-> 200 nl/min-300 µl/min Solvent reservoirs, Degasser, Gradient valve, Mixing vessel for delivery of the mobile phase, High-pressure pump,Switching valve injector, Sample injection loop, Pre-column (guard column), Analytical column, Detector, Waste or fraction collector. Analytical colmn • Filled column – Fully porous – Core shell • Monolithic • Silica based • Synthetic polymer based FILLED COLUMN Fully porous vs. Core-shell particles MONOLITHIC COLUMN • Polymer: increased pH range (0-14) STATIONARY PHASES Detectors (GC, LC) • • • • • Flame ionization detector (FID) Mass spectrometer (MS) UV detector Diode array detector (DAD or PDA) Fluorescence detector (FLD) 11-10-2011 Mayer Mátyás Forensic toxicology AUTOMATIZATION Thank you for your attention! 11-10-2011 Mayer Mátyás Forensic toxicology