Forensic Toxicology and Alcohol

1. Forensic Toxicologyand Alcohol

2. (1493-1541) Paracelsus “What is there that is not a poison? All things are poison and nothing without poison. Solely the dose determines that a thing is not a poison.”

3. What is Forensic Toxicology? Toxicology: Study of drugs and poisons and their adverse affects on the human system. Subfields include: pharmacokinetics (how the drug works on people) and pharmacodynamics (how people work on the drugs)

4. Species LD50 Guinea Pig 0.0006 Rabbit 0.115 Hamster 3.5 Monkey 0.07 Drugs and Toxic Substances • Dose - amount of substance that enters the body • LD50 - Dose necessary to kill 1/2 of the population • Acute Toxicity - effects are immediate • Chronic Toxicity - effects occur over an extended time • Species Specific: • Dioxin - polychlorinated organic compounds (many) • From burning chlorinated compounds • Agent orange • Paper industry • Toxicity varies with species

5. Toxic Substances Substance LD50 Aspirin 1750 Ethanol 1000 Morphine 500 Caffeine 200 Heroin 150 Lead 20 Cocaine 17.5 Cyanide 10 Nicotine 2 Strychnine 0.8 Batrachotoxin 0.002 LD50 Calculations: Cyanide (for 150 lb human - ~ 70 Kg) (70Kg)(10mg/Kg) = 700 mg (0.70g) Nicotine (e.g.; from Cigarettes) (70Kg)(2 mg/Kg) = 140 mg (0.14g) (note 1 cig. = ~ 2 mg Nicotine) Ethanol (70Kg)(1000mg/Kg) = 70,000mg (70g) Effect of Body Weight: Ethanol 150 lb Human = lethal dose = ~ 70 g 40 lb Dog = lethal dose = ~ 20 g

6. MSDS Sheets • Materials Safety Data Sheets - provide specific toxicological, chemical and physical data about a compound. • Physical Properties and Names • Chemical Reactivities • Incompatibilities • Safe Handling • Toxicology (symptoms and means of exposure) • Safety and First Aid

7. MSDS Sheets - Example NICOTINE (S)-3-(1-Methylpyrrolidin-2-yl)pyridine CAS # 54-11-5 3-(1-Methyl-2-pyrrolidinyl)pyridine RTECS # QS5250000 b-Pyridyl-a-N-methylpyrrolidine ICSC # 0519 1-Methyl-2-(3-pyridyl)pyrrolidine EC # 614-001-00-4 C10H14N2 Molecular mass: 162.2 TYPES OF ACUTE HAZARDS/ PREVENTION FIRST AID/ HAZARD SYMPTOMS FIRE FIGHTING FIRE Combustible. Gives off irritating NO open flames. Powder, alcohol-resistant or toxic fumes (or gases) in a fire. foam, water spray, carbon dioxide. EXPLOSION Above 95°C explosive vapor/air Above 95°C use a closed mixtures may be formed. system, ventilation. EXPOSURE PREVENT GENERATION OF MISTS! AVOID EXPOSURE OF (PREGNANT) WOMEN! IN ALL CASES CONSULT A DOCTOR! INHALATION Burning sensation. Nausea. Vomiting. Convulsions. Abdominal pain. Diarrhoea. Headache. Sweating. Weakness. Dizziness. Confusion. Ventilation, local exhaust, or breathing protection. Fresh air, rest. Refer for medical attention. SKIN MAY BE ABSORBED! Redness. Burning sensation Protective gloves. Protective clothing. Remove contaminated clothes. Rinse and then wash skin with water and soap. Refer for medical attention.

8. Forensic Toxicological Questions • Intentional or Accidental Poisoning? • What type of Poison? • Corrosive Poisons - Substances that actually destroy tissue outright • Metabolic Poisons - Affect biochemical mechanisms. • Drug Use - what drug, how much and when? • Activity while under the influence of drugs?

9. Common Terms • Analyte - the compound that you’re analyzing for. • Metabolite - Compound formed from the metabolism of a drug. Heroin quickly metabolizes to Morphine so Morphine is a metabolite of Heroin. Heroin Morphine

11. Journals • Journal of Analytical Toxicology • Journal of Forensic Science • Forensic Science International • Journal of Chromatography (B) • British Medical Journal • Drug and Alcohol Dependence • International Journal of Legal Medicine

12. Specimen Collection Each has it’s own set of problems and advantages Fluids: Organs: Blood Skin Urine Lungs Vitreous Humor Hair and Fingernails Oral Fluid Liver Semen Kidney Stomach Contents Bone Bile Other (heart, brain, etc.) Vitreous Humor - clear, gel-like mass that fills the space between the lens and the retina.

13. Sampling • Blood - It matters where you sample in the body • Blood taken near the liver or other solid organ may contain drug that has diffused from the organ into the blood post-mortem to give very high drug levels. Take blood from femoral artery because it’s far from organs. • Urine - tends to concentrate compounds. • Can’t be sure of body concentration since it is unknown how long it has been accumulating in the body. • Liver - concentrates and stores drugs for long times. • good for detection but problematic for concentrations.

14. Sampling • Bile - Useful for detection but not concentration. • Kidney - Similar to Bile. • Vitreous Humor - Correlates well with femoral blood for most compounds. • Hair and Fingernails - Good for some compounds with timelines possible. • Oral Fluids - Can be easily disguised. • Others - May be good for certain analyses but not generally good sources for many compounds.

15. Additional Matrices Vomit Meconium Earwax Amniotic Fluid Semen Umbilical Cord Blood Feces Milk Sweat

16. Specimen Collection: Case Examples • Deceased • Suspected drug related death • Road Traffic Death • Murder • Antecedent • Drink/drug driving • Drug Facilitated Sexual Assault (DFSA) • Professional/amateur athlete • Workplace drug testing Dependent on the type of case and what samples are available

17. Roles of Forensic Toxicology • Postmortem Forensic Toxicology • Human Performance Toxicology • Forensic Drug Testing

18. Postmortem Forensic Toxicology • Death Investigation • Medical Examiners Office. • NYS - elected corners or appointed Medical Examiners depending upon county. • Forensic Pathologist responsible for performing autopsy. • Forensic Toxicologist responsible for analyzing biological samples for poisons.

19. Specimen Collection • Essential to collect the appropriate specimens and that they are collected, packaged, transported, and stored correctly. • Pathologist is responsible for the collection of the human-derived specimens and may consult the toxicologist on unusual cases.

20. Specimen Collection • MOST IMPORTANT STEP!!!! • Obtain a Representative Sample: ‘A small portion of a material taken from a bulk specimen and selected in such a way that it possesses the essential characteristics of the bulk’. [E.g., easily possible to pick out a few crystals from a sample that do not reflect the majority of the sample - maybe that’s why they crystallized]

21. Sample Handling Storage Avoid - loss (due to volatility). - contamination (e.g., insufficient seal). Prevent - chemical interactions (e.g., Na and oil). - Degradation (refrigeratebiological samples).

22. Sample Preparation Homogenising - making the sample uniform: • grind (mortar and pestle) • blender • dissolution (water, solvents, acids)

23. Postmortem Forensic Toxicology • Blood Samples • Usually the most important specimen • Ideally 2 samples (25 mL each) • Femoral/jugular (peripheral site) - far as possible from solid organs • Heart blood • Trunk blood

24. Postmortem Toxicology • Vitreous humor samples - The clear, gel-like mass that fills the space between the lens and the retina. • Should be collected at all PM’s • Relatively stable matrix • Anatomically isolated area • Good correlation with blood • Easily obtained Retina - The delicate lining at the back of the eye that functions much like the film in a camera. It receives light through the lens in your eye, forms that light into images, and sends those images to the brain, enabling you to see.

25. Postmortem Toxicology • Urine samples: • All available sample should be collected. • Higher concentrations present than in other matrices, but not always. • Rapid tests available (both qualitative [what] and quantitative [how much] tests). • Not always available.

26. Postmorten Toxicology • Digestive System

27. Postmortem Forensic Toxicology • Bile samples • Alternative if urine unavailable • Accumulation of drugs • All available bile should be collected • Not an easy sample to analyze Bile is a thick digestive fluid secreted by the liver and stored in the gallbladder. It facilitates digestion by breaking down fats into fatty acids, which can be absorbed by the digestive tract.

28. Postmortem Forensic Toxicology • Liver samples: Drug metabolism occurs in the liver. Drugs may be present in higher concentrations. Metabolite Analysis.

29. Postmortem Forensic Toxicology • Other specimens: • Lung (volatiles) • Spleen (carbon monoxide) – blood unavailable • Stomach Contents (drug overdose) • Hair ( drug use history)

30. Human Performance Toxicology • The effects of drugs on skills, acquisition, learning, and performance. • Drugs may alter normal behavior by either enhancing or impairing performance. • Stimulant drugs can enhance performance – short term but used regularly can impair performance.

31. Human Performance Toxicology • Not only illicit drugs (e.g. heroin, cocaine, cannabis, etc… • Prescription medication can impair performance especially at the start of the treatment • E.g. antidepressants, muscle relaxants, etc.

32. Types of Abused Drugs • Narcotics - relief from pain and bring sleep. (now generally means a socially unacceptable drug). • Narcotics are analgesic - relieve pain by depressing nervous system (opium, morphine, heroin, codeine, opiates - oxycontin, methadone). • Hallucinogens - alter normal thoughts, perceptions and moods (PCP, LSD, mescaline, MDMA, ecstasy). • Depressants - depress functions of central nervous system, cause calm and bring about sleep (alcohol, barbituates). Tranquilizers are depressants. • Stimulants - increase alertness and activity (cocaine, amphetamines). • Steroids - promote muscle growth (androgen, testosterone, anabolic steroids).

33. Types of Abused Drugs • Narcotics - relief from pain and bring sleep. (now generally means a socially unacceptable drug). • Narcotics are analgesic - relieve pain by depressing nervous system (opium, morphine, heroin, codeine, opiates - oxycontin, methadone). • Cocaine (stimulant), Marijuana (hallucinogen), etc are Not classified as narcotics even though they are frequently called that. • Most narcotics are opium derived. • Opium from Poppy plants (sap is 4 - 21% opium). Opium Poppy

34. Hallucinogens • Hallucinogens - alter normal thoughts, perceptions and moods (PCP, LSD, mescaline, MDMA, ecstasy). • Marijuana most common (derived from Cannabis or “hemp” plant). • Derive resin from plant secretions that is extracted with organic solvents. • In use for 1000’s of years. • Brought to the US ca. 1920.

35. Caffeine Amphetamine Cocaine Labrium Valium Methaqualone Ethyl Alcohol Stimulants and Depressants

36. Steroids

37. Steroids

38. Human Performance Toxicology • Blood is the best specimen for determining impairment through drugs. • In contact with the central nervous system. • Hair/urine/sweat – becoming popular but unlikely that a quantitative relationship will exist.

39. Human Performance Toxicology • Alcohol and/or Drugs Involving Driving: • In addition to collecting the correct samples, additional information is essential: • Witness statements to the incident • Assessment of impairment at the scene • Field Sobriety tests • Drug Recognition Evaluation

40. Forensic Drug Testing - Where? • Workplace Drug Testing • Armed forces • Prisons • Private and public companies • Schools • International Olympic Committee • Athletes • Police Investigations • Arrestee • Drug Facilitated Sexual Assault (DFSA)

41. Forensic Toxicology • Sample Collection (uncontaminated) • Specimen Identification (locate at crime scene) • Storage • Chain of Custody • Analysis • Reporting

42. Forensic Toxicology • Storage • Fridge/freezer • Preservative (EDTA in blood) • Leak proof • Suitable size • Prevent contamination

43. Forensic Toxicology • Chain of Custody: • Keeping tract of the control of the sample and its handling from the time of collection until analysis done. • Very important to be admissible in court. • Ensure procedures have been followed, seals intact, signatures checked etc..

45. Analytical Methods • Qualitative Methods - What’s present? • GC-MS - Mass Spectrometry • Chemical Spot Tests - A test for a substance using reagents that together generate an easily observed color or some other physical change in the presence of the target substance. Spot tests for blood, semen or certain drugs can be done at a crime scene, but often the positive results are indicative only, and need more sophisticated testing for court evidence. • Quantitative Methods - How Much? • Spectrophotometric Methods - Infrared, UV-Vis., Nuclear Magnetic Resonance (NMR), others.

46. Forensic Analysis - GC

47. Immunoassay Immunoassay: A technique utilizing antibodies to bind specifically to targeted substances in a specimen in order to identify their presence. Antibody - Protein produced by the immune system of humans and higher animals in response to the presence of a specific antigen. Antigen - Substance that can trigger an immune response, resulting in production of an antibody as part of the body's defense against infection and disease. Many antigens are foreign proteins (those not found naturally in the body). An allergen is a special type of antigen which causes an antibody response.

48. Immunoassay TWO METHODS Immunoassay: A technique utilizing antibodies to bind specifically to targeted substances in a specimen in order to identify their presence. EMIT - Enzyme Multiplied Immunoassay Technique A competitive binding immunoassay that avoids the separation step. As Test antigen is increased it occupies more of the antibody molecules, so fewer of the enzyme labels are blocked. Substrate ---> product response increases.

49. Immunoassay TWO METHODS Immunoassay: A technique utilizing antibodies to bind specifically to targeted substances in a specimen in order to identify their presence. ELISA - Enzyme Linked Immunosorbent Assay - Wide application - diagnosis & research. Non-competitive "sandwich" form requires two antibody-recognition sites on test antigen.

50. Monoclonal Antibodies • Polyclonal vs Monoclonal • Immortal supply of anti-protein sera