Toxicology Procedures 10mL of blood in airtight container Add anticoagulant Add preservative 2 consecutive urine samples Some drugs take a while to show up in urine (1-3 days) Vitreous humor Hair samples

Toxicology Procedures Screening- quick test to narrow down possibilities color tests, TLC, GC, immunoassay Confirmation- determines exact identity GC/Mass Spec

Color Tests Marquis Test: Turns purple in the presence of Heroin, morphine, opium Turns orange-brown in presence of Amphetamines Dillie-Koppanyi Test: Turns violet-blue color in the presence of Barbiturates Duquenois-Levine: Test for marijuana –turns purple

Color Tests Van Urk Test: Scott Test: Three solutions Blue then pink then back to blue in the presence of Cocaine

More Analytical Tests Microcrystalline Tests: Identifies drug by using chemicals that reacts to produce characteristic crystals Chromatography: TLC, HPLC and gas – separate drugs/tentative ID Mass Spectrometry: chemical “ fingerprint ” no two drugs fragment the same

Postmortem Forensic Toxicology

Qualitative and quantitative analysis of drugs or poisons in biological specimens collected at autopsy Interpretation of findings in terms of: Physiological effect at time of death Behavioural effect at time of death

Quantitative vs. Qualitative Qualitative analysis – determines the presence or absence of a drug or poison in a submitted sample Quantitative analysis – determines the amount of drug or poison that is present in the submitted sample

Postmortem Forensic Toxicology Types of cases: Suspected drug intoxication cases Fire deaths Homicides Driver and pilot fatalities Therapeutic drug monitoring Sudden infant death (SIDS)

Samples of Forensic Interest

Issues in Specimen Collection Selection Multiple, varied sites of collection Collection Appropriate method of collection Adequate volumes for analysis Storage and handling Important to ensure analytical results are accurate and interpretations are sound

Typical autopsy specimens Blood Urine Stomach contents Bile Liver Hair Vitreous humor

Blood Antemortem  ideal blood sample Postmortem blood is not truly “blood” Anatomical site of collection at autopsy should be noted

Central sites Heart Peripheral sites Femoral Iliac Subclavian Other sites Head blood Hematoma blood Femoral Iliac Subclavian Heart

Hematoma Extravascular blood clot Protected from metabolism Analysis will indicate what drugs were present in the blood at the time of formation

Hematoma case example A 26 year old man was found dead at the bottom of a staircase. Death was due to physical injuries. Question as to alcohol use prior to fall down stairs No urine available at autopsy Alcohol not detected in femoral blood Alcohol in hematoma blood  150 mg/100 mL The deceased had been drinking prior to receiving the head trauma. The deceased had survived for several hours after the injury.

Hematoma Caution: There may be a delay between the incident which resulted in hematoma and the actual formation of the hematoma Therefore, this alcohol concentration does not necessarily indicate the BAC at the time of the fall down the stairs.

Urine Produced by the kidneys Blood filtered by the kidneys Stored in the bladder until voided Qualitative - the presence of a drug in the urine of an individual indicates that some time prior to death the drug or poison was present in the blood of the individual

Stomach contents Visual examination may reveal tablets Drugs that have been orally ingested may be detected in stomach contents Caution: drugs administered by other routes may also diffuse into stomach contents from the blood Generally qualitative: Stomach contents are not homogeneous Only a portion of stomach contents collected (unmixed?) Useful for directing further analysis

Liver Drug metabolism occurs in the liver Both parent compounds and metabolites may be present in higher concentrations in the liver than in the blood  ease of detection Limitation is that drugs are not uniformly distributed throughout the liver  confounds interpretation

Bile Digestive secretion Continuously produced by the liver Stored in the gallbladder Qualitative - the presence of a drug in the bile of an individual indicates that sometime prior to death, the individual was exposed to the drug

Vitreous humor Fluid that occupies the space between the lens and the retina of the eye. Sequestered from putrefaction, charring and trauma, microorganisms. Useful in cases where decomposition is advanced, body is exhumed or in fire deaths Limitation is blood:vitreous ratio may not be known

Hair Recent specimen of interest Metabolism does not occur in hair Can provide a historical record of drug or poison exposure Pros and cons of hair analysis still being uncovered  racial variability?

Case Example 30 year old woman, previously in good health Nausea, vomiting, diarrhea, rash, fever Weakness in hands and feet  Guillian Barre? Hospitalized with hypotension, seizures Misplaced laboratory result  Arsenic! Sequential hair analysis for arsenic showed chronic arsenic poisoning over 8 month period Poklis, A Abstract SOFT, Dearborn, Michigan.

Non-biological submissions Used to direct analysis of biologicals May indicate the nature of substances that may have been ingested, inhaled or injected Examples: Containers found at the scene Syringes Unidentified tablets or liquids

Chest Cavity Fluid Not readily definable Most likely to be collected if: Traumatic injury to the chest Advanced decomposition A “contaminated” blood sample, chest cavity fluid may contain fluids from stomach, heart, lungs etc.

Samples taken after embalming Methanol is a typical component of embalming fluid Most drugs are soluble in methanol Embalming process will essentially “ wash ” the vasculature and tissues Qualitative analysis can be performed on body tissues

Case Example A 72 year old woman, given meperidine to control pain following surgery, later died in hospital. The woman was in poor health and it is possible that death was due to natural causes. However, coroner requests toxicology to rule out inappropriate meperidine levels. BUT: Body had been embalmed Liver and spleen submitted

Storage and Handling

Proper specimen handling Identification of samples Continuity Contents Specimens delivered to lab without delay Specimens should be analyzed as soon as possible Storage areas should be secure

Storage and Handling Not feasible to analyze specimens immediately Sample should be in well-sealed container Sample containers must be sterile Use of preservatives and anti-coagulants Refrigeration vs. Freezing Both inhibit bacterial action; esp. freezing Freezing results in  prep time Freeze-thaw cycle may promote breakdown

Storage of Samples Preservative Sodium fluoride Anti-coagulants Sodium citrate Potassium oxalate EDTA Heparin Not imperative for postmortem blood samples

Determining analyses Case history Medical history Autopsy findings Symptomatology Experience of the toxicologist Amount of specimen available Nature of specimens available Policies of the organization

Pitfalls in Postmortem Forensic Toxicology

Decomposition Autolysis The breakdown of cellular material by enzymes Putrefaction A septic/infectious process The destruction of soft tissues by the action of bacteria and enzymes Traumatic deaths may demonstrate  putrefaction

Decomposition Fewer samples available for collection Quality of samples is diminished Putrefaction produces alcohols Ethanol Isopropanol Acetaldehyde n-propanol

Incomplete Distribution Site dependent differences in drug levels due to differential distribution of drugs at death Has been noted in rapid iv drug deaths Example: Intravenous injection of morphine between the toes Fatal amount of drug reaches the brain Full distribution of the morphine throughout the body has not occurred Femoral concentration > Heart concentration

Drug Stability Knowledge of a drug’s stability is necessary to facilitate interpretation of concentrations Breakdown of drugs may occur after death and during storage via non-enzymatic mechanisms Cocaine  Benzoylecgonine (Hydrolysis) LSD  degradation due to light sensitivity

Interpretation

Therapeutic, toxic or fatal? How do you know? Compare measured blood concentrations with concentrations reported in the literature: Clinical pharmacology studies Incidental drug findings Plasma  blood Consider case history: Symptoms observed by witnesses? Tolerance of the individual to the drug

Importance of History: Tolerance Drug concentrations in non-drug related deaths may overlap with reported drug concentrations in fatal drug intoxications Methadone example: Naïve users - deaths due to methadone are associated with blood levels > 0.02 mg/100 mL Patients on methadone maintenance – peak blood concentrations may range up to 0.09 mg/100 mL

Interpretation Acute vs. Chronic Ingestion: Can you tell? Parent:metabolite drug concentration ratio may be of assistance in differentiating between acute and chronic ingestion of a drug

Metabolites are produced when drugs are biotransformed (converted) into other chemicals, more easily excreted from the body Metabolite drug concentrations may be the more useful measure of exposure or toxicity Interpretation

The parent compound may be a prodrug or may have a shorter t 1/2 than the metabolite: Heroin  morphine Heroin – t½ is only 2-6 minutes long whereas the half-life of morphine is 2-3 hours. Heroin is rapidly converted to morphine (its active metabolite) in the body. Metabolites: Exposure

The metabolite may have  toxicity over the parent compound: Acetaminophen  N-Acetylbenzoquinoneimine Meperidine  normeperidine Methanol  formic acid Ethylene glycol  oxalic acid  calcium oxalate Metabolites: Toxicity