Forensic Toxicity Testing

CHAPTER 14 Forensic Toxicity Testing

I. Forensic toxicity testing A. Analytic chemistry melded with organ damage consistent with the compound and concentration found (consonance of data) 1. Most intentional poisoning deaths suicide; most unintentional poisoning deaths from overdose of opioids and cocaine; toxicity from ethanol, drugs of abuse, overuse of medications lead car crash causes; unintentional nonfatal poisonings causes by opioids benzodiazepines 2. Most exposures were oral 3. Sampling of tissues a. Living being is preferred as death results in tissue destruction, possible loss of toxicant, and redistribution of toxicant (from organ to plasma resulting in false poisoning indication if plasma is the key item utilized) For humans: take 15 mL of blood for ethanol/toxicants Take 30 mL urine for metabolites Hair samples (pulled not cut) for drugs of abuse (e.g., methamphetamine in child’s hair from house manufacturing this drug) → extraction with HCl → enzymatic treatment → → (Pronase or -glucuronidase) hot CH3OH extraction hot NaOH disintegration and extraction Metals → urine, blood, and hair samples possibly fingernails (As especially) Teeth for chronic exposure to Pb in children (1) Therapeutic drug monitoring (a) Required for highly lipophilic medications or pharmacokinetics, vary widely by patient (b) Also may be a function of a larger loading dose, long half-life, and narrow therapeutic range for action vs. toxicity (digitalis as loading dose needed to establish 0.5–1.5 or 2.0 ng/mL therapeutic range, 2 ng/mL toxicity, 1 h to achieve active plasma levels when 2 taken orally, and a t½ 160 h interacts with many medications including Ca -channel blockers, class III antiarrhythmic amiodarone, -blockers, and NSAIDs)—see Table 14-2

(c) Ctrough (also known as Cmin ) works for HIV medication atazanavir or cancer chemotherapy medication methotrexate—see figure

CONCEPTUALIZING TOXICOLOGY 14-1

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(d) For compounds such as the immunosuppressant mycophenolate mofetil used for kidney transplantation, the trough value doesn’t correlate with dose or therapeutic effect so concentration at 2 h (C2) following dosing sampled

96

Cmin Cav 72 AUC for this dose 48 Cdt τ = time Infinity between

Time (hours) Time 24 doses Loading dose AUC = 0 C tmax 0 max 0 Plasma concentration, C

b. For dead people or animals, antemortem or postmortem collection of as many fluids as possible to reach results (peripheral blood, aqueous humors, urine, stomach contents, etc.), analyzing for original compound metabolites: 50 g of brain, liver, kidney; 25 mL of heart blood; 10 mL of peripheral blood; and all available vitreous humors, bile, urine, and gastric contents For specific toxicants, lung, intestine, or other specimens may be taken from other organs or larvae feeding on corpses as was done for malathion. 4. Analysis of tissues (classic analytic chemistry) → → a. Acid digestion steam volatiles (acetaldehyde, acetone, CO, CN, CH3OH, CH3CH2OH, isopropanol, phenol, or anions such as Br, F, oxalate) Acid fraction → leaches metals → atomic absorption (AA) or inductively coupled plasma emission spectroscopy (ICP) b. Base digestion → aqueous phase-acidified organic solvent → cleanup of proteins → barbiturate analysis Base digestion → organic phase-acidified → aqueous (made basic and contains basic medications such as amphetamine) organic phases (contains neutral medications such as simvastatin) 5. Analysis of tissues (modern extraction linked to GC-MS, LC-MS, or LC-[tandem] MS[/MS]) a. Tissue represents the aqueous phase continuously extracted in the presence of inert diatomaceous earth by liquid–liquid chromatography extraction wells robotically; organic eluate is evaporated and reconstituted for electrospray (tandem) LC-MS/MS analysis. 6. Rapid screening tests—see Table 14-1 a. Drugs of abuse are detectable in urine up to a few hours to days following use, while peptide hormones are undetectable in urine; injected steroids may be detectable for a month or more following use. b. Certain standards of indications for human urine involve correct urine temperature, pH, creatinine, specific gravity, and human IgG. c. Adulterants involve changing pH, oxidizing agents, fixatives (to ruin immunoassays), while diuretics are used to remove metabolites more rapidly prior to testing. d. Hair samples have ability to confirm use of drugs of abuse up to 90 days following use. e. Usual postmortem examination for toxicants—Figure 14-1; first considerations are CO poisoning (COHb), CN (from fire inhalation or plant ingestion), drugs of abuse or prescription medications, hypoglycemia (insulin overdose or other causes), uncontrolled diabetes mellitus, ethanol

CONCEPTUALIZING TOXICOLOGY 14-1 (continued )

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→ Blood COHb, CN, HbA1c, drug analyses, ethanol Urine → drug (metabolites), ethanol, glucose Vitreous humor → ethanol, glucose Bile → RIA for drugs where urine can’t be collected Gastric contents → drugs Liver, skeletal muscle, spleen, lung, kidney, brain, heart → drugs, ethanol CSF (if enough available) → drugs, ethanol Problem is organ most protected on plane impact is kidney (80% available), while urine is not (80% unavailable)—see Figure 14-2. B. Signs/symptoms of exposure 1. Toxidromes—symptoms consistent with exposure to a given agent a. Ethanol use—ataxia, slurred speech, nystagmus on light exposure, etc. b. Hg—triad of tremors, erethism, and gingivitis c. Pediatric toxidromes and agents (1) Respiratory—difficult breathing for botulinum toxin, d-tubocurarine, organophosphates, carbamates, strychnine, CO, CN, metabolic acidosis, liver failure, heart failure, methemoglobinemia (metHb), salicylates

(2) Hypoxia—CO, NO3/NO2-induced metHb formation (of G-6-PDH deficiency), CN (3) Wheezing—-adrenergic antagonists, Cl (irritant) gas, hydrocarbons, isocyanates, organophosphates, carbamates, smoke inhalation, sulfites in food (4) Osmolarity gap—acetone, ethanol, ethyl ether, ethylene glycol, isopropanol, mannitol, methanol, renal failure, ketoacidosis (5) Anion gap—acetaminophen, -adrenergic medications, CO, CN, Fe, isoniazid, salicylates, theophylline, toxic alcohols, valproic acid (6) Neuromuscular—antipsychotics, metoclopramide, amphetamines, anticholinergics, antihistamines, cocaine, -hydroxybutyrate, SSRIs and tricyclic antidepressants, malignant hyperthermia, phencyclidine (PCP) (7) CNS depression—opiates, ethanol/organic solvents, sedative-hypnotics, tricyclic antidepressants (8) Coma—alcohols/organic solvents, anticholinergics, As, -adrenergic antagonists, cholinergic agents, CO, Pb, Li, opioids, PCP, phenothiazine antipsychotics, salicylates, sedative-hypnotics, tricyclic antidepressants (9) Miosis (pupil constriction)—opioids, cholinergic agents, clonidine (2 adrenergic agonist), nicotine, phenothiazines, PCP (10) Mydriasis (pupil dilation)—anticholinergics, glutethimide, meperidine, sympathomimetics, withdrawal from alcohol or opioids (11) Seizures—amphetamines, cocaine, caffeine, theophylline, tricyclic antidepressants, venlafaxine, phenothiazines and butyrophenones, camphor, organophosphates, carbamates, ethylene glycol, isoniazid, meperidine, methanol, salicylates, withdrawal from depressants (12) Serotonin syndrome—monoamine oxidase inhibitors (MAOIs) SSRIs (13) Sleepiness—antihistamines, any sedative hypnotic, alcohols, -hydroxybutyrate, tricyclic antidepressants, opioids, CO, CN, hypoglycemic agents (14) Withdrawal from depressants opposite to withdrawal from stimulants (effects of withdrawal opposite to action of agent) (15) Hyperthermia—serotonin syndrome, anticholinergics, MAOIs, metals, PCP, phenothiazines, salicylates, sympathomimetics, withdrawal from CNS depressants, malignant hyperthermia (halothane or succinyl choline)

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(16) Hypothermia— -blockers, CO, cholinergic agonists, ethanol, hypoglycemia-inducing agents, sedative-hypnotics (17) Cardiovascular (a) Hypertension—glycyrrhizin, mineralocorticoids, -1 agonist, Cd (b) Hypotension—CO, CN, hemorrhagic agents, Fe, opioids, nitrites, phenothiazine, sedative-hypnotics, tricyclic antidepressants, theophylline (c) Bradycardia—-blockers, Ca2-channel blockers, cardiac glycosides, clonidine, CN (high concentration), Li, nicotine, opioids, carbamates, organophosphates, parasympathomimetics (d) Tachycardia—amphetamines, antihistamines, caffeine, clonidine, cocaine, theophylline,

CO (lower concentrations), CN (lower concentrations), H2S, anticholinergics, ethanol, PCP, sympathomimetics, withdrawal from CNS depressants, theophylline, thyroid hormone (e) Torsade de pointes (dangerous ventricular arrhythmia)—amiodarone, As, chloroquine, quinine, quinidine, organophosphates, tricyclic antidepressants

(18) Hepatic—acetaldehyde, acetaminophen, aflatoxin, Amanita phalloides, CCl4, other chlorinated hydrocarbons, halothane, phenol, phosphorus, valproic acid (19) Renal—Hg, Au, Cd, statins via muscle wasting and creatinine formation (20) Skin—Sulfonamides, aromatic antiepileptics, lamotrigine, penicillins, doxycycline, nevirapine, strong acids and bases, anilines, CO, cyanide, strychnine, metals 2. Toxicoproteomics a. Develop biomarkers for organ specific toxicity b. Organ (e.g., liver) and plasma must be analyzed to detect proteins and peptides released or leaked from the organ c. Use five levels of analysis: (1) Match DNA microarrays with altered protein profiles (2) Compare affected pathways from gene transcripts and proteins responsible for activation or repression events (3) Posttranslational altered proteins analysis as reflective of gene expression of kinases, proteases, phosphorylases, conjugation enzymes (4) Protein trafficking and signaling pathway determination in nuclei, mitochondria, endoplasmic reticulum, plasma membrane and cytosol (5) Kinetic protein analysis of serum proteome over pretoxic, toxic, and recovery periods

CONCEPTUALIZING TOXICOLOGY 14-1 (continued )

This section includes forensic charts for each A lower oral dose may damage the liver, which chapter describing an organ system. The most stores metals. A lower dosage yet would likely likely toxicants that primarily damage each organ damage the kidney, as this is the excretory organ. are included in each chart. As tempting as it is to A lower dose might also cause central nervous give a “laundry list” of toxicants that damage an system (CNS) damage. This forensic approach organ, there usually is an organ through which indicates that toxicant damage to organs may the lethal effects are manifested. For example, not be a lethal or even extremely toxic event an extremely high concentration of heavy metals with irreversible damage. All toxicants that given orally may strip off the cells of the intesti- affect these systems are characterized based on nal tract and lead to hemorrhaging and death. the ability to do significant damage or have the

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toxicant altered in a way that damages another in 2005 (5,833 people) were suicides rather than organ (e.g., liver detoxication leading to kidney homicides (89 homicides). Ninety-five percent damage). This might appear to be a chapter on of all unintentional and undetermined deaths analytical chemistry, and in some ways it is an were related to opioid pain medication over- extension of this area. In another sense, there dose followed by the drugs of abuse cocaine and must be consonance of data. That means that heroin. Unintentional nonfatal poisonings came the amount or concentration of toxicant at the mainly from opioids followed by benzodiaze- target organ, the damage found, and the symp- pines (GABAergic sleeping pills). A more defined toms prior to death should all confirm the toxic- accounting for the year 2007 was a publication ity. For example, if a person had a high amount put forward from the American Association of of cyanide in the intestinal tract but showed no Poison Control Centers (Bronstein et al., 2008). cyanosis, this would not likely be the cause of In 2007, 2,482,041 human exposures were death. A blunt trauma to the head may be more reported (8.1/1,000 population). Nearly 65% of indicative of the brain damage that was found. those calls were for children. Greater than 78% A person found under water with no water in the were oral exposures. Most cases resulted in no lungs did not likely drown but died prior to that effect or minimal toxicity. Lethality was mostly point. Similarly, it is necessary to have data that experienced in adults over 19 years of age (0.2% consistently point to the cause of death. of the 860,692 cases for that age group). In 2007, 131,744 nonhuman exposures were reported, primarily for dogs (118,371) followed Most Likely Suspects by cats (11,818). The top 25 exposures reported The toxicants that are usually tested in clini- to U.S. poison control centers were analgesics cal or forensic toxicology laboratories are those (12.5%); cosmetics and personal care products most likely to kill or significantly affect func- (9.1%); household cleaning supplies (8.7%); tion in humans. These toxicants have govern- sedative-hypnotics and antipsychotic medica- ment programs to limit exposure such as drugs tions (6.2%), foreign bodies, toys, and miscella- of abuse, overdose of medications, ethanol, neous items (5.1%); topical preparations (4.5%); carbon monoxide, lead poisoning, and a series of cold and cough medicines (4.5%); antidepres- other toxicants. Most of the agents can be found sants (4.0%); pesticides (3.9%); cardiovascular in common use in the households of many medications (3.5%); alcohols (3.3%); antihista- Americans. The Centers for Disease Control mines (3.2%); food products and food poison- (CDC), in 2008 reported that 41,000 people ings (3.1%); bites and envenomations (3.1%); died from poisoning. For the first time since antimicrobials (2.7%); vitamins (2.7%); plants 1980, poisoning was the leading cause of injury (2.4%); hormones and hormone antagonists death and tripled over a 30 year period. Medi- (2.2%); gastrointestinal preparations (2.2%); cation and illegal drug deaths in that period hydrocarbons (2.0%); chemicals (2.0%); stimu- increased from a bare majority (60%) to almost lants and street drugs (1.9%); anticonvulsants the sole cause of poisoning deaths (90%; Warner, (1.7%); arts, crafts, and office supplies (1.6%); 2011). These mainly drug-related poisoning and fumes, gases, and vapors (1.6%). People deaths in the U.S. are still mainly uninten- over 19 years of age were most likely exposed to tional, while the remainder result from suicides cosmetics and personal care products (20.0%); or undetermined intent (CDC, 2015). Motor cleaning supplies (14.3%); analgesics (13.4%); vehicle crashes were the first in unintentional foreign bodies, toys, and miscellaneous items deaths, and those related to intoxication from (11.1%); topical preparations (10.1%); cold alcohol, drugs of abuse, overuse of medications, and cough medicines (7.6%); vitamins (5.7%); and so forth appear to lead that list. It is of pesticides (5.2%); plants (4.9%); antihista- interest that unintentional poisonings of people mines (4.6%); gastrointestinal preparations between the ages of 35 and 54 exceeded deaths (4.3%); antimicrobials (4.0%); arts, crafts, and from car accidents. The overwhelming majority office supplies (3.4%); hormones and hormone of the 18% of intentional deaths from poisoning antagonists (3.0%); cardiovascular medications

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(2.8%); electrolytes and minerals (2.8%); alcohols collected by a police officer for a person arrested (2.7%); food products and food poisonings for driving under the influence of ethanol on (2.3%); deodorizers (2.0%); dietary supplements, a weekend or holiday. However, increasing herbals, and homeopathic preparations (2.0%); numbers of children are having hair samples asthma therapies (1.9%); hydrocarbons (1.8%); pulled from their scalps using gloves to detect other or unknown nondrug substances (1.8%); methamphetamine as evidence against their sedative-hypnotics and antipsychotic medica- parents (for having a drug lab in home). Note tions (1.7%); and antidepressants (1.6%). Expo- that it is going to be important to match signs sures for children under 6 years of age involve and symptoms of exposure to sampling tech- cosmetics and personal care products (10.7%); niques and handling/storage of samples, and cleaning supplies (7.6%); analgesics (7.2%); finally methods of analysis. Forensic analysis foreign bodies, toys, and miscellaneous items means a legal analysis that will be scientifically (6.0%); topical preparations (5.4%); cold and sound, meet specifications for chain of evidence cough medicines (5.4%); vitamins (3.1%); pes- and chain of custody (knowing each person ticides (2.8%); plants (2.6%); antihistamines responsible for the sampling or sample through- (2.5%); gastrointestinal preparations (2.3%); arts, out the analysis), be free from contamination by crafts, and office supplies (2.2%); hormones and samplers and analysis group, have confirming hormone antagonists (1.8%); cardiovascular tests, and be consistent with medical knowledge medications (1.5%); electrolytes and minerals as to cause of poisoning or death. The sections (1.5%); alcohols (1.5%); food products and that follow examine each area to show the food poisoning (1.3%); deodorizers (1.2%); extent of scientific detective work that should dietary supplements, herbals, and homeo- ultimately yield a report on the agent or mixture pathic preparations (1.1%); asthma therapies of agents consonant with the observed toxicity. (1.0%), hydrocarbons (1.0%); other or unknown nondrug substances (1.0%); sedative-hypnotics and antipsychotic medications (0.9%); and antidepressants (0.9%). Signs/Symptoms of Exposure Which ones are the most lethal? For 2007, Some toxicities are easily diagnosed, because the most fatalities were reported for sedative- they come with a variety of unmistakable hypnotics and antipsychotic medications symptoms. Others are more difficult to assess, (377 people); opioids (331); antidepressants because the symptoms are common for a variety (220); acetaminophen in combination (208); of agents. An example of a toxicology assessment cardiovascular medications (203); stimulants is when a police officer stops a car for erratic and other street drugs (188); alcohols (170); driving, and then shines a light into the driver’s acetaminophen only (140); anticonvulsants eyes. If the eyes vibrate (nystagmus) in the light, (99); fumes, gases, and vapors (80); cyclic anti- the office will assume ethanol intoxication and depressants (80); muscle relaxants (70); antihis- test for ataxia via a walking test and having the tamines (69); aspirin alone (63)l chemicals (45); drive touching his finger to his nose. If the eyes unknown drug (44); other nonsteroidal anti- appear too constricted, the officer will assume inflammatory drugs (NSAIDs; 44 people died); that opiates may be involved. Toxidromes are a oral hypoglycemic (36); automotive, aircraft, set of symptoms associated with a given agent or and boat products (28); miscellaneous drugs agents that may be used to evaluate a poisoned (21); antihistamine/decongestants without animal or person. Some of the most common phenylpropanolamine (21); hormones and toxidromes are for stimulants, sedative-hypnotic hormone antagonists (20); anticoagulants (20); medications, opiates, anticholinergics, and cho- and diuretics (16). (Bronstein et al., 2012) linergic agents (Tomassoni et al., 2015). Certain A real forensic toxicologist is not a television toxidromes have been noted historically and in crime scene analyst looking at rare chemicals novels. For example, the mad hatter in Lewis used by a clever murderer. They usually are Carroll’s Alice’s Adventures in Wonderland has the employed to do head space analysis of blood syndrome associated with mercury poisoning

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due to spraying of felt hats with mercury nitrate mEq/L, this may be caused by acetamino- to preserve the fiber. The set of symptoms fit the phen overdose (as would liver failure and fictional mad hatter with excitability (erethism), jaundice), beta-adrenergic medications, gum disease (gingivitis), and tremor. Excessive carbon monoxide, cyanide, iron, isoniazid, salivation was also part of the syndrome. A set salicylates, theophylline, toxic alcohols, and of pediatric toxidromes is provided in the the antiseizure medication valproic acid. subsections that follow (Koren, 2007). Neuromuscular Respiratory ■ Acute movement disorder: Dystonia ■ Difficulty breathing: Neuromuscular (twisting and repetitive movements) blockade is achieved by botulinum may be caused antipsychotics and meto- toxin; neuromuscular blockers such as clopramide. Dyskinesia (difficulty with/ d-tubocurarine, organophosphates, and distortion of voluntary movements as in carbamates (AChE inhibition at high doses tic, chorea, spasm, or myoclonus) may be with tremor, muscle weakness, agitation, produced by amphetamines, anticholin- seizures, and coma, but at lower doses ergics, antihistamines, cocaine, gamma- induce vomiting, diarrhea, abdominal hydroxybutyrate, selective serotonin cramping, bradycardia, miosis, drooling, reuptake inhibitors (SSRIs), and tricyclic respiratory hypersecretion, and diaphore- antidepressants. Rigidity may be caused sis); strychnine; and tetanus. CNS depres- by malignant hyperthermia (dangerous sion occurs with opiates, ethanol (or other increase in temperature that is a reaction to alcohols), sedative-hypnotics, and tricyclic general anesthetic agents and succinylcho- antidepressants. Other factors that may line), neuroleptic malignant syndrome, and lead to labored breathing are carbon mon- phencyclidine. oxide, cyanide, metabolic acidosis, liver ■ Coma: Alcohols, anticholinergics, arse- failure, heart failure, methemoglobinemia, nic, beta-receptor antagonists, cholinergic and salicylates. agents, carbon monoxide, lead, lithium, ■ Hypoxia: carbon monoxide poisoning, opioids, phencyclidine, phenothiazine anti- nitrate poisoning or other methemoglo- psychotics, salicylates, sedative-hypnotics, binemia, hemolysis caused by glucose- and tricyclic antidepressants. 6-phophate dehydrogenase (G-6PDH) ■ Opioid overdose: CNS depression deficiency (favism), cyanide poisoning. (sedation and lethargy to coma), respira- ■ Wheezing: beta-antagonists, irritant tory depression, hypoxia, and miosis (no gases such as chlorine, hydrocarbons, iso- pupil constriction or miosis with other cyanates, organophosphates, carbamates, sedative-hypnotic agents). Miosis may also smoke inhalation, foodborne sulfites. be caused by cholinergic agents, clonidine (adrenergic alpha-2 receptor agonist that Changes in Osmolarity and Ion Balance decreases sympathetic outflow from the CNS), nicotine, phenothiazines, and phen- ■ Osmolarity gap: Because osmolarity should cyclidine (PCP). The opposite of miosis is be 290 mOsm/kg ( 2 [Na (mEq/L)] dilation or mydriasis caused by anticholin- glucose [mg/dL]/18 blood urea nitrogen ergics (belladonna alkaloids), glutethimide, [mg/dL]/2.8), alterations in osmolarity may meperidine, sympathomimetics (mimic be caused by acetone, ethanol, ethyl ether, action of norepinephrine and epinephrine), ethylene glycol, isopropyl alcohol, manni- and withdrawal from CNS depressants tol, methanol, renal failure, and ketoacido- such as alcohol or opiates. sis (type I diabetic and alcoholic). ■ Seizures: Amphetamines, cocaine, caffeine, ■ Anion gap: Calculated as Na ([Cl] theophylline, tricyclic antidepressants, venla- [HCO3 ]), where normal ranges from 8–12 faxine, phenothiazines and butyrophenones,

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camphor, organophosphates, carbamates, phenylephrine, and cadmium or other ethylene glycol, isoniazid, meperidine, meth- heavy or metals (or other toxicants) that anol, salicylates and any withdrawal from induce renal damage or lead to adrenal psychoactive drug (likely depressants— tumors. see withdrawal symptoms). ■ Hypotension: Carbon monoxide, cyanide, ■ Serotonin syndrome (hot and confused): hemorrhagic agents, iron, opioids, nitrites, Monoamine oxidase inhibitors (MAOIs) in phenothiazines, sedative-hypnotics, tricy- combination with SSRIs lead to akathisia- clic antidepressants, and theophylline. like restlessness, muscle twitches, myoc- ■ Cardiac: lonus, hyperreflexia, diaphoresis (excess ■ Bradycardia (too slow): Beta-receptor perspiration), penile erection, shivering, antagonists (especially beta-1 blockers), tremor, and with increased severity seizures calcium-channel blockers, cardiac glyco- and coma. Other agents that can increase sides (e.g., digitalis), clonidine, cyanide, temperature are anticholinergics (accompa- lithium, nicotine, opioids, carbamates nied by dry flushed skin, dry mouth, mydri- and organophosphates (AChE inhibi- asis, delirium, hallucinations, tachycardia, tors), and parasympathomimetics (mim- ileus or decreased peristalsis, urinary reten- ics action of acetylcholine). tion, and in most severe poisonings coma ■ (Atrial) tachycardia (too fast): Amphet- and respiratory arrest), MAOIs, metals, amines, antihistamines, caffeine, cloni- phencyclidine, phenothiazines (accompa- dine, cocaine, theophylline, carbon nied by muscle rigidity, metabolic acidosis, monoxide, cyanide, hydrogen sulfide, and confusion for neuroleptic overdose), anticholinergics (antihistamines, phe- salicylates, sympathomimetics, and with- nothiazines, tricyclics and atropine), drawal from CNS depressants such as ethanol, phencyclidine, sympathomi- ethanol and opioids. Decreased tempera- metics, withdrawal from any depressive ture or hypothermia can be caused by beta- psychoactive drug, theophylline, and blockers, carbon monoxide, cholinergic thyroid hormone. agonists, ethanol, hypoglycemia-inducing ■ Ventricular arrhythmias: Speeding from agents, and sedative-hypnotics. amphetamines, cocaine, caffeine, chlo- ■ Sleepiness: Antihistamines, any sedative- ral hydrate, aromatic hydrocarbons, hypnotic, alcohols, gamma-hydroxybu- anticholinergics and theophylline. Q-T tyrate, tricyclic antidepressants, opioids, prolongation that can lead to torsade carbon monoxide, cyanide, and hypoglyce- de pointes (polymorphic ventricular mic agents. tachycardia that may become deadly ■ Withdrawal symptoms: Withdrawal from ventricular fibrillation) may be caused depressants or toxicity from high levels of by the antiarrhythmic medication amio- stimulants leads to diaphoresis, diarrhea, darone, arsenic, chloroquine, quinine, fever, insomnia, hypertonia, hyperreflexia, quinidine, organophosphates, and tricy- lacrimation, respiratory distress, seizures, clic antidepressants. and tachycardia. Withdrawal from stimu- lants has the opposite effects such as bra- dycardia, depression, increased appetite (nicotine withdrawal), and sleepiness. Hepatic ■ Acetaldehyde (metabolite of ethanol), acet- Cardiovascular aminophen, aflatoxin, Amanita phalloides (death cap mushroom) or similar species, ■ Blood pressure: carbon tetrachloride, other chlorinated ■ Hypertension: Glycyrrhizin, miner- hydrocarbons that at lower doses may also alocorticoids, agents that may cause lead to kidney failure, halothane, phenol, tachycardia, alpha-1 agonists such as phosphorus, and valproic acid.

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Renal and monocrotaline serve as model agents for causing hepatic necrosis and inflammation. ■ Direct: Glomerulonephritis can be caused Male rats are treated with a vehicle as a control, by mercury and gold compounds as indi- a low dose to observe effects not expected to cated by proteinuria (Bigazzi, 1994). Heavy be associated with injury, and a high dose to metal exposure as assessed by cadmium cause injury. Rats are sampled at 6, 24, and and other metals found in the urine appear 48 hours following administration of the toxic to cause early kidney damage as indicated agent. These times are associated with changes by an increase in urinary N-acetyl- -D- in histopathology and serum chemistries glucosaminidase (Thomas et al., 2009). (alanine aminotransferase [ALT] and aspartate ■ Indirect: Statins have caused rhabdomyoly- aminotransferase [AST]) and account for the sis (muscle wasting) and ensuing renal fail- time necessary to overwhelm the reduced gluta- ure (Omar et al., 2001). thione content from reactive metabolites. Five levels of analysis are possible by this approach. First is matching gene alterations detected by Skin DNA microarrays with altered protein profiles. ■ Stevens-Johnson syndrome and toxic epi- Second is to compare affected pathways through dermal necrolysis: Sulfonamides, aromatic altered gene transcripts and proteins responsible antiepileptics, lamotrigine, penicillins, dox- for activation or repression events. Third is anal- ycycline, and nevirapine. Other skin prob- ysis of posttranslationally altered proteins as lems can be found with strong acids and they are related to modified genes encoding for bases (chemical burns), anilines (darken- kinases, proteases, phosphorylases, conjugation ing), carbon monoxide (cherry red color- enzymes, or others that facilitate those post- ation), cyanide (blue under finger nails, but translational processes. Fourth is a proteomic if skin exposed may find a redness and a determination of alterations of protein traffick- bitter almond-like smell), strychnine (dark ing and signaling pathways or subcellular site(s) face and neck), metals (certain metals give of injury in nuclei, mitochondria, endoplasmic a metallic luster to the skin), vanadium reticulum, plasma membrane, and cytosol. Fifth (green tongue). is the kinetic protein analysis of the changes in the serum proteome over the pretoxic, toxic, and recovery periods (Merrick, 2006). Toxicoproteomics A new field worth examining is toxicopro- teomics. Using model liver and kidney toxicants, Sampling Techniques biomarkers for organ-specific toxicity are being Once a presumed diagnosis has been made, the determined (Merrick and Witzmann, 2009). sampling should be made easier unless the tox- Samples can be subjected to two-dimensional icity proceeds to lethality unnoticed and with gel electrophoresis and mass spectrometry, no clear indication of cause. In that case, an liquid chromatography of protein digests that autopsy is performed. A living being is always are analyzed by tandem mass spectrometers, preferred, as death results in tissue destruction, retentate chromatography-mass spectrometry possible loss of toxicant, and redistribution of (laser-based mass spectrum), and antibody toxicant. For instance, if the time of death is arrays available for toxicoproteomic studies. known, the amount of cyanide may be extrapo- Analysis of the liver must involve both liver cells lated back to the time of death based on the and the plasma, because injured organs release length of time the sample remained in the peptides and proteins into the plasma. In the cadaver, length of storage time of the sample, case of the liver, some of the plasma proteins preservation of the sample by addition of are normally synthesized by the liver. Liver sodium fluoride, and storage conditions of the toxicants such as acetaminophen, bromoben- sample (McAllister et al., 2008). For example, zene, dichlorobenzenes, lipopolysaccharide, antemortem and postmortem collection of

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vitreous humor, femoral vein and artery, left and possible and many time sequences). Mechanical right heart ventricles indicated that pigs given trauma or inhalation of stomach contents may a high intravenous (IV) dose of morphine had complicate analysis (e.g., gunshot wounds, car significantly different free and total morphine accident). Tolerance or synergy due to prior or values before and after death and by site of injec- concurrent exposure may yield results that are tion over time. Free morphine concentrations initially not consistent with death (too low) or were higher in postmortem samples, but total inconsistent with life (too high, especially in morphine was similar to slightly lower in post- postmortem concentrations). For this reason, mortem samples. Antemortem and postmortem it is best to take as many fluids and tissues as values are not consistent based on IV dose. The possible postmortem, including peripheral femoral vein had the lowest values in either case, blood, aqueous humors, urine, stomach con- which is consistent with the site of metabolism tents, and so forth. Original compounds and (Crandall et al., 2006). In another example, two metabolites will also give some indication of antipsychotic drugs, haloperidol and thiorida- the total picture. As an example of the problems zine, administered intraperitoneally redistrib- associated with sample collection and reliability, ute from the rat lung to the blood following ethanol intoxication is a common poisoning in death as indicated by liquid chromatography— the population. Blood ethanol can be lost or tandem mass spectrometry. This is important, produced especially in the presence of signifi- because fatal arrhythmias may result from cant trauma, which happens often in alcohol- a high dose of these antipsychotics (Castaing related deaths (Flanagan and Connally, 2005). et al., 2006). A variety of pre- and postmortem What happens if only skeletal remains are found examinations revealed a variable redistribu- or the blood has been lost? One more recent tion of psychiatric medications into the blood publication indicates the possibility of bone as (Rodda and Drummer, 2006). Although earlier an indication of the presence of a medication reports (1970s) of redistribution following or toxicant. The concentrations of the medica- death are available in the literature, at least one tions tested were generally higher in the bone publication indicates many of the difficulties than in the blood and some were not found in of obtaining reliable samples in a critically ill the bone. Thus, interpretation should be done poisoned patient (Boyle et al., 2009). The prob- cautiously. The encouraging factor is that there lems of the nature of the poison or poisons— is at least one more possibility for the analyst gas, volatile liquid, corrosive chemical, metals, when considering forensic determination of anions and nonmetals, nonvolatile organic possible causes of death (McGrath and Jenkins, substances (organic strong acids, organic weak 2009). Sometimes the autopsy findings provide acids, organic bases, organic neutral chemi- a toxicity not indicated previously by animal cals, organic amphoteric compounds), or mix- data. Two suicides by acetaminophen yielded tures of compounds that may react with each rapid deaths without the usual hepatic centri- other or complex or disperse into living or dead lobular necrosis. Cardiac toxicity may cause tissue—is one factor. A second problem is asso- death at high concentrations of acetaminophen ciated with sample collection, transport, and in humans (Singer et al., 2007). storage, where little may be known of the toxic agent. The site where the victim is found may be remote or not ideally suited for sample collec- Live for Human Performance tion or storage (e.g., battlefield conditions, car The Society of Forensic Toxicologists (SOFT) accident, fire, explosion). The analytical meth- and American Academy of Forensic Sciences odology employed may not be optimal based on (AAFS) (2006) produced a document titled the facilities available in that part of the country “Forensic Toxicology Laboratory Guidelines,” or world. The circumstances of the exposure which describes in detail certified labora- may involve how the poison was encountered tory work in forensic toxicology including and for how long (multiple routes of exposure definitions, personnel, standard operating

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procedures, samples and receiving, security and Chrysomya rufifacies (Macquart) in a suspected chain of custody, analytical procedures, quality case of malathion poisoning (Gunatilake and assurance and quality control, review of data, Goff, 1989). reporting of results, interpretation of toxicol- ogy results, and safety. This section focuses on the sampling portion. SOFT and AAFS recom- mend taking 15 mL of blood for ethanol and Analysis Methodology other toxicants that are needed to assess perfor- mance. The “reasonable complete” drug screen Classical Analytical Chemistry Approach should only require 5 mL or less of blood. That The classical approach involves making an acid requires sensitive and specific methodology or base digest of the tissue. The acidified portion development by forensic toxicology laborato- may be subject to steam to release the volatile ries. Urine should have a minimum collection medications and poisons such as acetaldehyde, volume of 30 mL for analysis for detection of acetone, carbon monoxide, cyanide, ethanol, metabolites or the last tissue to indicate the isopropanol, methanol, phenol, and so forth or presence of an illegal agent. Hair samples may anions such as bromide, borate, fluoride, and be taken for exposure to drugs of abuse, but oxalate. The acid fraction leaches metals out laboratory ability to analyze should meet the of the tissues and can be analyzed by atomic Society of Hair Testing proficiency for detec- absorption or inductively coupled plasma emis- tion without false positives or negatives follow- sion spectroscopy. The basic extract is separated ing extraction in hydrochloric acid, enzymatic into an aqueous and an organic phase. The treatment (Pronase followed by liquid–liquid aqueous phase is acidified and organic solvent is extraction, or -glucuronidase treatment and added. This yields the acidic medications such as buffer extraction), hot methanol extraction the barbiturates (from barbituric acid). This step (with or without sonication), and hot NaOH can be preceded by a precipitation of protein or disintegration and extraction (Jurado and Sachs, similar cleanup procedure. The organic phase 2003). Urine, blood, and hair have been used of the organic extract is extracted with acid and for heavy metals and especially arsenic analysis. again separated into an aqueous and organic The fingernails prove to indicate the chronic phase. The organic phase contains the neutral occupational exposure of workers to arsenic medications such as simvastatin. The aqueous (Agahian et al., 1990). Lead concentrations in phase is made basic and contains the basic medi- teeth have been studied since the 1970s and cations such as amphetamine (the amine portion still serve to monitor environmental exposures of the catecholamine structure produces its in children in foreign countries (Arruda-Neto basic nature). In the past, volatiles were analyzed et al., 2009). by gas–liquid chromatography. The medications or street drugs were determined by thin-layer chromatography techniques. Organic extracts Postmortem of the tissues would also be used to analyze for SOFT and AAFS (2006) recommend taking hydrophobic compounds such as polycyclic aro- 50 grams of brain, liver, and kidney; 25 mL of matic hydrocarbons, polychlorinated biphenyls, heart blood; 10 mL of peripheral blood; and all and dioxins. If a chemical powder or unknown available of the vitreous humor, bile, urine, and liquid is found near a corpse or stomach or gastric contents as a start for all postmortem intestinal contents yield a liquid, pill, or com- toxicology analyses. For specific toxicants lung, pound, they may be subjected to tests such as intestine, or other specimens may be taken. boiling point, melting point, and other physical If a corpse has been deteriorating, even the determinations. These preparations may have a organisms that are assisting in its decomposi- chemical added to it to form crystals. Chemical tion can be sampled as was done for arthropod spot tests will be discussed later under rapid larvae Chrysomya megacephala (Fabricius) and screening tests. Chromatography is the next

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topic, as it is the method of choice if specificity Modern Extraction Linked to GC-MS, LC-MS, is required. The World Health Organization or LC-(Tandem) MS(/MS) (2015) recommends that analytical toxicol- ogy laboratories contain the following equip- The gold standard for toxicology analyses ment and have procedures to perform simple currently is an extraction procedure followed “spot” tests: Conway apparatus, Gutzeit appa- by GC-MS analysis. Not all toxicants are vol- ratus, direct-reading spectrophotometer, ultra- atile for gas chromatographic analysis, and violet (UV)/visible recording spectrophotometer, newer methods employ liquid chromatography thin-layer chromatography—qualitative, thin- coupled with one or two mass spectrometers layer chromatography—quantitative, gas (Maurer, 2007). Because the sample matrix may chromatography—packed columns, gas be complicated with a variety of contaminants chromatography—capillary columns, gas chro- in the extract, mass spectra can now have a stan- matography—flame ionization detection, gas dardized computer program to subtract out chromatography—nitrogen-phosphorus detec- interfering substances and provide evidence of tion, gas chromatography—electron-capture the structure for unknown toxicants not avail- detection, gas chromatography–mass spec- able in standard MS libraries (Stimpfl et al., trometry (GC-MS), high-performance liquid 2003). In June 1986, a poisoning epidemic in chromatography (HPLC)–UV detection, Sierra Leone was solved employing positive high-performance liquid chromatography– chemical ionization mass spectrometry and fluorescence detection, high-performance liquid nuclear magnetic resonance spectroscopy. An chromatography–mass spectrometry (LC-MS), unknown poison in bread samples proved to be high-performance liquid chromatography– the organophosphorus pesticide parathion (Hill electrochemical detection, high-performance et al., 1990). Note that two different methods of liquid chromatography–diode array UV detec- analysis were used as is recommended for deter- tion, capillary electrophoresis, atomic emission mination and confirmation of an unknown spectrometry, atomic absorption spectrometry toxicant. The development of LC-MS(/MS) has (flame), electrothermal atomic absorption spec- led to determination of unknown toxicants, trometry, inductively coupled plasma source and with the proper algorithms (e.g., SALSA spectrometry, radioimmunoassay—counting, [Stochastic Approach for Link-Structure Analy- enzyme immunoassay (e.g., enzyme-multiplied sis]) it can yield epoxide adducts of proteins. immunoassay technique), fluorescence immu- Hemoglobin adducts are likely, such as those noassay, enzyme-linked immunosorbent assay, formed on incubation with styrene oxide, eth- fluorimetry, and infrared spectrometry. Pure ylene oxide, and butadiene oxide (Badghisi standards are also required of the laboratory. and Liebler, 2002). The biological sample in Many of these techniques alone or in combi- this case represents the aqueous phase and the nation would not be trusted to identify an organic solvent the second phase that involve unknown. For example, SOFT/AAFS (2006) high-throughput liquid–liquid extraction (LLE) guidelines indicate that an identification by methodology by robotic liquid handling of a immunoassay followed by “confirmation” by 96-well LLE plate with inert diatomaceous earth flame ionization detectors and nitrogen-phos- particles for continuous and efficient extrac- phorus detectors only give a range of suspects tion of analytes between the aqueous biological based on retention time of the columns and will sample and the organic extraction solvent (Peng not hold up in a court of law. Mass spectrom- et al., 2001). etry (MS) gives structural features that may lead to identification of the unknown toxicant. The beam intensity for MS may be varied to change Rapid Screening Tests the amount of fragmentation of the compound, The older color tests are used heavily by the law and low intensities may yield more of the molec- enforcement agencies for trafficking in illegal ular ion, which is the organic molecule with a drugs. The U.S. Department of Justice, National positive charge due to removal of one electron. Institute of Justice has a Standards and Testing

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Program that has developed a list of Color Test 2 days. Amphetamines may be detected from Reagents/Kits for Preliminary Identification of 2–5 days after use. Clenbuterol can be found in Drugs of Abuse (NIJ Standard-0604.01), dis- urine 4–6 days following use. Nicotine may be played in Table 14-1. There are also commer- observed in urine 4–10 days after a person ceases cially available test kits for urine that measure smoking. A 5–7 day detection time is common 12 standard abused medications or street drugs to euphorics such as ecstasy, the opiates, the dis- and their metabolites using simple immunoas- sociative anesthetic agent ketamine, and meth- says to lateral flow-based immunoassays. For amphetamines. Cannabinoids such as THC are example, one commercially available kit uses detectable in urine for 5 days after one-time use sensitivities recommended by the National to as high as 48–63 days in those using mari- Institute on Drug Abuse to screen urine for the juana daily. Benzodiazepines may be detected following compounds and their metabolites in in urine from 7–10 days following cessation of urine: 25 ng/mL for phencyclidine, 50 ng/mL use. Oral anabolic steroids may be detected for for marijuana (THC); 100 ng/mL for oxyco- 14–28 days, while injected steroids are detect- done, 300 ng/mL for cocaine, methadone, bar- able 1–3 months following use. People have biturates, benzodiazepine, or propoxyphene; employed diuretics to decrease the detectable 500 mg/mL for MDMA (ecstasy), 1,000 mg/mL concentration of the medication, but a specific for amphetamine or methamphetamine; and gravity test and a test for diuretics indicate that 2,000 mg/mL for opiates. a person is trying to avoid detection of a banned Detection time is important in living beings. substance or indicate that use was below the Peptide hormones are undetectable in urine. legal limit for operating a motor vehicle or prac- Cocaine detection may range from 1–4 days ticing medicine. Specific gravity is also altered following administration. Propoxyphene can by salts or detergents. Other compounds that be detected from 6 hours to 2 days after use. may be attempted to be added to urine to avoid Short-acting barbiturates may be detectable for detection of banned substances or abused drugs

TABLE 14-1 Color Changes for Medications and Drug of Abuse Tests

Reagent (#) Color Change Drugs (form or solvent) Cobalt Thiocyanate (A.1) Brilliant greenish blue Benzphetamine HCl, brompheniramine maleate, chlordiazepoxide HCl, chlorpromazine HCl, doxepin HCl, hydrocodone tartrate, methadone HCl, methylphenidate HCl Strong greenish blue Cocaine HCl, diacetylmorphine HCl, ephedrine HCl, meperidine HCl, phencyclidine HCl, procaine HCl, propoxyphene HCl, pseudoephedrine HCl

Strong blue Quinine HCl (all in CHCl3) Dille-Koppanyi Reagent. Light purple Amobarbital, pentobarbital, phenobarbital, secobarbital

Modified (A.2) (all in CHCl3) Duquenois-Levine Reagent. Strong reddish purple to Mace (crystals) Modified (A.3) very light purple Pale reddish purple to light Nutmeg (extract) gray purplish red Light yellow green Tea (extract) Gray purplish blue to light Tetrahydrocannabinol, THC (in ethanol) purplish blue to deep purple

(continues)

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TABLE 14-1 Color Changes for Medications and Drug of Abuse Tests (continued )

Reagent (#) Color Change Drugs (form or solvent)

Mandelin Reagent (A.4) Moderate olive Acetaminophen (CHCl3) Grayish olive green Aspirin (powder)

Brilliant yellow green Benzphetamine HCl (CHCl3)

Strong orange Brompheniramine maleate (CHCl3), salt (crystals)

Dark olive Chlorpromazine HCl, codeine (both in CHCl3), Excedrin (powder)

Deep orange yellow Cocaine HCl (CHCl3) Strong yellow Contac (powder)

Moderate bluish green d-Amphetamine HCl (CHCl3)

Dark yellowish green d-Methamphetamine HCl (CHCl3)

Moderate reddish brown Diacetylmorphine HCl (CHCl3)

Dark olive brown Dimethoxy-meth HCl (CHCl3)

Dark reddish brown Doxepin HCl, propoxyphene HCl (both in CHCl3) Grayish olive Dristan (powder) Moderate olive green Mace (crystals)

Bluish black d,l-3,4-Methylenedioxyamphetamine HCl, MDA (CHCl3)

Dark yellowish brown Mescaline HCl (CHCl3)

Dark grayish blue Methadone HCl (CHCl3)

Very orange yellow Methaqualone (CHCl3)

Brilliant orange yellow Methylphenidate HCl (CHCl3)

Dark grayish reddish brown Morphine monohydrate (CHCl3)

Dark brown Opium (CHCl3)

Dark greenish yellow Oxycodone HCl (CHCl3)

Deep orange Procaine HCl (CHCl3)

Deep greenish yellow Quinine HCl (CHCl3) Marquis Reagent (A.5) Deep red Aspirin (powder)

Deep reddish brown Benzphetamine HCl (CHCl3)

Deep purplish red Chlorpromazine HCl, diacetylmorphine HCl (both in CHCl3)

Very dark purple Codeine (CHCl3)

Strong reddish orange d-Amphetamine HCl (CHCl3)

Deep reddish orange d-Methamphetamine HCl (CHCl3)

Dark reddish brown d-Amphetamine HCl, d-Methamphetamine HCl (both in CHCl3)

Moderate orange Dimethoxy-meth HCl (CHCl3)

Blackish red Doxepin HCl (CHCl3) Dark grayish red Dristan (powder) Dark red Excedrin (powder)

Olive black Lysergic acid diethylamide. LSD (CHCl3) Moderate yellow Mace (crystals) Dark reddish brown Dristan (powder)

(continues)

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TABLE 14-1 Color Changes for Medications and Drug of Abuse Tests (continued )

Reagent (#) Color Change Drugs (form or solvent)

Marquis Reagent (A.5) Black MDA (CHCl3) (continued) Deep brown Meperidine HCl (CHCl3)

Strong orange Mescaline HCl (CHCl3)

Light yellowish pink Methadone HCl (CHCl3)

Moderate orange yellow Methylphenidate HCl (CHCl3)

Very deep reddish purple Morphine monohydrate (CHCl3) Dark grayish reddish Opium (powder) Brown

Pale violet Oxycodone HCl (CHCl3)

Blackish purple Propoxyphene HCl (CHCl3) Dark brown Sugar (crystals)

Nitric Acid (A.6) Brilliant orange yellow Acetaminophen (CHCl3), Excedrin (powder),

Morphine monohydrate (CHCl3)

Light greenish yellow Codeine, MDA (both in CHCl3)

Pale yellow Diacetylmorphine HCl (CHCl3)

Very yellow Dimethoxy-meth HCl (CHCl3)

Brilliant yellow Doxepin HCl (CHCl3) Deep orange Dristan (powder)

Strong brown Lysergic acid diethylamide (LSD) (CHCl3) Moderate greenish yellow Mace (crystals)

Dark red Mescaline HCl (CHCl3) Dark orange yellow Opium (powder)

Brilliant yellow Oxycodone HCl (CHCl3)

Para-Dimethylamino- Deep purple LSD (CHCl3) Benzaldehyde (A.7) Ferric Chloride (A.8) Dark greenish yellow Acetaminophen (methanol) Deep orange Baking soda (powder) Very orange Chlorpromazine HCl (methanol) Moderate purplish blue Dristan, Excedrin (both powder) Dark green Morphine monohydrate (methanol) Froede Reagent (A.9) Grayish purple Aspirin (powder)

Very deep red Chlorpromazine HCl (CHCl3)

Very dark green Codeine (CHCl3) Moderate olive brown Contac (powder)

Deep purplish red Diacetylmorphine HCl, morphine monohydrate (both in CHCl3)

Very yellow green Dimethoxy-meth HCl (CHCl3)

Deep reddish brown Doxepin HCl (CHCl3) Light bluish green Dristan (powder)

(continues)

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TABLE 14-1 Color Changes for Medications and Drug of Abuse Tests (continued )

Reagent (#) Color Change Drugs (form or solvent) Froede Reagent (A.9) Brilliant blue Excedrin (powder) (continued) Moderate yellow green LSD (CHCl3) Light olive yellow Mace (crystals)

Greenish black MDA HCl (CHCl3) Brownish black Opium (powder)

Strong yellow Oxycodone HCl (CHCl3)

Dark grayish red Propoxyphene HCl (CHCl3) Brilliant yellow Sugar crystals

Mecke Reagent (A.10) Blackish red Chlorpromazine HCl (CHCl3)

Very dark bluish green Codeine, MDA HCl, morphine monohydrate (all in CHCl3) Moderate olive brown Contac (powder)

Deep bluish green Diacetylmorphine HCl (CHCl3)

Dark brown Dimethoxy-meth HCl (CHCl3)

Very dark red Doxepin HCl (CHCl3) Light olive brown Dristan (powder) Dark grayish yellow Excedrin (powder)

Dark bluish green Hydrocodone tartrate Excedrin (CHCl3)

Greenish black LSD (CHCl3) Dark grayish olive Mace (crystals)

Moderate olive Mescaline HCl, Oxycodone HCl (both in CHCl3) Brownish black Nutmeg (leaves) Olive black Opium (powder)

Deep reddish brown Propoxyphene HCl (CHCl3) Brilliant greenish yellow Sugar (crystals) Zwikker Reagent (A.11) Light blue Baking soda (powder) Light green Excedrin (powder)

Light purple Pentobarbital, phenobarbital, secobarbital (all in CHCl3) Moderate yellow green Tea (leaves) Moderate yellowish green Tobacco (leaves)

Simon’s Reagent (A.12) Dark blue d-Amphetamine HCl, MDMA HCl (both in CHCl3)

Deep blue Dimethoxy-meth HCl (CHCl3)

Pale violet Methylphenidate HCl (CHCl3)

Modified from National Institute of Justice Law Enforcement and Corrections Standards and Testing Program. 2000. Color test reagents/kits for preliminary identification of drugs of abuse (NIJ Standard 0604.01). Washington, DC: U.S. Department of Justice, Office of Justice Programs, National Institute of Justice. http://www.ncjrs.gov/pdffiles1/nij/183258.pdf

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are acids and baking soda (change pH and Confirmatory Tests can be detected by pH as well) and bleach to Confirmatory test usually require greater sen- oxidize compounds or test components (odor sitivity and usually employ GC-MS or LC-MS and color test can detect these). If urine tamper- as the gold standard for identification. For ing is suspected, human physiological values of example, confirmatory tests require the sen- urine temperature (32.2–37.8ºC), pH (4.5–8.0), sitivity for marijuana at 15 ng/mL, cocaine at and creatinine ( 20mg/dL) should be found. 150 ng/mL, and amphetamines at 500 ng/mL. If urinary creatinine is 20 mg/dL, it is diluted However, phencyclidine and opiates remain urine. Below 5 mg/dL creatinine, the sample at the screening values of 25 ng/mL and is unlikely to be human urine. Specific gravity 2,000 ng/L, respectively. should be 1.003 g/mL and human IgG should be 0.5 mg/L. Some adulterants commonly used are glutaraldehyde (fixative that interferes with immunoassays and can be detected by Flow Chart of a Typical gas chromatography), nitrite (oxidizing agent that can be detected colorimetrically), and pyri- Postmortem Examination dinium chlorochromate (oxidizing agent that An example where postmortems are common can be detected by color, atomic absorption, or are crashes involving air transportation. The gas chromatography; Jaffee et al., 2008). Some Federal Aviation Administration (FAA) has “blocking agents” are more mythology than difficulty obtaining uniform samples from effective. For example, probenecid is a medica- crashes, because explosions and other effects tion that decreases penicillin excretion. It does of impact may make recovery of certain bio- not work to retard anabolic steroid excretion logical fluids impossible. Note the flow chart in and is also a banned substance in athletic com- Figure 14-1. It is clear that esoteric toxicants petition even if it could do so. It is more diffi- are not considered in the first analysis. Clearly, cult to alter professionally drawn blood samples, carbon monoxide poisoning is a primary which are mostly useful for detection of toxi- concern when a fire occurs in an enclosed cants and essential for therapeutic drug moni- plane or in vehicles powered by a combustion toring. Hair samples have increased longevity engine as indicated by the test for carboxyhe- for substances and may detect use of drugs such moglobin (COHb). That is also a concern in as amphetamines, marijuana, cocaine, opiates, northern climates. The state of Minnesota now and phencyclidine for as long as 90 days follow- requires dwellings to contain carbon monoxide ing exposure. The combination of presence of detectors by bedrooms in houses with furnaces a substance, its concentration at a given time that use combustion for heating purposes or following use, and its redistribution following contain a gas stove. Cyanide is another toxicant death (determining minimum body burden may that occurs during a fire, and it may occur natu- be more important in this case) is important, rally in certain plants and has been employed in because the presence may indicate abuse, and poisonings that could account for a plane crash. excess leading to death may determine that a Alcohol or other volatile substances may render homicide was committed. This is especially true the pilot less capable of handling an aircraft for injected substances that can only be legally safely due to the CNS effects of volatile sol- obtained and administered by a medical practi- vents. Additionally, other possibly abused street tioner. The investigation of the death of a very drugs or prescription medications are of general famous pop star headed in that direction as the concern during investigation of the reason for a number of medications and their concentra- plane or other crash involving the U.S. Depart- tions were determined following autopsy (physi- ment of Transportation. Glucose is important, cian convicted of involuntary manslaughter due because hypoglycemia may account for uncon- to administration of a fatal dose of the anes- sciousness. Additionally, uncontrolled diabe- thetic agent propofol). tes mellitus is a concern for pilots as indicated

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• A/V • Batch preparation Specimens Accessioning for COHb • Bile • Solid tissues • Case number assignment • CN– • Blood – Liver – Muscle • Internal chain-of-custody • Drug screening • Urine – Lung – Brain • Specimen chain-of-custody • Glucose analyses • Vitreous humor – Spleen – Heart • HbA1c

Blood Urine/vitreous humor Solid tissues • A/V • Drugs • A/V • Glucose • Abused drugs only – • CN • HbA1c • Drugs • Urine • A/V and drugs • COHb • Bile

Analysis result review

If negative, report If positive, confirmatory/ Batch preparation Analysis result review is generated quantitative analyses in and analyses and report generation specimens are requested

Whole case review • Clerical review • Accessioning • Report dissemination • QA/QC levels – AAI – ME • Scientific – NTSB – RFS • Report signing

FIGURE 14-1 A Flowchart for a Typical Toxicological Processing of Postmortem Biological Samples http://www.hf.faa.gov/docs/508/docs/cami/0214.pdf

both by the glucose measurement and the use (RIA) when urine cannot be collected. Gastric of the hemoglobin (Hb) A1c test which deter- contents (100 mL) are used as necessary for mines whether Hb is irreversibly glycated at the drug analyses. Ethanol and drug analyses are N- terminal valine of the β-chain (Nakanishi also performed on 500 g of liver; 300 g of skel- et al., 2003). The Civil Aerospace Medical Insti- etal muscle; 150 g of spleen; 100 g of lung, tute laboratory optimally requests 40 mL of kidney, and brain; and 50 g of heart. Spinal blood in a green-top tube for COHb, blood fluid will be sampled for the amount available CN ,HbA1c, and drug analyses. The same and tested for ethanol and perhaps drugs if amount of blood in a grey-top tube is used for enough is present. The problem is that all these ethanol and drug analyses. Urine (100 mL opti- tissues may not be available or only available mally) is collected for drug, ethanol, and glucose at suboptimal (adequate or less) volumes. This analyses. Vitreous humor (2 mL optimally) is demonstrated in Figure 14-2. The kidneys is sampled for ethanol and glucose testing. are found at the most protected portion (most Bile (10 mL) is analyzed by radioimmunoassay dorsal) of the abdominal area and therefore had

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Heart Brain Muscle Spleen Kidney Lung Liver Gastric contents Bile Spinal fluid Vitreous fluid Urine Blood

09010 20 30 40 50 60 70 80 100 CAMI cases (%)

Optimal Adequate Inadequate

FIGURE 14-2 Optimal, Adequate, and Inadequate Amounts of Various Postmortem Biological Specimens Received at CAMI from the 1,891 Cases (Fatalities) of the United States Fatal Aviation Accidents that Occurred During 1996–2000 http://www.hf.faa.gov/docs/508/docs/cami/0214.pdf

80% of the cases having optimal to adequate amounts of this tissue for examination for Therapeutic Drug (Medication) the years 1996–2000 (Chaturvedi et al., 2002). Uncontaminated spinal fluid is hardest to Monitoring in Living Human obtain, with less than 10% of the cases having Patients optimal volumes for analyses. Urine is very On occasion, certain drugs carry a high danger useful in detecting metabolites of chemicals of toxicity (small therapeutic index or LD / that may have already been excreted, but unfor- 50 ED ) and generally are so lipophilic that they tunately appears to be inadequately available 50 require extensive monitoring. Other reasons for in more than 80% of the crashes. Blood is also following plasma concentrations are that the heavily relied upon in toxicology for current pharmacokinetics vary widely by patient, plasma levels of toxicants, the control of diabetes mel- concentration is a better measure than dose for litus, and the clear indication of succumbing to therapeutic and adverse effects, there is a plasma fire (COHb and CN). Unfortunately, optimal concentration that must be achieved to ensure and adequate samples were only available in proper onset of therapy (and must be kept close slightly more than 60% of the cases. It is inter- to that value), or the therapeutic effects are dif- esting to note that gastric contents were more ficult to monitor. The last two points are similar likely to be in optimal adequate volumes than to the determination of the international nor- blood. This is why it is important to consider all malized ratio (INR), which is used to determine possible recoverable uncontaminated samples anticoagulant therapy with a medication such and what might be gained from their analyses.

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as warfarin (Anand and Yusuf, 2003). The target below which no therapeutic value is indicated INR is 2–3. If the coagulation is too rapid, then for the medication alone. This trough concen- dangerous clots may form. If coagulation is too tration works well for antibiotics and antivirals,

low, hemorrhage may occur. This is specifically as the Ctrough for the HIV medication atazana- not therapeutic drug monitoring as the effect vir, for example, is associated with efficacy (de rather than the concentration of the medication Requena et al., 2005). Other medications may is monitored. When effect is not well measured, indeed change this value significantly, as is the then plasma concentration may be the next best case for certain combinations of protease inhibi- option. Proper dosing is not possible without tors used to treat HIV infection (Guiard-Schmid measuring resultant plasma concentrations. The et al., 2003). For anti-epileptic medications, the journal Therapeutic Drug Monitoring is devoted to plasma concentration of lamotrigine varies four- this area. fold when comparing joint anti-seizure therapy A number of factors must be considered with the metabolic enzyme inhibitor valproic regarding monitoring of medications. These acid as opposed to inducers phenytoin or car- include compliance (whether the patient is bamazepine (Morris et al., 1998). The high peak taking the medication as prescribed) and age, concentration should represent a toxic thresh- because neonates, children, and the elderly old of sorts that would endanger the patient absorb, metabolize, and excrete differently than and have less therapeutic value. Note that for adults. The gender of the patient and pregnancy the cancer chemotherapy agent methotrexate, play important roles in pharmacokinetics/toxi- only the trough may be relevant. This indicates cokinetics. Hepatic disease may hamper metabo- that all concentrations that are therapeutic are lism. Renal disease can influence half-life and toxic as well, but hopefully more so to cancer clearance. Cardiovascular disease can decrease cells. Toxicity to normal tissues is to be moni- or affect circulation and blood pressure, which tored carefully, so this may obviate the need for is important regarding assumptions of speed of a peak value. Some compounds have controver- action, organ distribution, or filtration resulting sial values or published articles indicating the in excretion. Respiratory disease affects circu- lack of need for therapeutic drug monitoring lation, blood pH, and is especially important for certain medications. Rapid metabolism of in anesthetic gases and excretion of volatile the medication may lead to a difficult match components of the medication. Other medica- between therapeutic effect and plasma concen- tions clearly may interact in a toxic fashion with tration of the unaltered drug. This is true for the medication or alter metabolism or elimi- the immunosuppressant purine anti-metabolite nation rate. Additionally, tolerance and depen- azathioprine (Lin et al., 1980). Also, binding to dence must be considered. There are a variety the plasma protein albumin in the blood makes of environmental factors that influence drug the compound unavailable for activity. Certain metabolism. Genetic polymorphisms of drug compounds such as the anti-seizure medica- metabolism also influence plasma concentra- tion valproic acid and the anti-inflammatory tions. To do medication monitoring properly, salicylate have non-linear protein binding in the doctor takes a history that includes the their therapeutic range (Birkett, 1994). Other patient’s age, weight, sex, their genetic history of compounds have uncertain therapeutic value, disease, the nature of their current disease, med- such as may be the case for a person who has ications taken, doses, medication schedule, and had HIV long enough to experience mutations for the specific medication(s) for which plasma in the virus that renders the protease inhibi- concentrations must be determined the time tor less effective. Some compounds may be of last dose, time of specimen, and the clini- well described by their area under the curve cal status relevant to the drug used (e.g., blood value, which requires many points and appears pressure, ECG, EEG, plasma liver enzymes). to be impractical. One such compound is the Consider Table 14-2, where therapeutic ranges immunosuppressant mycophenolate mofetil, are reported for a variety of medications. The which is used in kidney transplant patients.

lowest value is the trough concentration (Ctrough) The selection of a sampling time is difficult

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TABLE 14-2 Therapeutic Drug Monitoring Based on Class of Drugs, Concentration Range, Monitoring Method, and Interaction with Other Medications

Class of Medication Generic Name Therapeutic Plasma Concentration Range Aminoglycoside antibiotics Amikacin 15–25 g/mL Gentamicin 5–10 g/mL Tobramycin 5–10 g/mL Other toxic antibiotics Chloramphenicol 10–20 g/mL Vancomycin 5–10 g/mL or 10–20 g/mL trough (30 min prior to next dose controversial) Anti-arrhythmics Amiodarone 0.5–2.0 g/mL Digoxin 0.5–2.0 ng/mL Digitoxin 10–30 ng/mL Flecainide 0.2–1.0 g/mL Lidocaine 1.5–5.0 g/mL Mexiletine 0.5–2.0 g/mL Procainamide 4.0–8.0 g/mL N-Acetyl procainamide (metabolite) 10–20 g/mL Quinidine 2.0–5.0 g/mL Anti-arthritis or Salicylates 100–250 g/mL anti-inflammatory Antiepileptics Carbamazepine 5–12 g/mL Ethosuximide 40–100 g/mL Gabapentin 2–10 g/mL Lamotrigine 3–14 g/mL Phenobarbital 10–30 g/mL Phenytoin 10–20 g/mL Valproic acid 50–100 g/mL Bronchodilators Caffeine 10–20 g/mL Theophylline 10–20 g/mL Cancer chemotherapy Methotrexate > 0.01 mol/L HIV treatments—protease Atazanavir 150–850 ng/mL inhibitors Indinavir 0.15 g/mL trough, 10 g/mL peak Lopinavir 3–8 g/mL Nelfinavir 0.7–1.0 g/mL (trough) Ritonavir 1.1–6.3 g/mL (trough) Saquinavir 100 ng/mL (trough minimum for wt virus) Immunosuppressants Azathioprine 20–90 ng/mL at peak (mercaptopurine metabolite) Cyclosporine C2 (conc 2 hours after dosing) 800 ng/dL Mofetil mycophenolate 1–3.5 g/mL (C2 trough) Sirolimus 4–20 ng/mL (C2 trough less on initiation, more for maintenance) Tacrolimus 5–20 ng/mL Psychiatric medications Lithium 0.8–1.2 mEq/L Amitriptyline 120–150 ng/mL Desipramine 150–300 ng/mL Doxepin 50–200 ng/mL

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for this medication as the trough value has 2. What samples should be taken for: little correlation with the dose or therapeutic (a) a child living in a house manufactur- effect. The plasma concentration 2 (C2) hours ing methamphetamine but not taking following dosing may be the best measure for any of the drug, (b) monitoring the level this medication (Jirasiritham et al., 2004). Note of digitalis in a patient, (c) a living person this C2 approach is also used for the immu- to show that they had used illegal drugs nosuppressant agent cyclosporine (Marcén et in the recent past, (d) a corpse buried in al., 2005). The methods for assessment of the the ground that might have been poisoned plasma concentration include HPLC, GC/MS with an organophosphate nerve agent, (or tandem MS), and automated immunoas- and (e) chronic exposure of a child to pica says. Both accuracy and cost effectiveness must (Pb in paint chips)? be considered for large-scale clinical use (Touw 3. Why not test for original toxicant, medica- et al., 2005). Many of the automated immunoas- tions, or peptide hormones in urine? say units focus on digoxin (ng/mL) and theoph- 4. In plane crash cases, why not use the urine ylline (mg/mL). Digitalis has difficult onset to as this fluid is most likely to contain effects (1 hour post administration) and a very metabolites of drugs, ethanol, and other narrow therapeutic range (0.5–1.5 or 2.0 ng/mL, prescription medications used before the a long half-life (160 hours) and interacts with crash as well as whether the person had many medications, so it must be monitored diabetes mellitus under control? often and carefully. Any overdose may affect 5. In therapeutic drug monitoring, when the heart and nervous system severely (3Na , is Ctrough appropriate to use for dos- 2K-ATPase inhibition), so a digitalis antibody ing versus C2 versus establishing a range is used to avoid plasma concentrations in excess with maximum and minimum plasma of 2.0 ng/mL. Automated methods in therapeu- concentrations? tic drug monitoring reflect clearly established guidelines for use in a widespread and diverse population. Other medications have contro- References versial aspects of when to measure or whether to measure at all. That is an easier decision to Agahian B, Lee JS, Nelson JH, Johns RE. 1990. Arsenic make if the therapeutic activity is more easily levels in fingernails as a biological indicator of exposure to arsenic. Am Ind Hyg Assoc J. 51:646–651. determined than previously thought when mon- Anand S, Yusuf S. 2003.Oral anticoagulants in itoring was considered. Additionally, the toxicity patients with coronary artery disease. J Am Coll may be easily observed and does not endanger Cardiol. 41:62S–69S. the patient more than the adverse effects of Arruda-Neto JD, de Oliveira MC, Sarkis JE, Bordini P, other medications that are not monitored by Manso-Guevara MV, Garcia F, Prado GR, Krug FJ, plasma concentrations. This is the reason that Mesa J, Bittencourt-Oliveira MC, Garcia C, some doctors in rural areas use the effect of digi- Rodrigues TE, Shtejer K, Genofre GC. 2009. Study of environmental burden of lead in children using talis on heart rate alone as an inexpensive alter- teeth as bioindicator. Environ Int. 35:614–618. native to testing. Badghisi H, Liebler DC. 2002. Sequence mapping of epoxide adducts in human hemoglobin with LC-tandem MS and the SALSA algorithm. Questions Chem Res Toxicol. 15:799–805 Bigazzi PE. 1994. Autoimmunity and heavy metals. 1. How would you use to toxidromes to Lupus. 3:449–453. discern the difference between someone Birkett DJ. 1994. Pharmacokinetics made easy 9: non- driving under the influence of alcohol linear pharmacokinetics. Aust Prescr. 17:36–38. Boyle JS, Bechtel LK, Holstege CP. 2009. Management (ethanol) versus Vicodin (hydrocodone of the critically poisoned patient. Scand J Trauma opioid acetaminophen)? How would Resusc Emerg Med. 17:29. CO poisoning look different from CN Bronstein AC, Spyker DA, Cantilena LR Jr, Green JL, poisoning? Rumack BH, Heard SE. 2008. 2007 annual

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