Forensic Chemistry and Toxicology

Forensic Chemistry and Toxicology

Toxicology Formed from the Greek words toxicos and logos, toxicology is the study of the symptoms, mechanisms, treatments and detection of poisoning 2

Vocabulary Forensic toxicology centers on the determination of toxic substances in human tissues, organs and body fluids such as urine and blood, and the subsequent determination of the role any toxic agents may have contributed to or caused death.

Toxicology Toxin is any material that is considered to have a life threatening effect on a living organism. Poison is a subgroup of toxins - comes in many forms Gaseous Liquid Solid Animals Minerals Vegetables Enters Body 1 of 3 ways: Ingested Inhaled Absorbed into the skin

Toxicology Types of toxicologists
Descriptive Toxicologist: Performs toxicity test to evaluate the risk that exposure pose to humans Mechanistic Toxicologist: attempts to determine how substances exert deleterious effects on living organisms. Regulatory Toxicology: Determines whether or not a substance has low enough risk to justify making it available to the public. Forensic Toxicology is a subspecialty

Provide Answers to “?” Forensic Toxicologists:
Provide answers to questions that may be asked during the investigation or at a subsequent legal hearing. Was a poison involved in the case? If so, what was the nature of the poison how was it administered was it at a potentially lethal concentration?

Forensic Toxicology What do they Investigate? Accidental Poisoning
Drug Abuse/Overdose Suicidal/Homicidal Poisoning *75% of the evidence evaluated in the crime lab is drug related!

16th Century Physician Paracelsus
“All substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy.”

Vocabulary of the Chemist
Poison: a substance taken in sufficient quantity to cause ill health or death

Vocabulary 2 classes of toxicity: acute and chronic.
Acute toxicity refers to effects that occur shortly after a single exposure or small number of closely spaced exposures. Chronic toxicity refers to delayed effects that occur after long term repeated exposures. 10

Common Toxins / Poisons
Acids(HNO3, HCl, H2SO4) Burns around mouth, lips, nose Aniline(hypnotics, nitrobenzene) Skin of face & neck quite dark As(metals, Hg, Cu, Pb) Severe, unexplained diarrhea Atropine(Belladonna), Scopolamine Pupil of eyes dilated Bases(lye, potash, OH-) Carbolic Acid (phenols) Odor of disinfectant CO Skin is bright cherry red HCN Red skin, odor of peach, Quick Food poisoning Vomiting, abdominal pain Metallic compounds Diarrhea, vomiting, abdominal pain Nicotine Convulsions Opiates Pupil of eyes contracted Oxalic acid (P-) Odor of garlic NaF Convulsion Strychnine Convulsion, dark face & neck

Poisonings Children under 6 account for most of the reported poisonings/year About 700 deaths by poisoning are reported each year Approximately 2 million cases are voluntarily reported to poison control centers each year However adults account for the majority of deaths by poisoning most of which is intentional rather than accidental

Toxins/Poisons Most Reported 1 - Household cleaning supplies
2 - Analgesics (aspirin, acetaminophen) 3 - Cosmetics 4 - Cough & cold remedies 5 - Plant scrapes & insect bites 6 - Pesticides 7 - Topical creams & lotions 8 - Hydrocarbons i.e. gasoline, kerosene 9 - Anti-micro bacterial soaps 10 - Sedatives/hypnotics/anti-psychotics 11 - Food poisoning 12 - Alcohol

Toxins/Poisons Most Frequent Deaths 1 – Antidepressant medications
2 - Analgesics (aspirin, acetaminophen) 3 - Street drugs 4 - Cardiovascular drugs 5 - Alcohol 6 - Gases and fumes 7 - Asthma therapies 8 - Industrial chemicals 9 - Pesticides 10 - Household cleaning supplies 11 - Anticonvulsant medications 12 - Food, plants, & insects

Pharmacokinetics & Pharmacodynamics
Pharmacokinetics is the relationship between drug conc. In the body & time after administration of the drug. Form, frequency & route all have an effect on this Pharmacodynamics is the study of the biochemical & physiological effects of a drug and its mechanism of action.

Drug Absorption from the GI tract is dependent on the rate & amount of intact drug and the rate that it reaches circulation after oral administration. Rate is affected by: Solubility Absorption across membrane Gastric emptying Metabolic Rate

Structure of GI Tract The GI tract has three major regions: the stomach, small intestine and the large intestine. Stomach – the mucosa contains many folds that increase the surface area available for drug absorption. The high blood supply and the fact that a drug can potentially reside in the stomach for several hours can provide optimum conditions for the absorption of acidic drugs.

Structure of GI Tract Small Intestine - the most important GI site for drug absorption. The intestinal mucosa provides an extremely large surface area; villi and microvilli aids drug absorption (about 30’ long in an adult) Large Intestine - like the stomach, lacks the villi and microvilli of the small intestine; serves as a site for the absorption of drug that has not been completely absorbed in the small intestine. (about 6’ long in an adult)

Mechanisms of Drug Transport Across the GI Barrier
The majority of drugs cross the membrane by passive diffusion. Passive Diffusion - Drug transport rate is determined by the physical and chemical properties of the drug, and its concentration gradient across the membrane. Drug entering the blood stream will be carried away from the site of absorption by the gastrointestinal blood supply and will become diluted by: Distribution in a large volume of blood. Distribution into body tissue. Metabolism and excretion. Protein binding in the blood.

Physiological Factors Influencing Drug Absorption
Surface area of the absorption site pH of the GI fluids Gastric emptying rate: can be stimulated by hunger, anxiety, body position and liquid consumption; Fatty foods, high bulk diet, depression and various drugs and alcohol retard it. Intestinal motility Drug stability in the GI system Dietary components Disease states

Drug Metabolism Metabolism is the fundamental mechanism of drug elimination. Metabolism aids drug excretion processes and may affect the pharmacological response of a drug by altering its potency and/or duration of action Metabolism can occur in the plasma as well as organs other than the liver such as the GI tract, kidneys and lungs.

Factors Affecting Metabolism
Age Young children and elderly people usually have a lower metabolic capacity Disease Diseases can affect all of the ADME processes. Liver and kidney diseases probably have the greatest effect on drug concentrations by affecting metabolism and excretion.

Factors Affecting Metabolism
Weight The weight of a patient affects drug concentrations in the blood since it determines the volume into which the drug is distributed. Drug metabolism tends to be faster in males than in females. Genetic Factors Gender and race differences in enzyme activity can affect drug metabolism Diet Diet may influence the metabolism of some drugs. Exposure to other drugs (particularly alcohol) markedly affects the metabolism of certain drugs.

Excretion of Drugs The excretion of drugs and metabolites terminates their activity and presence in the body. Elimination can occur by various routes. The kidney plays a major role with the excretion of drugs and/or their metabolites into the urine. Drugs may also be excreted in the feces, bile, lungs sweat, saliva and breast milk.

What is a drug? A drug can be defined as a natural or synthetic substance that is used to produce physiological or psychological effects in humans or other higher order animals. Narcotic drugs are analgesics, meaning they relieve pain by a depressing action on the central nervous system. This effects functions such as blood pressure, pulse rate and breathing rate. The regular use of a narcotic drug will invariably lead to physical dependence. The most common source for these narcotic drugs is opium, extracted from poppies.

Illicit Drugs “NIDA 9” Methamphetamine Cocaine Opiate Benzodiazepam
THC Methadone Barbiturates PCP CNS Depressants: Narcotics (opiates) Sedatives, hypnotics (PCP, Quaalude) Tranquilizers (Valium) CNS Stimulants Cocaine Amphetamines Hallucinogens Mescaline, psilocybin, LSD, PCP THC Steroids Inhalants

Illicit Drug Use The hazards of illicit drug use are the increase in infections, disease, and overdose Medical complications that are commonly seen among addicts are brain, skin, and lung abscesses; inflammation of the lining of the heart, hepatitis, and AIDS.

Signs of an Overdose Respiratory depression cold clammy skin Confusion
Convulsions Severe drowsiness Constricted pupils

Hallucinogens A class of drugs is hallucinogens; marijuana is the most well-known member of this class. Hallucinogens cause marked changes in normal thought processes, perceptions, and moods. Marijuana is the most controversial drug in this class because its long-term effects on health are still largely unknown.

Abused Drug Timetables Hallucinogens/Opiates
Drug/Commercial & Street Names Administered/Detection in Urine* Cannabinoids Marijuana Blunt, dope, ganja, grass, herb, joints, Mary Jane, pot, reefer, sinsemilla, skunk, weed Swallowed, Smoked 14 days - 11weeks

Hallucinogins Cannabinoids – Marijuana
The chemical substance largely responsible for the hallucinogenic properties of marijuana is known as tetrahydrocannabinol, or THC. The THC content of Cannabis varies in different parts of the plant, generally decreasing in the following sequence: resin, flowers, leaves, with little THC in the stem, roots, or seeds. The THC-rich resin is known as hashish. Marijuana does not cause physical dependency the risk of harm is in heavy, long-term use.

Hallucinogens Drug/Commercial & Street Names Administered/Detection in Urine* Hashish Boom, chronic, gangster, hash, hash oil, hemp Swallowed, Smoked 14 days - 11weeks

Other Hallucinogens Other hallucinogens include LSD, mescaline, PCP, psilocybin, & MDMA (Ecstasy). LSD is synthesized from lysergic acid, & can cause hallucinations that can last for 12 hours. Phencyclidine, or PCP, is often synthesized in clandestine laboratories & is often smoked, ingested, or sniffed. PCP is often mixed with other drugs, such as LSD, or amphetamine, & is sold as a powder (“angle dust”), capsule, or tablet. Oral intake of PCP first leads to feelings of strength and invulnerability, which may turn to depression, tendencies toward violence, & suicide

Opiates Morphine is readily extracted from opium and is used to synthesize heroin. Addicts frequently dissolve heroin in water by heating it in a spoon, and then inject in the skin. Heroin produces a “high” that is accompanied by drowsiness and a sense of well-being that generally last for three to four hours. Codeine is also present in opium, but it is usually prepared synthetically from morphine.

Opiates OxyContin, with the active ingredient oxycodone, is not derived from opium or morphine, but does have the same physiological effects on the body as do opium narcotics. OxyContin is prescribed to a million patients for treatment of chronic pain. Methadone is another well-known synthetic opiate. Methadone, which is pharmacologically related to heroin, appears to eliminate the addict’s desire for heroin while producing minimal side effects.

Abused Drugs Timetable Depressants/Barbituates
Drug/Commercial & Street Names Administered/Detection in Urine* Depressants Barbiturates Amytal, Nembutal, Seconal, Phenobarbital: barbs, reds, red birds, phennies, tooies, yellows, yellow jackests Injected, swallowed days

Depressants Depressants are another class of drugs.
Depressants are substances used to depress the functions of the central nervous system. Depressants calm irritability and anxiety and may induce sleep. These include alcohol (ethanol), barbiturates, tranquilizers, and various substances that can be sniffed, such as airplane glue, model cement, or aerosol gas propellants such as freon.

Depressants Alcohol (ethyl alcohol) enters the body’s bloodstream and quickly travels to the brain, where it acts to suppress the brain’s control of thought processes and muscle coordination. Barbiturates, or “downers,” are normally taken orally and create a feeling of well-being, relax the body, and produce sleep. Tranquilizers, unlike barbiturates, produce a relaxing tranquility without impairment of high-thinking faculties or inducing sleep. Sniffing has immediate effects such as exhilaration, but impairs judgment and may cause liver, heart, and brain damage, or even death.

Abused Drugs Timetable
Drug/Commercial & Street Names Administered/Detection in Urine* Benzodiazepine Anesthetics/PCP Ativan, Halcion, Librium, Phencyclidine: angel Valium, Xanax: candy, dust, downers, boat, hog, love boat, sleeping pills, peace tanks pill Injected, swallowed Injected, swallowed, smoked 1-6 weeks days

Drug/Commercial & Street Names Administered/Detection in Urine* Opioids & Morphine/Codeine Empirin with Codeine, Fiorinal with Codeine, Robitussin A-C, Tylenol with Codeine: Captain Cody, Cody, schoolboy, doors & fours, loads, pancakes & syrup Injected, swallowed days

Drug/Commercial & Street Names Administered/Detection in Urine* Fentanyl Actiq, Duragesic, Sublimaze: Apache, China girl, China white, dance fever, friend, goodfella, jackpot, murder 8, TNT, Tango and Cash Injected, smoked, snorted hours

Heroine (first synthesized In 1874) diacetylmorphine: brown sugar, dope, H, horse, junk, skag, skunk, smack, white horse Injected, smoked, snorted days Morphine Roxanol, Duramorph: M, Miss Emma, monkey, white stuff Injected, swallowed, smoked days

Heroin Usage In 1974 estimated users were 246,000.
Between the years there were an estimate of 28,000-80,000 new users Between 1995 and 2001 the number of new users was greater than 100,000 Today there has been a reported 3.7 million people in the US alone who reported using heroin at least once in their lifetime.

Opium Laudanum, Paregoric: big O, black stuff, block, gum, hop Swallowed, smoked days Oxycodone Oxycontine: Oxy, O.C., killer Swallowed, snorted, injected hours OXY Hydrocodone Bitartrate Vicodin: vike, Watson-387 Swallowed days

Oxycodone In tons of Oxycodone were manufactured for sale in the United States tons were manufactured The tablet is either taken orally or crushed and sniffed or dissolved in water and injected This drug is often stolen and prescriptions are often forged

Hydrocodone It is used as a cough suppressant and analgesic
DEA has this drug listed as the most frequently encountered opiate pharmaceutical that is submitted for drug evidence to federal, state and local forensic laboratories. Abusers obtain the is drug by theft, doctor shopping, fraudulent prescriptions, and fake call in prescriptions

Methadone Dolophine, Methadone Swallowed, Injected days Buprenorphine Subutex, Buprenex, Temgesic, Suboxone Swallowed, Injected days

Dextropropoxyphene Related to Morphine First marketed in 1957
Used to help moderate to mild pain There is 150 tons produced in the United States annually 25 million prescriptions have been written. This drug is among the top 10 drugs reported by the medical examiner in drug abuse deaths.

Stimulants: Amphetamine Biphetamine, Dexedrine: bennies, black beauties, crosses, hearts, LA turnaround, speed, truck driver, uppers Injected, swallowed, smoked, snorted 1-3 days Methamphetamine Desoxyn: chalk, crank, crystal, fire, glass, go fast, ice, meth, speed; Injected, swallowed, smoked, snorted days

Stimulants The drug classification of stimulants includes amphetamines, sometimes known as “uppers” or “speed,” and cocaine, which in its free-base form is known as crack. Stimulants are substances taken to increase alertness or activity, followed by a decrease in fatigue and a loss of appetite.

Stimulants Amphetamine and methamphetamine, often injected intravenously, causes an initial “rush,” followed by an intense feeling of pleasure. Followed by a period of exhaustion and a prolonged period of depression. Cocaine, extracted from the leaves of Erythroxylin coca, causes increased alertness and vigor, accompanied by the suppression of hunger, fatigue, and boredom. Crack is cocaine mixed with baking soda and water, then heated. Crack is often smoked in glass pipes, and, like cocaine, stimulates the brain’s pleasure center.

Cocaine Cocaine hydrochloride: blow, bump, C, candy, Charlie, coke, crack, flake, rock, snow, toot Injected, smoked, snorted days

Date Rape Drugs Substances that are often used as club drugs include, but are not limited to, MDMA (Ecstasy), GHB (gamma hydroxybutyrate), Rohypnol (“Roofies”), ketamine, and methamphetamine. GHB and Rohypnol are central nervous system depressants that are often connected with drug-facilitated sexual assault, rape, and robbery.

Date Rape Drugs Ecstasy Adam, clarity, ecstasy, lover's speed, peace, STP, X, XTC Swallowed Referred to as party drugs Are commonly found at bars, night clubs, raves and techno parties. Causes a comma in minutes Associated with sexual assaults

Date Rape Drugs Methylenedioxymethamphetamine, also known as MDMA or Ecstasy, is a synthetic mind-altering drug that exhibits many hallucinogenic and amphetamine-like effects. Ecstasy enhances self-awareness and decreases inhibitions; however, seizures, muscle breakdown, stroke, kidney failure, and cardiovascular system failure often accompany chronic abuse.

Date Rape Drugs Ketamine is primarily used as a veterinary animal anesthetic that in humans causes euphoria and hallucinations. Ketamine can also cause impaired motor functions, high blood pressure, amnesia, and mild respiratory depression.

Nicotine Cigarettes, cigars, smokeless tobacco, snuff, spit tobacco, bidis, chew Smoked, Snorted, taken in snuff & spit tobacco days Alcohol Alcohol Beer, Wine, Liquor Swallowed Hrs- 2 days *Detection time in urine is an average and can vary greatly. Detection time can vary due to length and amount of use. **These are tests that UriTox carries. Please be sure to select the appropriate test. Different panels may have more then one combination of tests.

Alcohol Related Deaths
Of the 40,000 auto accidents in the United States 50% are drunk drivers 60% of pedestrians killed have high blood alcohol levels

Toxicology of Alcohol The analysis of alcohol exemplifies the primary objective of forensic toxicology—the detection and isolation of drugs in the body for the purpose of determining their influence on human behavior. Alcohol, or ethyl alcohol, is a colorless liquid normally diluted with water and consumed as a beverage. Like any depressant, alcohol principally effects the central nervous system, particularly the brain.

Alcohol Levels Alcohol appears in the blood within minutes after it has been taken by mouth & slowly increases in concentration while it is being absorbed from the stomach & the small intestine into the bloodstream. When all alcohol has been absorbed, a max alcohol level is reached in the blood; and the post-absorption period begins. The alcohol concentration slowly decreases until a zero level is again reached.

Alcohol Levels Factors affecting outcome on individual consumption:
Time taken to consume the drink Alcohol content Amount consumed Food presence in the stomach The extent to which an individual may be under the influence of alcohol is usually determined by either measuring the quantity of alcohol present in the blood system or by measuring the alcohol content in the breath.

Alcohol Levels Elimination of alcohol throughout the body is accomplished through oxidation & excretion. Oxidation takes place almost entirely in the liver, while alcohol is excreted unchanged in the breath, urine, and perspiration. Experimental evidence has verified that the amount of alcohol exhaled in the breath is in direct proportion to the blood concentration.

Alcohol & the Circulatory System
Humans have a closed circulatory system consisting of a heart, arteries, veins, and capillaries. Alcohol is absorbed from the stomach and small intestines into the blood stream. Alcohol is carried to the liver where the process of its destruction starts. Blood, carrying alcohol, moves to the heart and is pumped to the lungs. In the lungs, carbon dioxide and alcohol leave the blood and oxygen enters the blood in the air sacs known as alveoli. Then the CO2 & alcohol are exhaled during breathing.

Alcohol Breath Testers
Breath testers that operate on the principle of IR light absorption are becoming increasingly popular within the law enforcement community. Many types of breath testers are designed to capture a set volume of breath. Captured breath is exposed to IR light. It’s the degree of the interaction of the light with alcohol in the captured breath sample that allows the instrument to measure a blood alc. conc. in breath.

Alcohol Field Testing Law enforcement officers typically use field sobriety tests to estimate a motorist’s degree of physical impairment by alcohol and whether or not an evidential test for alcohol is justified. The horizontal gaze nystagmus test, walk and turn, and the one-leg stand are all considered reliable and effective psychophysical tests. A portable, handheld, roadside breath tester may be used to determine a preliminary breath-alcohol content.

Alcohol and the Law The AMA. & the National Safety Council have been able to exert considerable influence in convincing the states to establish uniform & reasonable blood-alcohol standards. Between 1939 and 1964 a person having a blood-alcohol level in excess of 0.15 percent w/v was to be considered under the influence, which was lowered to 0.10 percent by 1965. In 1972 the impairment level was recommended to be lowered again to 0.08 percent w/v.

Alcohol and Law Starting in 2003, states adopted the 0.08 percent per se level. To prevent a person’s refusal to take a test for alcohol consumption, the National Highway Traffic Safety Administration recommended an “implied consent” law. Adopted by all states by 1973, this law states that the operation of a motor vehicle on a public highway automatically carries with it the stipulation that a driver will submit for a test for alcohol intoxication if requested or be subject to loss of the license.

Collection and Preservation of Blood for Alcohol Testing
Blood must always be drawn under medically accepted conditions by a qualified individual. It is important to apply a nonalcoholic disinfectant before the suspect’s skin is penetrated with a sterile needle or lancet. Once blood is removed from an individual, it is best preserved sealed in an airtight container after adding an anticoagulant and a preservative.

Field Testing Law enforcement officers typically use field sobriety tests to estimate a motorist’s degree of physical impairment by alcohol and whether or not an evidential test for alcohol is justified. The horizontal gaze nystagmus test, walk and turn, and the one-leg stand are all considered reliable and effective psychophysical tests. A portable, handheld, roadside breath tester may be used to determine a preliminary breath-alcohol content.

Anabolic Steroids These are synthetic compounds that are chemically related to the male sex hormone testosterone. Anabolic steroids are often abused by individuals who are interested in accelerating muscle growth. Side effects include unpredictable effects on mood and personality, depression, diminished sex drive, halting bone growth, and liver cancer.

Drug Control Laws The U.S. federal law known as the Controlled Substances Act will serve to illustrate a legal drug-classification system created to prevent and control drug abuse This federal law establishes five schedules of classification for controlled dangerous substances on the basis of a drug’s: potential for abuse potential for physical and psychological dependence medical value

Schedules of Classification
Schedule I drugs have a high potential for abuse and have no currently accepted medical use such as heroin, marijuana, methaqualone and LSD.

Schedule II drugs have a high potential for abuse and have medical use with severe restrictions such as cocaine, PCP, and most amphetamine and barbiturate prescriptions.

Schedule III drugs have less potential for abuse and a currently accepted medical use such as all barbiturate prescriptions not covered under Schedule II, codeine, and anabolic steroids.

Schedule of Classification
Schedule IV drugs have a low potential for abuse and have a current medical use such as darvon, phenobarbital, and some tranquilizers such as diazepam (valium) and chlordiazepoxide (librium). Schedule V drugs must show low abuse potential and have medical use such as opiate drug mixtures that contain nonnarcotic medicinal ingredients.

Collection & Preservation
Generally common sense is the best guide, keeping in mind that the package must prevent the loss of the contents and/or cross-contamination. Often the original container in which the drug was seized will suffice. All packages must be marked with information that is sufficient to ensure identification by the officer in the future and establish the chain of custody.

Collecting for the Lab Who does what?
First responder prepares specimen for lab Controlled Substance Analyst Poison Analyst Controlled Substances/Poisons are Forensic Toxicology Show Video

Role of the Toxicologist
The toxicologist is not dealing with drugs at the concentration levels found in powders and pills, having been dissipated and distributed throughout the body. The body is an active chemistry laboratory as few substances enter and completely leave the body in the same chemical state. Last, when and if the toxicologist has surmounted all of these obstacles, he or she must be prepared to assess the toxicity of the drug or poison.

Drug Identification The challenge or difficulty of forensic drug identification comes in selecting analytical procedures that will ensure a specific identification of a drug. This plan, or scheme of analysis, is divided into two phases. Screening test that is nonspecific and preliminary in nature to reduce the possibilities to a manageable number. Confirmation test that is a single test that specifically identifies a substance.

Screening Tests A screening test is normally employed to provide the analyst with quick insight into the likelihood that a specimen contains a drug substance. Positive results arising from a screening test are considered to be tentative at best and must be verified with a confirmation test. The most widely used screening tests are thin-layer chromatography, gas chromatography, and immunoassay.

Types of Toxicologist Tests Screening Tests
Physical Tests: boiling point, melting point, density, and refractive index Crystal Tests: treatment with a chemical reagent to produce crystals

Types of Toxicologist Tests Screening Tests
Chemical Spot Tests: treatment with a chemical reagent to produce color changes Chromatography (thin-layer or gas): used to separate components of a mixture

Confirmatory Tests Gas chromatography/mass spectrometry GC/MS is generally accepted as the confirmation test of choice. The GC separates the sample into its components, while the MS represents a unique “fingerprint” pattern that can be used for identification. Once the drug is extracted and identified, the toxicologist may be required to provide an opinion on the drug’s effect on an individual’s natural performance or physical state.

*Detection time in urine is an average and can vary greatly. Detection time can vary due to length and amount of use. Different testing panels may have more then one combination of tests. SPOT TEST Methamphetamine - Test Kit Marquis Reagent Sodium Nitro-prusside

The Analytical Scheme The forensic toxicologist must devise an analytical scheme that will successfully detect, isolate, and specifically identify toxic drug substances. Once the drug has been extracted from appropriate biological fluids, tissues, and organs, the forensic toxicologist can proceed to identify the drug substance present. Drug extraction is generally based on a large number of drugs being either acidic or basic. The strategy used for identifying abused drugs entails a two-step approach: screening and confirmation.

Analytical Instrumentation
Specific/ Non-specific: Stimulant vs. Depressant Exact ID of the drug Destructive/Non-destructive: Sample consumed, further testing Instrumentation: GC/MS FTIR Gas chromatography/mass spectroscopy Fourier Transform Infrared Spectrophotometer

Chromatography Chromatography is a means of separating and tentatively identifying the components of a mixture. The theory of chromatography is based on the observation that chemical substances have a tendency to partially escape into the surrounding environment when dissolved in a liquid or when absorbed on a solid surface. Those materials that have a preference for the moving phase will slowly pull ahead and separate from those substances that prefer to remain in the stationary phase.

TLC TLC uses a solid stationary phase usually coated onto a glass plate and a mobile liquid phase to separate the components of the mixture. The liquid will slowly rise up the plate by capillary action causing the sample to become distributed between the stationary phase and the moving liquid phase. Because most compounds are colorless, the materials must be visualized by placing the plates under ultraviolet light or spraying the plate with a chemical reagent. The distance a spot travels up a thin-layer plate can be assigned a numerical value known as the Rf value.

GC/MS Destructive, Specific
Chromatography: selective separation of components of a sample GC separates MS identifies and quantitates *Forensic Labs use GC methods to test for blood alcohols Stationary/Mobile Phases of chromatography

GC/MS Chromatography: selective separation of components of a sample
Stationary/Mobile Phases of chromatography

Gas Chromatography In GC: 2 phases The moving phase.
The stationary phase After a mixture has traversed the length of the column, it will emerge separated into its components. The written record of this separation is called a chromatogram. The time required for a component to go through a GC column is known as retention time.

Gas Chromatography Sample diluted in solvent
Injected into capillary column Heat vaporizes sample, travels through column Detector records time it takes to move through column (retention time) Data (chromatogram) compared to known substances Separates based on volatility

Carrier Gas: Helium Capillary: glass tube (silica)…stationary phase Operating conditions (temperatures of injector, column, and detector; carrier gas flow rate, and column stationary phase)

Gas Chromatography – Chromatograms Two drug spectrums

Theory of Light Light is described as a continuous wave.
When white light passes though a prism, it is dispersed into a continuous spectrum of colors. Waves are described in terms such as: Wavelength, the distance between two successive crests (or one trough to the next trough). Frequency, the number of crests (or troughs) passing any one given point per unit of time. The electromagnetic spectrum is the entire range of radiation energy from the most energetic cosmic rays to the least energetic radio waves. Visible light is only a small part of the electromagnetic spectrum.

Spectrophotometry Just as a substance can absorb visible light to produce color, many of the invisible radiations of the electromagnetic spectrum are likewise absorbed. Spectrophotometry, an important analytical tool, measures the quantity of radiation that a particular material absorbs as a function of wavelength and frequency.

UVand IR Spectrophotometry
Most forensic laboratories use UV and IR spectrophotometers to characterize chemical compounds. The simplicity of the UV spectrum facilitates its use as a tool for determining a material’s probable identity. May not provide a definitive result. The IR spectrum provides a far more complex pattern. Different materials always have distinctively different IR spectra; each IR spectrum is therefore equivalent to a “fingerprint” of that substance.

Mass Spectrometry In the mass spectrometer, a beam of high-energy electrons collide with a material, producing positively charged ions. These positive ions almost instantaneously decompose into numerous fragments, which are separated according to their masses. The unique feature of mass spectrometry is that under carefully controlled conditions, no two substances produce the same fragmentation pattern.

GC and Mass A direct connection between the GC column and the mass spectrometer allows each component to flow into the mass spectrometer as it emerges from the GC. The separation of a mixture’s components is first accomplished by the GC. Then, fragmentation of each component by high-energy electrons in the mass spectrometer, will produce a distinct pattern, somewhat like a “fingerprint”, of the substance being examined.

FTIR Non-destructive, specific
Visible light is made of electromagnetic waves, seen as colors…IR is in the spectrum, we just can’t see Works by measuring degree of absorption of IR waves of certain frequencies Works only with pure samples Percentage transmittance

FTIR - Printouts Cocaine Heroin

Drug Recognition Expert (DRE)
During the 1970s, the Los Angeles Police Department developed clinical and psychophysical examinations that a trained police officer could use to identify and differentiate between types of drug impairment. This program has evolved into a national program to train police as drug recognition experts. Normally, a three- to five-month training program is required to certify an officer as a drug recognition expert (DRE). The DRE program incorporates standardized methods for examining suspects to determine whether they have taken one or more drugs.

Toxicity Water can cause fatal electrolyte imbalances
Arsenic and cyanide are harmless in small doses Psychogenic polydipsia in certain forms of schizophrenia

Marsh Arsenic Test Developed by James Marsh in 1836
When Zn and acid react with arsenic, gas released that deposits metallic arsenic when burned Use of arsenic for poisoning so prevalent that nicknamed “inheritance powder”

Specimens for Analysis
Adipose Tissue Insecticides, Thiopental Bile Codeine, Morphine Blood Alcohols, CO Brain Volatile Poisons Kidney Heavy metal Liver Most Toxicants Lung Methadone, Gases, Inhalants Stomach/Intestine All toxicants taken orally Contents Urine Most Toxicants Vitreous Humor Digoxin, electrolytes, glucose

Detecting Drugs in Hair
Drugs present in blood diffuse through the capillary walls into the base of the hair and become permanently entrapped in the hair’s hardening protein structure. As the hair continues to grow, the drug’s location on the hair shaft becomes a historical marker for delineating drug intake. Given that the average human head hair grows at the rate of 1 centimeter per month, analyzing segments of hair for drug content may define the timeline for drug use.

Drug Identification The challenge or difficulty of forensic drug identification comes in selecting analytical procedures that will ensure a specific identification of a drug. This plan, or scheme of analysis, is divided into two phases. Screening test that is nonspecific and preliminary in nature to reduce the possibilities to a manageable number. Confirmation test that is a single test that specifically identifies a substance.

Preliminary Analysis The unknown substance may be any one of a thousand or more commonly encountered drugs, the analyst employs screening tests to reduce these possibilities to a small and manageable number. This objective is often accomplished by subjecting the material to a series of color tests that will produce characteristic colors for commonly encountered illicit drugs. Microcrystalline tests can also be used to identify specific drug substances by studying the size and shape of crystals formed

Conformational Determination
Once this preliminary analysis is completed, a conformational determination is pursued. Forensic chemists will employ a specific test to identify a drug substance to the exclusion of all other known chemical substances. Typically infrared spectrophotometry or gas chromatography-mass spectrometry is used to specifically identify a drug substance.

Qualitative vs. Quantitative
Another consideration in selecting an analytical technique is the need for either a qualitative or a quantitative determination. The former relates just to the identity of the material, whereas the latter requires the determination of the percent composition of the components of a mixture.

The Screening Step A screening test is normally employed to provide the analyst with quick insight into the likelihood that a specimen contains a drug substance. Positive results arising from a screening test are considered to be tentative at best and must be verified with a confirmation test. The most widely used screening tests are thin-layer chromatography, gas chromatography, and immunoassay.

The Confirmation Step Gas chromatography/mass spectrometry is generally accepted as the confirmation test of choice. The GC separates the sample into its components, while the MS represents a unique “fingerprint” pattern that can be used for identification. Once the drug is extracted and identified, the toxicologist may be required to provide an opinion on the drug’s effect on an individual’s natural performance or physical state.

Nondrug Poisons Heavy metals such as arsenic, bismuth, antimony, mercury, and thallium are only occasionally encountered because severe environmental protection regulations restrict their availability to the general public. Carbon monoxide is one of the most common poisons encountered in a forensic laboratory. To measure the concentration of carbon monoxide in the blood spectrophotometric methods determine the amount of carboxyhemoglobin relative to oxyhemoglobin or total hemoglobin; or a volume of blood can be treated with a reagent to liberate the carbon monoxide, which is then measured by gas chromatography.

Significance of Findings
Once a drug is found and identified, the toxicologist determines its influence on the behavior of the individual. For many drugs, blood concentration levels are readily known and are used to estimate the pharmacological effects of the drug on the individual. When dealing with a living person, the toxicologist has the added benefit of knowing what a police officer may have observed about an individual’s behavior and motor skills.

ELISA Template – Confirmatory testing
Uses antibodies and enzymes to bind to drug of interest Qualitative, not quantitative Specific Enzyme linked immunosorbent assay Antibody: immunologic molecule Enzyme: protein that catalyzes a reaction

Well Plates – basic tests
Come pre-coated with specific drug antigens Add sample Add conjugate, incubate Antibody-enzyme complex that binds primary antibody For drug testing, drugs will occupy that spot so the conjugate cannot bind Wash plate, add substrate (produces color change if bound to conjugate) Add stop, stops reaction from continuing, read plate with UV-Vis

Testing Sample Sources
What types of samples do forensic toxicologist take for analysis? Urine Blood Hair Maggots Bacteria Other Anthrax body fluids

Results: A clear well indicates the presence of the drug (The drug bound to the plate, prevented conjugate from binding so substrate couldn’t bind and produce a color change) A yellow well indicates the absence of the drug (conjugate bound to plate, substrate bound and produced color change) Negative Calibrators, +/- Controls, and Cutoff Calibrator

How many test + on this plate?

Video Segment: Love Thy Neighbor
Dr. Michael Baden Board-certified, Forensic Pathologist George Trepal Love Thy Neighbor In 1988, 42-year-old Peggy Carr, a two-time divorcee on her third marriage, was hospitalized in Florida & eventually diagnosed with Thallium poisoning; she later slipped into a coma and died. The woman's two sons also became gravely ill from Thallium poisoning. In scouring the family's home, police found several empty Coke bottles that tested positive for Thallium.

Forensic Chemistry and Toxicology

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Forensic Chemistry and Toxicology

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