Analytical Toxicology Fall 2007

Analytical Toxicology Fall 2007
Forensic Toxicology Analytical Toxicology Fall 2007

Forensic Toxicology Toxicology is defined as the study of the adverse effects of chemicals on living organisms. Forensic toxicology is defined as the application of toxicology for the purposes of the law.

Forensic Toxicology Postmortem forensic toxicology.
Human performance toxicology. Forensic drug testing.

History Ancient Egyptians and Grecians reported poisonings due to herbs, plants and food. Opium, arsenic and hydrocyanic acid were used throughout Europe during the middle ages. Although the study of the science of toxic substances and poisons began in the early 1800’s, knowledge of poisons and poisonings has been around for thousands of years. Greeks used hemlock as a means of state sponsored execution. Socrates was the most famous poisoning case by this method.

History Philippus Theophrastus Aureolus Bombastus von Hohenheim (or Paracelsus) observed that any substance could be a poison, depending on its dose “ What is there that is not poison? All things are poison and nothing without poison. Solely the dose determines that a thing is not a poison” Paracelsus ( ), more properly Phillippus Theophrastus Aureolus Bombastus von Hohenheim, was born in Einsiedeln, Switzerland in 1493, one year after Columbus' first voyage to the New World. He was a contemporary of Copernicus, Martin Luther, Leonardo da Vinci and a host of other figures we associate with the shattering of medieval thought and the birth of the modern world.

History In 1814, M.J.B. Orfila, the chairman of the legal medicine department at the Sorbonne in France, published a book entitled Traite des poisons ou Toxicologie Generale. This was the first attempt to systematically study and classify poisons. Six classes of poisons were established, based mainly on their toxic effects. He also isolated arsenic from a variety of postmortem specimens and was the first to articulate the fact that poisons must be absorbed, or enter the blood, to manifest their toxic effects.

History In 1851, Stas developed the first effective method for extracting alkaloids from biological specimens. This was modified several years later by Otto, which enabled the isolation of purer alkaloid substances. This became known as the Stas-Otto method and remains the basis for drug extraction to this day.

History In the U.S., forensic toxicology did not develop until the early 20th century. Dr. Alexander Gettler is considered this country’s first forensic toxicologist. In the U.S., forensic toxicology did not develop until the early 20th century, with the change of the coroner system in New York to a medical examiner system. The first forensic toxicologist was Dr. Alexander Gettler, who directed the laboratory for 41 years and trained the first generation of forensic toxicologists in the country.

Postmortem Forensic Toxicology
Suspected drug intoxication cases Homicides Arson fire deaths Motor vehicle fatalities Deaths due to natural causes Drug intoxications are not readily diagnosed at autopsy. In intravenous drug deaths, there may be a recent injection site observable; oral intoxications may be inferred by a large amount of unabsorbed tablet fragments in the stomach contents. Otherwise, the only other anatomic findings are pulmonary congestion and edema. Scene investigation may indicate the causative agent or agents in some instances. Nevertheless, the function of the toxicology laboratory is to identify the substances present in the biologic specimens. Once these substances are identified, they are quantitated in appropriate specimens to determine whether these drugs caused or contributed to death.

Death Investigations Coroner Medical Examiner A coroner may be elected by the people or appointed by a governmental authority. A medical examiner is appointed, usually by the health department. A coroner is not required to have any particular training or experience in medicine. Conversely, a medical examiner must be a physician, usually a pathologist, with specific training in forensic medicine.

Specimens Blood – from the heart and from the femoral or jugular veins Vitreous humor Urine Bile Liver Other – lung, spleen, stomach contents or brain Approximately mL of blood should be collected. Blood from subdural or epidural clots should also be collected. These specimens could be useful when there is some period between an event and death. Vitreous humor displays good stability and resides in an anatomically isolated area. Therefore it is more resistant to putrefactive changes than are other specimens.

Analytical Process Separation Identification Confirmation Quantitation Separation: The initial step in the process for postmortem specimens is analyte separation. Except for some drug classes that can be analyzed directly in urine specimens, the analytes of interest usually require separation from the biological matrix. For example, volatile substances can be separated from an aqueous matrix by heating the specimen in a sealed container at 60‑80oC. The gaseous phase above the matrix layer will contain volatile substances which can be sampled and analyzed.

Analytes Volatiles (Carbon Monoxide, Cyanide, and Alcohols) Drugs Metals Carbon monoxide (CO) is produced by the incomplete combustion of organic material. CO is the causative agent in many fire deaths. In general fire deaths can be explained by thermal injuries, smoke and soot inhalation or a combination of the two. CO binds with great affinity to hemoglobin (Hb) forming carboxyhemoglobin (CO-Hb) and reducing the blood's oxygen carrying capabilities. The unit of measurement of CO is "percent CO saturation" and is defined as the percent of total Hb which is CO-Hb.

EHTANOL (1.787 min.) 2-PROPANOL (2.804 min.) ACETALDEHYDE (1.414 min.)
MEK(ISTD) (5.584 min.) ACETONE (2.462 min.) 6

Drugs One Comprehensive Approach: The most involved aspect of postmortem forensic toxicology is the analysis of drugs. One difference between the postmortem laboratory from other forensic toxicology laboratories is the need to have methodolgies for therapeutic and abused drugs. The methods should be established for identification, confirmation and quantitation of both types of drugs.

Metals Aluminum Arsenic Iron Mercury Lead Thallium

Metals (continued) Analysis Colorimetric Graphite Furnace Atomic Absorption Spectrometry Inductively Coupled Plasma – Mass Spectrometry Neutron Activation Spectrometry The uncomplicated Reinsch Test methodology was developed by the German chemist Reinsch and is still used today in forensic toxicology It is a qualitative screening test for an overdose of arsenic, antimony, bismuth, or mercury. This method lacks sensitivity but can usually determine an overdose. A positive test requires confirmation of the metal in question. Arsenic can be quantitatively determined by colorimetry using the Gutzeit method.

Human Performance Toxicology
Human performance toxicology is also referred to as behavioral toxicology. It is the study of human performance under the influence of drugs. This branch of forensic toxicology is concerned with the relationship between the presence of a drug and associated behavioral changes. It is generally accepted that there is a dose-effect relationship between drugs that elicit behavioral changes and those changes; elucidation and quantification of such a relationship is a significant role of the behavioral toxicologist.

Ethanol and driving History Behavioral effects Specimens

Ethanol Toxicology Types of alcohol Ethanol (ethyl alcohol)
Methanol (methyl alcohol) Isopropanol Ethylene glycol (NHTSA) - alcohol was involved in 41% of fatal automobile crashes and 7% of all crashes in Deaths due to the physiological effects of chronic alcoholism are frequently seen in coroners’ and medical examiners’ cases. The loss of productivity due to alcohol abuse impacts both large and small businesses. Ethanol is the most commonly encountered toxic substance in forensic toxicology. Methanol, or wood alcohol, is a commonly used solvent and reagent in the chemical industry

Ethanol Toxicology Ethanol production Fermentation of sugar or starch
Can only achieve 20% ethanol Distillation Distilled alcoholic beverages are usually 40 to 50% ethanol by volume ( proof) Fermentation stops at higher % of ethanol (above 20%). Since ethanol boils at a lower temperature than water, ethanol can be separated from an aqueous matrix by heating to a temperature above the boiling point of ethanol, but below the boiling point of water and collecting the ethanol vapors. In addition to ethanol and water, alcoholic beverages contain other substances broadly classified as congeners. Congeners may be other volatile alcohols, as well as aldehydes and ketones obtained from the fermented product. Other congeners may also be extracted from the containers in which the distillate is aged. Congeners account for the different taste, smell and color of different alcoholic beverages.

Ethanol Pharmacokinetics
Absorption Means of absorption Dermal Inhalation IV Oral Gastrointestinal tract Presence of food. Absorption is mostly by diffusion in the gastrointestinal tract (75% through the small intestines) The presence of food has two major effects on the blood ethanol concentration versus time curve. The peak ethanol concentration occurs earlier on an empty stomach as opposed to a full stomach. In addition, the magnitude of the peak ethanol concentration is higher on an empty stomach than on a full stomach. Food competes with ethanol for absorption sites in the small intestine, thus delaying absorption of ethanol. The elimination of ethanol is independent of ethanol concentration such that a greater proportion of the absorbed ethanol is being eliminated when food is present. When a single dose of ethanol is consumed on an empty stomach, the peak ethanol concentration will be reached within one hour. 30 minutes after last drink.

Distribution Gastrointestinal tract Portal vein Liver Heart Lung Body

Elimination 5-10% in the urine Saliva, expired air and sweat Liver (enzymatic oxidation to acetaldehyde, acetic acid and carbon dioxide) Greater than 90% of the ethanol is eliminated by the liver. The average rate of elimination is 15 mg/dL per hour for men and 18 mg/dl per hour for women. Alcoholics have a greater elimination than social drinkers or non-drinkers. Athletic individuals who do not drink have the slowest ethanol elimination rates.

Ethanol Effects on the Body
Cardiovascular system Central nervous system Gastrointestinal tract After moderate ethanol consumption, there is no significant effect on blood pressure, cardiac output and cardiac contractile force CNS Depressant. At high enough levels the respiratory system center in the brain may become depressed sufficiently to cause coma and death. Consumption of alcohol in combination with meals stimulates the production of gastric juices rich in acid. Concentrated ethanol in the stomach can irritate mucosal membranes which may lead to gastritis.

Ethanol Effects on the Body
Kidney Liver In kidney, alcohol acts a a diuretic. Acute alcohol ingestion has little effect on hepatic function. Long term, regular consumption of alcohol will damage the liver: 1st - accumulation of fat in liver 2nd - formation of acetaldehyde, which has a toxic effect on the liver. Continued chronic abuse of alcohol will lead to liver cirrhosis, liver failure and death.

Breath Ethanol Testing
Theory Henry’s law Ethanol in breath Vs ethanol in blood 2100 to 1 ratio 2300 to 1 ratio The distribution of ethanol between blood and alveolar air obeys Henry’s law, which states that at a given temperature, there is a direct relationship between the amount of a volatile substance (ethanol) dissolved in a liquid (blood) and the amount of the substance in the vapor (alveolar air) above the solution. Most breathalyzers are calibrated so that the reading of the breathalyzer (g/2100 mL) is equivalent to amount of ethanol in blood. Actual value is 2300 to 1, so breathalyzer levels are underestimating the amount of blood alcohol concentrations.

Types of analyzers Chemical Reaction of ethanol with potassium dichromate/sulfuric acid solution Colored solution that results is measured spectrophotometrically IR spectrophotometry Electrochemical oxidation - fuel cell

IR Spectrophotometry Based on absorbance of light by the ethanol molecule Mainstay in evidential breath testing devices Electrochemical Oxidation Oxidation of ethanol to acetic acid Also used in evidential breath testing

Blood Ethanol Testing Chemical Screening Quantitative
Disadvantage - aldehydes and ketones will interfere with the test Potassium dichromate/sulfuric acid. Oxidation reaction with a color change from yellow (dichromate ion) to green (chromic ion)

Blood Ethanol Testing Enzymatic
Conversion of NAD to NADH by ethanol (serum, urine and whole blood) Measured spectrophotometrically at nm Same reaction with a blue dye (thiazoyl blue) (serum, urine, fresh blood and postmortem blood) Measured with a fluorometer

Blood Ethanol Testing Gas Chromatography
ACETALDEHYDE (1.414 min.) EHTANOL (1.787 min.) 2-PROPANOL (2.804 min.) MEK(ISTD) (5.584 min.) ACETONE (2.462 min.) 6 Blood Ethanol Testing Gas Chromatography Can measure ethanol in a wide range of specimens Can distinguish ethanol from other alcohols, aldehydes and ketones Two common methods Head space Direct injection

Assessment of Ethanol Impairment
In a British study: Detectable deterioration of drivers at between 30 – 50 mg/dL Obvious deterioration observed at between 60 – 100 mg/dL In another British study: Pilots exhibited impairment at 40 mg/dL

Blood alcohol concentration: 10-50 mg/dL: Impairment detectable by special tests mg/dL: Beginning of sensory-motor impairment mg/dL: Sensory-motor incoordination; impaired balance mg/dL: Increased muscular incoordination; apathy; lethargy

Blood alcohol concentration: mg/dL: Impaired consciousness; sleep; stupor mg/dL: Complete unconsciousness; coma 450 and greater mg/dL: Death from respiratory arrest

Drugs and driving DEC program Drug recognition expert Toxicologist Prosecution Throughout this century numerous studies have been performed enabling the forensic toxicologist to correlate blood ethanol concentration with behavior. At a given blood ethanol concentration, certain behaviors or a range of behavior can be expected without direct observation of an individual providing supporting evidence of impairment. These studies have permitted legislative bodies and regulatory agencies to enact laws and regulations prohibiting individuals from performing certain activities at or above a specified blood ethanol concentration (e.g., DUI/DWI laws). Unfortunately, an analogous situation does not exist with other drugs of abuse.

Drug Recognition Evaluation - 12 Step Process Breath alcohol test Interview of the arresting officer. Preliminary examination of the suspect. Examination of the eyes. Divided attention psychophysical tests. Vital signs examination. The breath alcohol test is used to determine if the observed impairment is a result of the consumption of alcohol and if the degree of impairment is consistent with the level of alcohol. A low or negative breath alcohol result may be the first indication the DRE has that other impairing drugs are present. The interview of the arresting officer allows the DRE to develop a fuller understanding of the suspect and to gain the advantage of important information the suspect may have revealed to the officer at the scene early in the arrest process.

Drug Recognition Evaluation (continued) Dark room examination. Examination of muscle tone. Examination for injection sites. Suspect’s statements and other observations. Opinion of the evaluator. Toxicological examination.

Drug Recognition Evaluation

Drug Recognition Evaluation (continued) Toxicology Type of Testing Specimens Blood and/or urine specimens collected at the culmination of the DRE evaluation are submitted to a toxicology laboratory for evaluation. The opinion of the DRE concerning the suspect’s state of impairment and the category of drug responsible for that impairment is a subjective evaluation. The determination of the presence of an impairing drug in the suspect’s specimens by the toxicology laboratory provides objective scientific support of the DRE’s opinion.

Drug Recognition Evaluation (continued) Drug Class Effects Central Nervous System Depressants Central Nervous System Stimulants Hallucinogens Phencyclidine Narcotic Analgesics Inhalants Cannabis The DRE is trained to observe behavior and collect physiologic data in an effort to determine if observed impairment is secondary to drug effects. As a consequence of numerous evaluations, the DRE begins to look for a combination of behaviors and physiological indicators that may suggest that an individual is under the influence of a particular drug or one of the members of a particular class of drugs. Each of the seven categories of drugs is associated with a set of observable and measurable signs. It is important to note that the drug categories employed in the DEC program do not directly correspond to traditional drug classes. The drugs that fall into each category are placed there on the basis of the signs they generate during the various examinations of the DEC process and not on their pharmacological properties.

Forensic Drug Testing Introduction History Rationale Military
Criminal justice system Public sector Private sector Rationale Also called forensic drug urinalysis

Forensic Drug Testing Uses in the workplace: Pre-employment screening
Post-accident testing Return to Work testing “For Cause” testing Random testing

Forensic Drug Testing Military Experience

Forensic Drug Testing President Ronald Reagan 1986
Executive Order No Objective: To develop a “drug-free” workplace.

Mandatory Guidelines for Federal Workplace Drug Testing Programs
Forensic Drug Testing Mandatory Guidelines for Federal Workplace Drug Testing Programs

Forensic Drug Testing Mandatory Guidelines Laboratory Accreditation
Specified Menu & Cutoffs Proficiency Testing Inspections External Blind Controls Corrective Actions

Forensic Drug Testing Programs:
Federal Dept. of Health & Human Services (DHHS) National Institute on Drug Abuse (NIDA) Substance Abuse & Mental Health Services Administration (SAMHSA)

Forensic Drug Testing Programs: Department of Transportation (DOT)
Nuclear Regulatory Commission (NRC) College of American Pathologists (CAP) Forensic Urine Drug Testing (FUDT)

Forensic Drug Testing Specimen Collection
External Custody & Control Form Unobserved/Observed Collection Toilet Water (Off or Blue) Sink Water (Off or Cold) No purses or bags No large coats Minimum Volume Check Temperature Split specimen Specimen Bottle Seal

Standard Operating Procedures SOP
Forensic Drug Testing Standard Operating Procedures SOP

Forensic Drug Testing Test Menus – SAMHSA Screen for Drugs:
Amphetamines Cannabinoids Cocaine Metabolite Opiates Phencyclidine (Alcohol)

Forensic Drug Testing Test Menus – SAMHSA Drugs Confirmed:
Amphetamine, Methamphetamine Delta-9-Tetrahydrocannabinol Carboxylic Acid (THCA) Benzoylecgonine Codeine, Morphine, 6-Acetylmorphine Phencyclidine (Alcohol)

Pyridine, Glutaraldehyde, Bleach, Soap, Chromium
Forensic Drug Testing Test Menus – SAMHSA Specimen Validity: Temperature pH Creatinine Specific Gravity Nitrite Pyridine, Glutaraldehyde, Bleach, Soap, Chromium

Forensic Drug Testing Test Menus – CAP & non-DOT Drugs: “NIDA-5” plus
Barbiturates, benzodiazepines, methadone, propoxyphene MDA, MDMA Others?

Test Menus – CAP Validity: Same as SAMHSA
Forensic Drug Testing Test Menus – CAP Validity: Same as SAMHSA

Forensic Drug Testing Test Procedures SAMHSA / DOT
Screening: Immunoassay (Emit, RIA, FPIA, CEDIA) Confirmation: GC/MS Alcohol: Alcohol Screening Device Evidential Breath Tester GC

Forensic Drug Testing Test Procedures CAP & Non-DOT
Screening: Immunoassay (Emit, RIA, FPIA, CEDIA) Automated Clinical Analyzer (EtOH) Alcohol: Alcohol Screening Device Confirmation: GC, GC/MS Alcohol: Evidential Breath Tester GC

Forensic Drug Testing Cutoff Concentration
That concentration below which the analytical result must be reported as “Negative” A cutoff concentration is specified for screening analysis and for confirmation analysis for each analyte.

Cutoff Concentrations (ng/mL)
Forensic Drug Testing Cutoff Concentrations (ng/mL) Phencyclidine Screening 25 Confirmation 25

Forensic Drug Testing Cutoff Concentrations (ng/mL) Screening Cocaine metabolites 300 Confirmation Benzoylecgonine 150

Forensic Drug Testing Cutoff Concentrations (ng/mL) Screening
Marijuana metabolites 50 Confirmation Delta-9-THCA 15

Forensic Drug Testing Cutoff Concentrations (ng/mL) Screening Amphetamines Confirmation Amphetamine 500 Methamphetamine (and Amphetamine 200)

Forensic Drug Testing Cutoff Concentrations (ng/mL) Screening Opiates Confirmation Codeine Morphine (Test for 6-AM 10)

Security of Laboratory Facility
Forensic Drug Testing Security of Laboratory Facility

(Specimen and aliquots)
Forensic Drug Testing Chain of Custody External Custody and Control Form Internal Chain of Custody Forms (Specimen and aliquots)

Forensic Drug Testing Specimen Storage
Refrigerated while analyses in process; Discarded if negative; Frozen ( -20 o C) > 1 year if positive.

Validation of: Methods Instruments Reagents
Forensic Drug Testing Validation of: Methods Instruments Reagents

“Negative” if either screening or confirmation gives negative result.
Forensic Drug Testing Reporting Results “Negative” if either screening or confirmation gives negative result. “Positive” only if positive result for both screening and confirmation. “Test Not Performed”

Forensic Drug Testing Reporting Results Rejected for Testing
(Give Remark) Fatal Flaw Uncorrected Flaw Specimen Unsuitable Specimen Adulterated Specimen Substituted

Forensic Drug Testing Reporting Results
If specimen donor requests, “split” specimen may be sent to another certified laboratory to retest for that substance reported positive.

Forensic Drug Testing Quality Control / Quality Assurance
Screening Quality Control Confirmation Quality Control Proficiency Testing

Forensic Drug Testing Screening Quality Control Calibrator / Cutoff
Controls: Open - Blank, Negative, Positive, Blinds (external and internal)

Forensic Drug Testing Confirmation Quality Control Calibrators:
Single vs. Multiple point Controls: Open - Blank, Negative, Positive, Blinds (external and internal) Carryover

Samples submitted by an outside agency.
Forensic Drug Testing Proficiency Tests Samples submitted by an outside agency. May be negative; May be positive.

Forensic Drug Testing Proficiency Tests No false positives
Identify 90% of drug challenges Quaititate 80% within +/- 20% or +/- 2 SD of target value.

Depends on size of laboratory
Forensic Drug Testing Inspections Semi-Annual Number of Inspectors & Number of Days Depends on size of laboratory Review SOP, talk to staff, watch sample processing, look at lots of case records, personnel records, proficiency test records, security records, instrument logs, etc., etc., etc.

Forensic Drug Testing Personnel New Titles: Responsible Person (RP)
Certifying Scientist Medical Review Officer (MRO) Collector Accessioner Records Custodian

Analytical Toxicology Fall 2007

Similar presentations

Presentation on theme: "Analytical Toxicology Fall 2007"— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

Analytical Toxicology Fall 2007

Similar presentations

Presentation on theme: "Analytical Toxicology Fall 2007"— Presentation transcript:

Similar presentations

About project

Feedback