Chapter 2 Atoms and Elements

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Girard Chapter 12
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What is Toxicology?

• Toxicological examinations involve the
  identification and often quantitation of drugs
  toxic materials in the human body
• The toxicologist examines body fluids and/or
  organs for the presence of drugs and poisons.
• The role of the forensic toxicologist is limited
  to matters pertaining to violations of criminal
  law
• determination of the amount of alcohol
• identification of substances causing unnatural
  death

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Role of the Toxicologist

• Postmorteum Analysis
• Postmortem Drug Testing
• Poisonings
• Human Performance Toxicology
• Blood Alcohol Testing
• Workplace Drug Testing
• Drug Abuse

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Questions Addressed by Toxicology

• Is a drug or poison present? What is it?
• How much of the substance is present? Is its
  concentration in the body sufficient to cause
  death?
• How was the drug/poison administered? Where did
  it enter the body?
• How long ago was it administered?

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Postmortem Forensic Toxicology

• 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, or organs, the
  forensic toxicologist can proceed to identify the
  drug present.
• Drug extraction is generally based on a large
  number of drugs being either acidic or basic.

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Postmortem Forensic Toxicology

• Collect sample of all body fluids
• Collect samples from organs and tissues
• A forensic toxicologist must also understand how
  the body processes these molecules
• Toxicological analysis must start as soon as
  possible after a persons death

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Samples Collected at Autopsy

• Specimen collection--fluids
• Blood All available up to 100 mL
• Urine 100 ml (no preservative)
• Bile All available
• Vitreous humor All available
• Gastric contents 50 grams
• Specimen collection--soft tissue
• Liver 100 grams
• Brain 100-200 grams
• Kidney 50 grams
• Lung 50 grams
• Spleen 50 grams

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A Short History of Poisons

• 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.
• 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.

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General Classes of Poisons

• Gases (HCN, CO)
• Metallic Poisons (ions of As, Cd, Pb, Hg, Tl, Be)
• Non-metallic poisons (NaCN, NaF, strong acids and
  bases)
• Non-volatile Organics
• Alkaloids (strychnine, atropine)
• Pesticides (organophosphates)
• Neurotoxins (snake or other venom)

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Carbon Monoxide (CO)

• Normal level 1-3 (up to 10 in smokers)
• Fatal level at autopsy considered to be 50 for
  a healthy middle-aged male
• CO prevents O2 binding to Hemoglobin in blood and
  this leads to suffocation
• CO victims have cherry pink color since
  hemoglobin with CO is darker red than normal

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Alkaloids

• Nitrogen containing organic bases
• Found in Plants and fungi
• Atropine (deadly nightshade)
• Strychnine
• Death from muscle over-stimulation respiratory
  failure
• Spasms and convulsions

Strychnine
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Metal Poisons

• Symptoms
• Vomiting
• Diarrhea
• Death can occur within 24 hrs or it can be given
  over long period of time with a cumulative
  effect.
• Analysis techniques for metal poisons
• Colorimetric/Spectrophotometric
• Atomic Absorption Spectrophotometry
• Inductively Coupled Plasma Mass Spectrometry
• Neutron Activation Spectrometry

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Toxicological Analysis

• Without supportive evidence, such as the victims
  symptoms, a postmortem pathological examination,
  or an examination of the victims personal
  effects, the toxicologist is forced to use
  general screening procedures with the hope of
  narrowing thousands of possibilities to one.
• Furthermore, the body is an active chemistry
  laboratory as few substances enter and completely
  leave the body in the same chemical state.

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Analysis of Toxins

• Separation by chromatography or extraction
• Identification of drug classes (screening)
• Confirmation
• Quantitation

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Extraction of Toxins

• Gases analyze for blood gases or test saliva
• Metal and non-metal ions grind and extract
  tissues with water
• Drugs are either acidic or basic
• Acidic drugs (barbiturates) can be extracted with
  acidic buffer solutions and organic solvents
• Basic drugs (alkaloids) can be extracted with
  basic buffer solutions and organic solvents

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The Tox Screen

• 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.

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The Tox Screen
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Screening Techniques

• Color tests
• Immunoassay
• Chromatography
• a. thin-layer chromatography (TLC)
• b. gas chromatography (GC)
• c. high performance liquid chromatography (HPLC)
• Spectroscopy
• a. UV light absorption
• b. infrared

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Immunoassay

• Most drugs dont react with antibodies. However
  by linking the drug to a protein, this can be
  injected into an animal that will then produce
  antibodies to the drug-protein complex.
• The antibodies are harvested and will react with
  different classes of drugs.
• These assays can be done in a competitive or
  non-competitive manner.
• Non-competitive there is a direct drug-antibody
  reaction that produces a color change.

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Competitive Immunoassay

• Works by adding a urine or blood plasma sample to
  the antibody mixture.
• To the immunoassay mixture is added a
  drug-protein antigen that will immediately react
  with the antibodies to change color.
• When there is high enough concentration of a drug
  in the urine so that it ties up the antibodies,
  then they dont react with the drug-protein
  antigen and they dont turn color, and the test
  is positive.

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Confirmation/Quantization Step

• Gas chromatography/mass spectrometry is generally
  accepted as the confirmation test of choice.
• Once the drug is extracted, identified and
  confirmed, the toxicologist may be required to
  provide an opinion on the drugs effect on an
  individuals physical state.
• To do this the quantity of the drug must be
  known. Quantitative organic or inorganic analysis
  is used to determine the exact concentration
  (GC-MS, visible or IR spectrophotometry, etc.)

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Example Analysis of Cannabis

• Cannabis contains the active chemical
  Tetrahydrocannabinol (THC) and creates 2 active
  metabolites.
• Can be detected in blood plasma for at least 72
  before being complete broken down to metabolites.
• Radioimmunoassay is first used to determine if
  THC is present.

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Analysis of Cannabis

• Extraction is then used since the acidic THC
  phenol groups allows them to be removes from a
  hexane solution by reacting them with aqueous
  base.
• GC/MS via selective ion monitoring with a
  standard THC-2H3 added to the sample allows
  calibration curves to be drawn and concentration
  to be determined

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Toxicology of Alcohol
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Human Performance Testing

• Most common human performance tests are those to
  determine if someone is driving a car under the
  influence of alcohol or drugs
• Amount of alcohol in a persons body is expressed
  as blood alcohol concentration (BAC)
• Studies have shown a direct relationship between
  increased BAC and increased risk of accidents

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Alcohol Morbidity Statistics

• half of traffic injuries involve alcohol
• 1/3 of fatally injured passengers pedestrians
  have elevated blood alcohol levels
• half of homicides involve alcohol
• 1/2 to 1/3 of suicides involve alcohol
• CDC estimates 30,000 unintentional injury deaths
  are directly attributable to alcohol

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Pharmacology of Alcohol

• Alcohol, or ethyl alcohol, is a colorless liquid
  completely miscible with water and consumed as a
  beverage.
• Like any depressant, alcohol principally effects
  the central nervous system, particularly the
  brain.
• The degree to which the CNS function is impaired
  is directly proportional to the concentration of
  alcohol in the blood and brain.

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Alcohol and the Law

• In 1939 the first drunk driver law was passed
  using BAC (blood alcohol concentration) to
  determine sobriety.
• 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 1964 the use of the Breathalyzer to determine
  BAC was validated.
• In 1972 the impairment level was recommended to
  be lowered again to 0.08 percent w/v.

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Alcohol Driving
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Alcohol and the Law

• In 1973, to prevent a persons refusal to take a
  test for alcohol consumption, the National
  Highway Traffic Safety Administration recommended
  an implied consent law.
• 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.
• Starting in 2003, states that have not adopted
  the 0.08 percent level will lose part of their
  federal funds for highway construction.

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Alcohol Driving
BAC levels that negatively affect driving
behaviors
Relative Fatality Risk (drivers in a single
vehicle crash)
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Alcohol and the Law

• Needed for Prosecution of DUI
• Probable cause (to aid in determining impairment
  if BAC is 0.05 but
• Field Sobriety Test
• Breathalyzer results and calibrations
• Medical records if hospital blood draw is to be
  used
• Toxicology report

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Stages of Alcohol Intoxication
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Blood Alcohol Concentration

• Alcohol appears in the blood within minutes after
  it is ingested and slowly increases in
  concentration while it is being absorbed from the
  stomach and the small intestine.
• When all the alcohol has been absorbed, a maximum
  alcohol level is reached in the blood and the
  post absorption period begins. Then the alcohol
  concentration slowly decreases until a zero level
  is again reached.
• Factors such as time taken to consume the drink,
  the alcohol content, the amount consumed, and
  food present in the stomach determine the rate at
  which alcohol is absorbed.

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Blood Alcohol Concentration

• This shows the time of alcohol absorption is the
  about he same regardless of the amount but is
  less on a full stomach.
• This shows the rate of BAC declines over time as
  a result of metabolism excretion.

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Blood Alcohol Concentration

• Curve A
• drinking 2 oz alcohol each hr for 4 hours
• BAC increase is cumulative because alcohol is
  consumed faster than it can be metabolized
• Curve B
• drinking 8 oz all at once

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Blood Alcohol Concentration
Open circles 1 oz of 100 proof whiskey every
hour Red circles 2 oz of 100 proof whiskey every
hour
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Blood Alcohol Concentration
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Predicting Blood Alcohol Levels
To use this diagram, lay a straightedge across
your weight and the number of ounces of liquor
youve consumed on an empty or full stomach. The
point where the edge hits the right-hand column
is your maximum blood-alcohol level. The rate of
elimination of alcohol from the bloodstream is
approximately 0.015 percent per hour. Therefore,
to calculate your actual blood-alcohol level,
subtract 0.015 from the number in the right-hand
column for each hour from the start of drinking.
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Calculations of Blood Alcohol Concentration (BAC)

• We can calculate the BAC (mg alcohol/100mL blood)
  at time of an accident (Ca) from the BAC a some
  later time (Ct).
• Ca Ct ?t, where t is time in hours and ?
  12.5-25 mg/100 mLh (avg. 18.7)
• An accident occurred at 3 AM and a blood alcohol
  sample taken at the scene at 5 AM. The BAC at
  that time was found to be 60 mg/100mL or 0.06.
  If witnesses saw the suspect consume his last
  drink at 1 AM, what was his BAC at the time of
  the accident?

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Blood Alcohol Concentration

• No two people respond exactly the same to equal
  amounts of alcohol.
• People develop tolerance with chronic use of
  alcohol.
• Acute tolerance to alcohol can develop in a short
  period of time.
• The BAC is determined by measuring the quantity
  present in the blood or the content in the
  breath.

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What Happens to Alcohol in the Body?

• When an alcoholic beverage is swallowed, it is
  diluted by stomach juices quickly distributed
  throughout the body
• Alcohol does not require digestion before its
  absorption into the bloodstream
• some diffuses into bloodstream directly through
  the stomach wall
• remainder passes into the small intestine where
  it is rapidly absorbed circulated
• Elimination of alcohol from the body is
  accomplished through oxidation by the liver or
  excretion in breath urine or perspiration

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What Happens to Alcohol in the Body?

• 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 carbon dioxide and alcohol are exhaled
  during breathing.
• The blood also carries alcohol to the liver to be
  destroyed.

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What Happens to Alcohol in the Body?
45
What Happens to Alcohol in the Body?
The respiratory system. The trachea connects the
nose and mouth to the bronchial tubes. The
bronchial tubes divide into numerous branches
that terminate in the alveoli sacs in the lungs.
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Alcohol Absorption

• Fasting individual
• 20-25 of a dose of alcohol is absorbed from the
  stomach
• 75-80 is absorbed from the small intestine
• peak blood alcohol concentrations occur in
  0.5-2.0 hrs
• Non-fasting individuals
• presence of food in stomach (especially fatty
  foods) delays absorption
• peak alcohol concentrations 1.0-6.0 hrs

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Alcohol Distribution

• Alcohol has a high affinity for water
• It is diffused in the body in proportion to the
  water content of the various tissues organs
• greater concentration in blood brain
• lesser concentration in fat muscle
• Absorbed alcohol is greatly diluted by the
  aqueous body fluids
• BloodSerum 11.18
• BloodBrain 10.75
• BloodBreath 21001
• BloodSaliva 11.12

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Elimination

• Liver eliminates 95 of the alcohol through
  enzymatic oxidation to acetaldehyde then to
  acetic acid then to carbon dioxide water.
• Remaining 5 eliminated through excretion in
  breath, urine, sweat, feces, milk saliva.
• 0.5 oz (15 mL) alcohol eliminated per hr.

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Field Testing for Sobriety

• Law enforcement officers typically use field
  sobriety tests to estimate a motorists degree of
  physical impairment by alcohol and whether or not
  an evidential test for alcohol is justified.
• The nystagmus test (irregular eye tracking), 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.

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The Breathalyzer

• Developed due to greater practicality of testing
  breath rather than extracting blood.
• The distribution of alcohol between the blood
  alveolar air as stated in Henry's Law.
• Henrys Law When a volatile chemical (alcohol)
  is dissolved in a liquid (blood) and brought to
  equilibrium with air (alveolar air), there is a
  fixed ratio between the concentration of the
  volatile compound (alcohol) in air (alveolar
  breath) and its concentration in the liquid
  (blood). This ration is constant for a given
  temperature.
• At body temperature, the bloodbreath ratio is
  21001

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The Breathalyzer

• Breathalyser-Invented in 1954 by R.F.Borkenstein
  is used to collect measure the alcohol content
  of alveolar breath.
• Amount of breath collected was 52.5 mL or 1/40 of
  2100 mL. Since 1 mL of blood has the same alcohol
  concentration of 2100 mL of alveolar air.
• The alveolar air is passed into a glass ampoule
  that has 3mL of 0.025 potassium dichromate
  0.025 silver nitrate in sulfuric acid and water.
  The alcohol dissolves in the dichromate solution
  and is oxidized to acetic acid.

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The Breathalyzer

• In the oxidation process potassium dichromate is
  also destroyed. It is the loss of dichromate that
  is measured by the breathalyzer and is related to
  quantity of alcohol.

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Alcosensor

• A fuel cell breathalyzer uses a platinum surface
  to oxidize the fuel (breath alcohol) and create
  two electrons per molecule of alcohol.
• Atmospheric oxygen is also reduced and uses one
  electron per molecule.
• The extra electrons produce an electrical current
  proportional to the quantity of alcohol present.

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Intoxilyzer

• An Infra-red breathalyzers that uses a
  photoelectric detector to give a measurement of
  the amount of the absorption of IR light by the
  alcohol in the captured breath sample.
• False positive can come from acetone (nail polish
  remover), toluene (paint thinner) and
  acetaldehyde (smoking)

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Blood Ethanol Testing by GC

• Gas chromatography is the most widely used
  approach for determining alcohol levels in blood.
• Blood must always be drawn under medically
  accepted conditions by a qualified individual.
• It is important that a nonalcoholic disinfectant
  be applied before the suspects skin is
  penetrated with a sterile needle or lancet.
• Once blood is removed, it is preserved by sealing
  it in an airtight container with an anticoagulant
  and a preservative and stored in a refrigerator.

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Blood Ethanol Testing by GC

• Can measure ethanol in a wide range of specimens
• Can distinguish ethanol from other alcohols,
  aldehydes and ketones by retention time match
  with standards or when MS confirms chemical
  identity
• Two common methods of sampling
• Head space
• Direct injection of serum

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Blood Ethanol Testing by GC

• The areas under the GC peaks can be measured and
  a calibration graph is used to determine the BAC.