Analytical and Forensic Toxicology - ACMT · Chromatography Separation and Detection •Encompasses...
Transcript of Analytical and Forensic Toxicology - ACMT · Chromatography Separation and Detection •Encompasses...
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Analytical and Forensic Toxicology
Evan S. Schwarz, MDWashington University
St. Louis, MOBarnes Jewish Hospital
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Special Thanks!
• Howard Greller
• Jeffrey Brent
• Adhi Sharma
• Chuck McCay
• Kent Olson
It was the break they had been waiting for; prints left at the crime scene
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Content• Core Content of Medical Toxicology
• Part 5: Analytical and Forensic Toxicology
• LLSA Articles
• Post-mortem Toxicology: What the dead can and cannot tell us. Clin Tox 2003;41(1):47-56.
• Is this urine really negative? J of Sub Abus Treat 207;33:33-42.
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Laboratories
• Clinical Laboratory Improvement Amendments of 1988 (CLIA)
• Medical Lab Testing governed by federal regulations since 1992
• Regulations apply to all lab testing of human specimens for medical purposes
• All tests require the possession of an appropriate certificate
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Testing
• Waived
• Moderate complexity
• High complexity
ModerateRecord KeepingWritten ProceduresLaboratory DirectorCompetency TestingProficiency TestingControlsInspection
HighQualified onset supervisorDaily review of all results
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Magic Answer Box?
• Low volume, often old methodologies
• Limits to every technique
• Implies confirmation or exclusion
• More toxic xenobiotics than named diseases
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Magic Answer Box?
• Low volume, often old methodologies
• Limits to every technique
• Implies confirmation or exclusion
• More toxic xenobiotics than named diseases
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Basics of analysis
• Screening
• Confirmation
• Separation
• Detection
• Quantification
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Assay Methods
• Spot & spectrochemical
• Immunoassays
• Non-competitive
• Competitive
• Enzyme-multiplied immunoassay (EMIT)
• Magnetic microparticle chemiluminescent competitive immunosassay
• Microparticle capture immunoassay
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Assay Methods
• Chromatography
• Thin layer liquid (TLC)
• High performance (pressure) liquid (HPLC)
• Gas chromatography (GC)
• Mass spectroscopy (MS)
• Inductively coupled plasma mass spectroscopy
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Testing
“You’re fired, Jack. The lab results just came back, and you tested positive for Coke.”
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Spot tests • Simple
• Rapid Reaction
• Color change
• Ferric chloride for salicylate
• Unreliable (FP & FN)
• Visual interpretation
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Spot Testing
• Salicylates
• Ferric chloride
• Mercuric chloride
• Meixner Test
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Spectrochemical
• Sophisticated spot tests
• Chemical reaction to form light-absorbing substance
• Carefully controlled
• Spectrophotometer vs eyeball
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Co-oximetry
• Spectrophotometry used to measure various forms of hemoglobin
• Measurement of light absorbance at multiple wavelengths allows allows several hemoglobin species to be quantified
• Need more wavelengths than types of hemoglobin
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Spectrochemical
• Older tests waited until complete conversion
• Modern tests measure rate of conversion
• Initial phase, rate is constant and proportional to the initial concentration of the analyte
• Nonreacting substances that absorb light
• Don’t affect results
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Spectrochemical
• Produce light absorbing substances
• Ex: High concentrations of lactate
• Inhibit the assay reaction or that consume reagents
• Ex: ascorbic acid in oxidation reactions
Oh, a drug test. That’s a relief.I thought you were going to test
my ethics.
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Spectrochemical
• Improve Selectivity
• Enzymes that can catalyze highly selective reactions
• Assays for ethanol use alcohol dehydrogenase
• Rate of change of NAD+ to NADH
• Rate of increase in light absorption is proportional to ethanol concentration
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Immunoassays
• Created due to need to measure a very low concentraiton of an analyte
• Quick, inexpensive
• Combination of
• High affinity
• High selectivity
• 2 common types
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Immunoassays• Non-competitive
• Analyte is sandwiched b/w 2 antibodies
• Difficult with small drugs
• Competitive
• Analyte from specimen competes for number of Ab binding sites with a labeled version of the analyte
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Non-competitive“Sandwich” assay
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Competitive“Inverse” assay
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Competitive
From Goldfranks 9th edition
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Immunoassays
• Nonisotopic immunoassays are common
• Limited to those in high demand
• Lots of effort, high development cost
• Low production cost
• Homogenous immunoassays
• Measure differences in bound and free labels
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Immunoassays• Old - radio immunoassays
• New - spectrophotometric
• Enzyme multiplied immunoassay technique (EMIT)
• Fluorescence polarization immunoassay (FPIA)
• Kinetic inhibition of microparticles in solution (KIMS)
• Cloned enzyme donor immunoassay (CEDIA)
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EMIT
Goldfranks 9th edition
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Magnetic Microparticle
Goldfranks 9th edition
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ChromatographySeparation and Detection
•Encompasses several related techniques where analyte specificity is achieved by physical separation
•Partition analytes between a stationary and mobile phase
•Stationary: very fine particles aranged in a thin layer
•Mobile (moving): phase flows thru spaces between particles
•After separation--need a detection phase
•However, can have provisional identification based on their characteristic velocities, distance traveled, or time to traverse the chromatography column
•Not specific
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Rf
• Retention time
• Time required to traverse the column
• Retardation (planar) or retention (column)
• Separation based on polarity, affinity, solubility, etc.
• Standard for each analyte
Rf =
migration distance of substancemigration distance of solvent front
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Thin Layer Chromatography
• Extracts dissolved in a solvent
• Placed on thin layer of silica gel
• Plates placed vertically in closed tank
• Solvent drawn upwards thru the gel
• Xenobiotics are drawn up the gel
• Hydrophobic rapidly
• Hydrophilic slowly
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TLC
Goldfranks 9th edition
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Thin Layer Chromatography
• Fast, easy, inexpensive
• Polar silica medium
• Low sensitivity (~ 1000 ug/L)
• Low specificity
• Used in drug screens
• Requires large amount of material
• Done on urine, gastric aspirate
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TLC Drawbacks
• Multiple steps
• Slow, labor intensive
• Interpretation of spots
• Difficult to quantitate
• Used to demonstrate the presence of a drug
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High-Performance Liquid Chromatography• Stationary phase packed in a column
• Mobile phase pumped through under pressure
• Allows better separation in less time
• Identification by retention time in a column
• Also use ultraviolet spectroscopy
• Measuring light absorbance allows the amount of the xenobiotic to be determined
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Reverse phase chromatography
• Non-polar stationary phase and hydrophilic mobile phase
• Polar out first, non-polar retained
• Hydrophobic stationary
• Hydrophilic mobile phase
• Most common HPLC technique
• TLC can be done this way too
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High-Performance Liquid Chromatography• Expensive, complex, fast
• Separation under pressure
• Quantitation, detection > 10 ug/L (10 fold difference)
• Low specificity for same class
• One at a time; narrow range of polarity
• Measure serum concentration for which no immunoassay is available
• Can’t analyze multiple specimens
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HPLC
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Gas Chromatography• Similar to HPLC except the moving phase is a gas
• Nitrogen, helium
• Low flow resistance of gas
• High flow rates
• Less time
• Temperature gradient allows multiple xenobiotics to be analyzed at once
• Partitioning depends on natural volatility
• Temp < 572°F (300°C)
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Gas Chromatography
• Expensive
• Screening or confirmatory
• High specificity and sensitivity
• Quantitation and broad screening
• Temperature gradient
• Analyte volatilized in injector, column kept hot
• Heat labile cannot be assessed
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Gas Chromatography
• Substances have characteristic retention times
• Column outflow detectors
• Flame-ionization - organic compounds
• Most common detector
• Nitrogen-phosphorous - N or P compounds (many drugs)
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Mass Spectrometry
• Can serve as highly sensitive GC detector
• Analyte separated from the gas carrier and then filtered to a detector
• Extremely high specificity
• Unparalleled ID of organic chemicals
• Shoots electrons → fragment ions
• Characteristic of the parent molecule
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Mass Spectrometry
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MS Limits
• Cannot separate enantiomers
• D-methamphetamine (DOA)
• L-methamphetamine (inhalers)
• Vick’s inhalers, metabolite of selegiline
• “L” = legal
• Can separate by a chiral analysis
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LC/MS/MS
• High sensitivity and specificity extended by the related hybrid technique of liquid chromatography/tandem mass spectrometry
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GC/MS
• Expensive equipment
• Most common gold standard, in ~1 hour
• Most specific and sensitive
• Sensitivity 2 – 10 ug/L
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Relative ComparisonMethod Speed Cost
Spot Test Fast $
Spectrochemical Medium $
Immunoassay Medium $$
TLC Slow $$
HPLC Medium $$
GC Medium $$
GC/MS Slow $$$
LC/MS/MS Medium $$$$*Reproduced from Goldfranks 9th edition
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Hair Analysis
• Hair grows ~1 cm/month
• Can utilize any methodology
• Contamination is greatest flaw
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Drug Screens
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NIDA-5
• Amphetamines
• Cannabinoids
• Cocaine
• Opiates
• Phencyclidine
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Cocaine
• Highly specific
• Benzoylecgonine
• Inactive metabolite
• No false positives
• Acute: 2-3 days
• Chronic: 1 week
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Tetrahydrocannabinol
• Tetrahydrocannabinoic acid
• Inactive metabolite
• Occassional use: 3 days
• Chronic use: > 1 month
• 11-OH THC
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The “second hand smoking” defense
• Studies of passive exposure
• Confined areas
• Mixed results
• Cutoffs: 20 ng/ml
• Levels: ~6 ng/ml
• NIDA 50 ng/ml
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THC
• Dronabinol
• Efavirenz
• NSAIDs
• Promethazine
• Riboflavin
• Ethacryinc acid
False Positives
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• Morphine, codeine, heroin
• Cross reactivity depends on assay
• Synthetics show little or no cross-reactivity
• Specific directed assays available
Opioids
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“Poppy Seed” Defense
6-MAM
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Challenges
• Dextrorphan – major metabolite
• Levorphanol – “L” enantiomer, also opioid
• Can’t differentiate optical enantiomers by MS
Dextromethorphan
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Methadone
• Quetiapine
• Doxylamine
• Olanzapine
• Diphenhydramine & Verapamil metabolites
False Positives
*Tests for other sythetic and semisynthetics are available
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PCP
• Phencyclidine
• False Positives
• DXM, Ketamine
• Diphenhydramine
• Venlafaxine, bupropion
• Metabolites of PCP
• False Negatives?
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Benzodiazepines• Metabolized to
oxazepam
• Chlordiazepoxide
• Diazepam
• Temazepam
• Glucuronides (lorazepam)
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Amphetamines
• Amphetamine assay plagued with false positives
• Fails to detect “designer” amphetamines
• Bupropioin (cathinone)
• Pseudoephedrine
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Nasal Inhalers
• Can contain l-methamphetamine
• less potent isomer of d-methamphetamine
• Both turn immunoassays positive
• Difficult to distinguish with mass spec
• Optical enantiomers
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TCA Challenges
• Cross react with ringed xenobiotics
• Carbamazepine
• Phenothiazines
• Diphenhydramine
• Major challenge is timing
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Substance Screening Confirmatory
Cocaine (met) 300 ng/ml 150 ng/ml
Opiate (met) 2000 ng/ml 2000 ng/ml
Amphetamines 1000 ng/ml 500 ng/ml
THC (met) 50ng/ml; 1000 ng/ml THCA 15 ng/ml THCA
PCP 25 ng/ml 25 ng/ml
Federal Cutoffs (ng/mL)
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Adulteration• 2004: First mandatory guidelines for federal
workplace testing (SAMHSA)
• Tampering
• In vivo adulteration
• In vitro adulteration
• Urine substitution
• Little evidence to support any product working consistently
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Specimen Validity• Appearance
• Temperature
• 90-100oF
• pH testing
• pH 3-11
• Specific gravity
• Cr < 20 mg/dl
• > 1.003
• Creatinine
• > 20 ppm
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In Vivo Adulteration“Ingest prior to urination”
• Primary mechanisms: dilution and excretion
• Water and diuretics
• Fool visual inspection
• Interfere with creatinine level checks
• Water, Naturally Klean herbal tea, golden seal, HCTZ
• B-vitamins, riboflavin, creatinine
• Riboflavin may cause fluorescent urine
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In Vitro Adulteration“Add after urination”
• Interfere with an immunoassay or convert a target drug to a different compound
• Sold under many names but contain:
• Glutaraldehyde
• Sodium or potassium nitrate
• Pyridinium chlorochromate
• Peroxide/peroxidase
• Household products, too
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In Vitro Adulteration“Add after urination”
• Glutaraldehyde: interferes with immunoassay, denatures
• Nitrite: decreased ion concentration
• Pyridinium: decrease in pH levels
• Peroxidase: oxidizing drugs and metabolites
• Bleach: decreases detection
• Vinegar: interferes with detection; lowers pH
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Others
• Acids and bases (i.e. lemon juice, NaOH)
• Oxidizing agents (i.e. bleach, peroxide)
• Denaturants (i.e. glutaraldehyde)
• Eyedrops (i.e. benzalkonium chloride)
• Reduces binding of immunoassay
• Soap (created FP and FN)
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Urine SubstitutionOntario Smith
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Cholinesterases
• Butyrylcholinesterase (plasma cholinesterase)
• Metabolizes cocaine, succinylcholine
• Falls first, recovers first
• Red Cell Acetylcholinesterase
• Reflects activity at the NMJ
• Low concentrations in people
• Serve as markers for poisonings
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Cholinesterases
• Malnutrition
• Hereditary deficiency
• Iron deficiency anemia
• Hepatic disease
• Chronic Illness
• Succ, codeine, morpine
• Pernicious anemia
• Antidepressants
• Antimalarials
• Hemoglobinopathies
• Inc with oral contraceptives
Red CellButrylcholinesterase
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Anion Gap Reliability
• MUDPILES
• Cyanide, CO, Acetaminophen, Toluene, Theophylline, Hydrogen Sulfide
• Increase unmeasured anions
• Dehydration, sodium salts, antibiotics
• Decreased unmeasured cations
• Mag, Ca, and K
AG = Na+ - (Cl- + HCO3-)
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Anion Gap Reliability
• Hypercalcemia
• Hypermagnesemia
• Hyperkalemia
• Lithium
• Multiple myeloma
• Hypoalbuminemia
• Bromism
• Iodism
• Nitrate excess
Low Anion Gap
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Forensic Toxicology
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Forensics
• Aid medical / legal investigation of death, poisoning, and drug use
• Concern is not legal outcome, but obtaining and interpreting results
• Chain of Custody
• List everyone who handled a specimen (special couriers)
• Where specimen was at any given time
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DEA Schedule• Controlled Substances Act (1970)
• I - High abuse potential, no medical use
• Heroin, PCP, LSD, GHB, MDMA, etc.
• II - High abuse potential, but has medical use
• Most opioids, barbiturates, methylphenidate, etc.
• No refills
• III - Ketamine, buprenorphine, GHB
• IV - BZD, long acting barbiturates, modafinil
• V - Codeine cough suppressants, pregabalin, diphenoxylate
GHB
GHB
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Medical Review Officer
• Licensed physician
• “Expert in drug and alcohol testing and the application of federal regulations to the process.”
• Consultant
• Business, industry, labor, government or academia
• Relating to prevention, detection and control of drug abuse in the workplace
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Drug Abuse Testing
• Strict, invariable procedures, federally mandated (DOT)
• Certified lab, separate from all other testing
• MRO interpretation required
• COAT-PE
• ‘SAMHSA 5’ - cocaine, opioids, amphetamine, THC, PCP
• ETOH breath testing
• Screening, then confirmatory if + by cutoffs
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Post Mortem Toxicology
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Post Mortem Changes“Necrokinetics”
• Postmortem interval
• Defined by the degree of decomposition
• Decomposition
• Autolysis: enzymes are released and chemicals move down gradients
• Putrefacation: digestion by bacteria
• Anthropophagia: feeding on remains
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Specimens• Blood
• Reported as “blood concentrations”
• From femoral or subclavian (low glucose)
• Right heart blood (elevated glucose)
• Vitreous
• Avascular, acellular so well protected
• Aqueous content > blood
• Urine
• Bladder serves as a “reservoir”
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Interpretation Confounders
• Postmortem redistribution
• Postmortem metabolism
• Continuous absorption
• Xenobiotic stability
• Chemical interactions
• Expected clinical effects
• Comorbid, tolerance, genetics
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Other sources
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Redistribution Doesn’t Occur
Leikin JB. Clin Tox 2003;41(1):47-56.
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Legal Ethanol
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Legal Ethanol
• State determines own legal driving limit
• DWI, DUI, DWAI, etc.
• Zero tolerance
• “Illegal per se”
• What is “drunk”?
• ~ 150 mg/dL
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The Barman’s Paradox
• Legally drunk 50-80 mg/dL or 0.05-0.08 g/dL [%]
• Serving to intoxicated is prohibited
• No better at determining “drunk”
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Blood Ethanol Testing
• The law = whole blood
• The lab = serum / plasma
• [serum] = [plasma]
• ETOH does not enter RBCs well
• [serum]/[blood] ~ 1.15
• The lab measures higher than the law
• [Breath ethanol] mmol/L X 2100 = [Blood ethanol] mmol/L
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The Clues1. Wide turns
2. Straddling / driving on lane marker
3. Nearly striking object or another vehicle
4. Weaving / swerving
5. Going too slow (> 10 MPH below speed limit)
6. Stopping inappropriately / without cause
7. Following too closely
8. Erratic braking
9. Driving into opposing / crossing traffic
10.Slow response to traffic signals
*NHTSA Top 10 in descending order of probability of intoxication
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Goldfranks 9th edition
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What’s in a Drink?
• 10 oz beer (5%)
• 4 oz of wine (12%)
• 1 oz liquor (50%)
• All ~ 15 g ETOH
• 100-125 mg/kg/hr
• Avg adult: 7-10 g/hr or 15-20 mg/dL/hr
• Tolerance: 30 mg/dL/hr
Approximately
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Laboratory Methods
• Enzymatic
• ADH + ETOH = NADH (340 nm)
• FP with elevated lactate (lactate converted to pyruvate increases NADH formation)
• GC
• Can detect other volatiles (“toxic”)
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The Numbers
• Specific gravity 0.8 g/ml
• Vd 0.6 L/kg
• 1-1.5 shot(s) = 30 ml
• mmol/L = (mg/dL)/4.6
• % = grams/100 ml
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Sample Calculation50 kg man drinks 1 shot of 80 proof alcohol
1.5 oz (30 ml/oz) = 45 ml 40% of 45 ml = 18 ml
18 ml X 0.8 g/ml = 16 g which is 16,000 mg
50 kg (0.6 L/kg) = 30 L (10 dL /L) = 300 dL
16,000 mg/300 dL = 80 mg/dL
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“Breathanol”
• [expired air] surragote for [blood] (sort of)
• Henry’s law
• [ethanol] remains constant throughout respiratory tract
• Mean breath:blood ratio 1:2100 used in forensics
• Many confounders
• [Breath ethanol] mmol/L X 2100 = [Blood ethanol] mmol/L
• Breath units underestimate BAC
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References1. Goldfranks 9th Edition
2. Jaffee WB et al. Is this urine really negative? A systematic review of tampering methods in UDS and testing. J of Subs Abu Treat 2007;33:33-42.
3. Leikin JB, et al. Post-mortem toxicology: What the dead can and cannot tell us. Clin Tox 2003;41(1):47-56.
4. Shannon: Haddad and Winchester’s Clinical Management of Poisoning and Drug Overdose, 4th edition 2007
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Photo References1. http://www.offthemark.com/cartoons/1998-12-22.gif
2. http://www.ecardica.com/ecards/postcards%5Cfunny%20pictures/drug_testing_at_pepsi.jpg
3. http://www.flickr.com/photos/cornellfungi/2828851149/sizes/z/in/photostream/
4. http://www.cartoonstock.com/newscartoons/cartoonists/jlv/lowres/jlvn386l.jpg
5. http://cdn1.images.videobash.com/photos/000/022/977/22977.jpg
6. http://26.media.tumblr.com/tumblr_lykxob8LmS1qfcutbo1_500.jpg
7. http://www.thcfinder.com/uploads/files/marijuana-mcdonalds.jpg
8. http://www.cannabisculture.com/v2/files/images/2493-homer-phish.jpg
9. http://www.myfreewallpapers.net/movies/pages/the-good-the-bad-and-the-ugly.shtml
10. http://www.sodahead.com/entertainment/bristol-palin-gets-her-own-reality-show-laughable-or-legit/question-1778221/?page=4&link=ibaf&q=meth+mouth&imgurl=http://farm4.static.flickr.com/3063/2780829321_9f3f10177b.jpg
11. http://images.teamsugar.com/files/upl0/1/13254/08_2008/headline.preview.jpg
12. http://www.cdc.gov/Features/AlcoholConsumption/
13. http://sharetv.org/shows/seinfeld/episodes/346227
14. http://ourweed.com/funny-marijuana-pictures/
15. http://cdn.bleacherreport.net/images_root/slides/photos/000/840/267/52000365_display_image.jpg?1302153389
16. http://www.4aceswholesale.com/cart/images/New-Whizzinator-w.jpg
17. http://theestreet.com/wp-content/uploads/2012/01/entourage.jpg
18. http://thebeautybrains.com/2007/08/16/the-scandalous-secret-beauty-companies-dont-want-you-to-know/
19. http://2pep.com/funny%20pics/cool_funniest_hilarious_pictures/super_funny_hilarious_pictures_of_25_epic_drunk_shaming_photos_20090603_1483976130.jpg
20. http://www.cartoonstock.com/newscartoons/cartoonists/mba/lowres/mban1080l.jpg
21. http://www.thecrimsoncrow.com/wp-content/uploads/2011/05/heroin-up-his-butt-billboard-12835-1306154078-11.jpg
22. http://images3.wikia.nocookie.net/__cb20111221073446/simpsons/images/7/7b/Eliza_Simpson_Detective.png
23. http://amazingdata.com/mediadata57/Image/crazy_funny_amazing_shocking_awesome_sexy_200907311639075608.jpg
24. http://mimg.ugo.com/201103/3/8/7/176783/cuts/charlie-sheen-crazy-eyes_288x288.png