Transcript Clinical and Forensic Toxicology

Clinical and Forensic Toxicology
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
Chief of Clinical Chemistry & Toxicology
Toxicology Disciplines
• Industrial Toxicology
– Toxic exposures in the workplace
– Product testing
•
•
•
•
•
Molecular Toxicology
Veterinary Toxicology
Environmental Toxicology
Clinical Toxicology
Forensic Toxicology
Clinical Toxicology
• The branch of toxicology that is concerned
with human poisoning
– Drug overdoses
• Pharmaceuticals
• Drugs of abuse
– Toxic exposures
• Environmental
• Occupational
• Accidental
The Top Ten Poisoning. . .
Exposures
1. Cleaning Products
2. Analgesics
3. Cosmetics
4. Plants
5. Cough/Cold Preparations
6. Hydrocarbons
7. Bites
8. Topicals
9. Foreign Bodies
10. Chemicals
Deaths
1. Antidepressants
2. Analgesics
3. Sedative/Hypnotics
4. Street Drugs
5. Cardiovascular Drugs
6. Alcohols
7. Fumes
8. Chemicals
9. Asthma Medications
10. Cleaning Products
Plasma drug concentration
Pharmacokinetics
Peak plasma concentration
Ct  C 0 e  kt
t1/2

Time 

Pharmacokinetics Summary
Ct = C0 e
-kt
 Ct 
ln   = -kt
 C0 
C 
ln  0  = kt
 Ct 
 C0 
ln 
 = ln (2) = kt1/2 = 0.693
 0.5 C 0 
k=
0.693
t 1/2
Pharmacokinetics Summary
C
Ct  C0 e


  0.693t 
 t



1
2


Pharmacokinetics Summary
 0.693 
Clearance = k  V d = 
  V d
 t 1/2 
C
The Autonomic Nervous System
Sympathetic
Parasympathetic
(Thoracocolumbar)
Norepinephrine
(Craniosacral)
Acetylcholine
Muscarinic


GI
Vascular smooth
muscle
1
Cardiovascular
2
Smooth muscle
Insulin release
Gluconeogenesis
Nicotinic
Cholinergic Poisoning
• Organophosphates, some mushrooms (Group III,
clytocybe and inocybe species), betel nuts,
pilocarpine, carbachol, acetylcholine
• Miosis, vasodilatation, bronchial secretions,
bradycardia, increased bowel motility, urination,
sweating
• Mnemonic: DUMB BELS (Diarrhea, Urination,
Miosis, Bradycardia, Bronchorrhea, Emesis,
Lacrimation, Salivation)
• Atropine + pralidoxamine is antidote
• Laboratory monitors AChE activity
Anticholinergic Poisoning
• Belladonna, Jimsonweed, antihistamines,
phenothiazines, certain mushrooms (Group V),
scopolamine, tricylics, OTC sleeping pills
• Mnemonic: Red as a beet, dry as a bone, mad as a
hatter, hot as a stone, bowel and bladder lose their
tone, and the heart runs alone.
• Physostigmine is antidote
Sympathetic Poisoning
• : vasoconstriction, pupillary dilitation, coronary
artery dilitation, decreased bowel motility, bladdar
contraction
• 1: tachycardia
• 2: smooth muscle dilatation, insulin release,
lipolysis, renin release, gluconeogenesis (miosis,
vasodilatation, bronchodilatation, hyperglycemia,
decreased bowel motility, bladder relaxation)
Nicotinic Poisoning
• Insecticides, tobacco, black widow spider
venom
• Tachycardia, hypertension, muscle
fasciculations, weakness, paralysis
• d-turbocurarine is antidote
Ethanol
• Most common (by far) toxic exposure
• Often associated with:
– Trauma
– Loss of consciousness
– Other drug exposure
• Frequently involves medico-legal
interventions
Ethanol Pharmacodynamics
CNS impairment 
Loss of consciousness
Staggering gait
Slurred speech
Impaired motor coordination
Loss of inhibition
0
0.05
0.10
0.15
0.20
0.25
Blood alcohol concentration (mg/dL, %)
0.30
0.35
Ethanol distribution
Cells
82% H2O
Serum
95% H2O
EtOH
Serum (or plasma) ethanol is 5 – 15% higher than whole blood ethanol
Enzymatic Ethanol Methods
ADH
CH3CHO
CH3CH2OH
ADH
CH3CHO
CH3CH2OH
+
NAD NAD
+
NADH
NADH
• ADH is selective, but not specific for
ethanol
• Other enzymes that involve NADH can
potentially interfere
Non-ethanol Alcohol Poisoning
• Alcohol toxicity is primarily related to
metabolites
–
–
–
–
Ethanol  Acetaldehyde  Acetate
Isopropanol  Acetone
Methanol  Formaldehyde  Formic acid
Ethylene Glycol  Oxalate and Hippuric acid
• Non-ethanol alcohol exposures can be
detected by an increase in the osmol gap
The Osmol Gap
Calculated Osmolality:
Gluc BUN
2  Na 

18
2.8

The Osmol Gap
• Colligative properties depend on the
number of of dissolved particles
– Boiling point
– Freezing point
• Osmolality is usually determined by
freezing point depression
• The difference between the calculated and
measured osmolality is the Osmol Gap
The Osmol Gap
Alcohol
Ethanol
Toxic
mg/dL
300
MW
46
Add to serum osmol
(mosm/mg/dL)
0.22
Methanol
50
32
0.31
Isopropanol
200
60
0.17
Ethylene glycol
50
60
0.17
Osmol Gap Mnemonic
Methanol
Ethanol
Diuretics (glycerol, mannitol, sorbitol)
Isopropanol
Ethylene glycol
Analgesic Poisoning
Ibuprofen
19%
Exposures
Aspirin
17%
Acetaminophen
64%
Ibuprofen
4%
Fatalities
Aspirin
34%
Acetaminophen
62%
Salicylate Poisoning
• Toxic symptoms develop at serum
concentrations exceeding 250 mg/L
• Serum concentrations exceeding 1000 mg/L
can be fatal
• Symptoms are tinnitus, hyperventilation,
respiratory failure, convulsions, coma
• Lab results reveal mixed metabolic
acidosis/respiratory alkalosis
• Acidification of urine enhances elimination
Acetaminophen Poisoning
• Toxic symptoms develop at serum
concentrations exceeding 100 mg/L
• Serum concentrations exceeding 450-500
mg/L result in severe liver damage
• Symptoms may not appear until hepatic
failure is evident and irreversible
• Antidote is N-acetylcysteine
Acetaminophen Metabolism
NHCOCH3
NHCOCH3
Hepatic Glucuronyl transferase
Microsomal mixed
function oxidases
OH
Glucurinide
NCOCH3
NHCOCH3
Glutathione conjugation
Glutathione
OH
O
Imidoquinone (toxic)
Carbon Monoxide Poisoning
• Can be deliberate or accidental
– CO is odorless
• CO binds irreversibly to hemoglobin,
displacing oxygen
• CO-Hb (carboxyhemoglobin) can be
measured on a co-oximeter
– Different max than O2-Hb (oxyhemoglobin)
• Hyperbaric oxygen may be indicated
Metal Poisonings
• Iron is most common (particularly in kids)
– Ferritin
– Deferoxamine is antidote
• Arsenic is most notorious
– Acute vs. chronic
– Inorganic vs. arsine gas
– BAL (dimercaprol) is antidote
Metal Poisonings
• Lead
–
–
–
–
Most cases in children exposed to lead paint
Blood lead >10 g/L is considered risk
Monitor with -aminolevulinic acid dehydratase
EDTA is antidote
• Mercury
– Organic vs. inorganic
– Neurotoxic, nephrotoxic, teratogenic
Spot Tests for Metal Poisoning
• Reinsch Test: Copper wire turns. . .
–
–
–
–
Shiny silver: Mercury
Dull black: Arsenic
Shiny black: Bismuth
Dark purple sheen: Antimony
• Gutzeit test for arsenic
– Acidification produces arsine gas, which discolors
silver nitrate paper
• Iron reacts with potassium ferricyanide and
ferrous sulfate to produce Prussian Blue
Thin Layer Chromatography
TLC Stains
• Ninhydrin: 1o or 2o amines (sympathomimetics)
• Mercuric Sulfate: barbiturates, glutethimide,
phenytoin (white ppt)
• Diphenylcarbazone: same as mercuric sulfate (blue or
purple spot)
• Iodoplatinate: 3o amines
• Dragendorf's reagent: methaqualone
• UV absorption at 254 nm: benzodiazepines,
barbiturates, methaqualone
• Fluorescence at 366: Benzodiazepines, quinine,
quinidine
Medical vs. forensic drug testing
• Patient consent not
required
• Identity of specimen is
presumed
• Screening result is
sufficient for medical
decision
• Results are used for
medical evaluation
• Subject must consent to be
tested
• Identity of specimen must
be proved
• Only confirmed results
can be considered positive
• Results are used for legal
action
Illegal Drug Use in the U.S.
(1998 Household Survey)
• 13.6 million Americans use illicit drugs
– 25 million in 1979
• 8.3% of youths age 12-17 use marijuana
– 14.2% in 1979
• 1.8 million Americans use cocaine
– 5.7 million in 1985
Types of drugs used
Percent using in previous 30 days
7
6
5
4
3
2
1
0
All drugs
THC
PsyRx
Cocaine
LSD, etc.
Inhalants
History of workplace drug testing
• 1960s – 1970s: The Department of Defense begins
testing military personnel for illegal drug use.
• 1986: President Reagan establishes the “Federal
Drug-Free Workplace”.
• 1988: Mandatory Guidelines for Federal
Workplace Drug Testing Programs is published in
the Federal Register.
The “NIDA” program
• NIDA (now SAMHSA) requirements for
drug testing were drafted by Research
Triangle Institute
• The RTI established the National Laboratory
Certification Program (NLCP)
• Drug testing for federal agencies (DOT,
NRC, etc.) must be performed in a NLCPcertified laboratory
Florida Drug-Free Workplace
• The Florida HRS (now AHCA) established a
drug-free workplace program in 1990
• Specifications for the State of Florida
program are similar to federal requirements,
but there are notable differences
• Employees of Florida Drug-Free Workplacecompliant businesses must be tested in
AHCA-licensed laboratories
Screening vs. Confirmation
•
•
•
•
•
Low cost
Fast
Semi-quantitative
High sensitivity
Low specificity
•
•
•
•
•
High cost
Slow
Quantitative
High sensitivity
High specificity
A confirmatory method should . . .
• Utilize the most accurate (specific) testing method
available
• Have sensitivity equal to or better than the
screening method
• Be economically feasible
• Be simple enough to standardize across many
laboratories
• Produce results that are legally defensible
GC/Mass Spectrometry
Detector
Injector
Ionizer
Mass Filter
GC Column
100
90
Data System
80
70
60
50
40
30
20
10
0
40
50
60
70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
Electron impact ionization
Power supply
Filament
From GC
Focusing
lens
e+ +
To MS
+
eIon volume
(or source)
(-)
Collector
(+)
The “Right Hand Rule”
Direction of current
Direction of
magnetic
field
Magnetic sector mass spectrometer
+
From ion source
To detector
Quadrupole mass spectrometer
+
From ion source
To detector
Electron multiplier
Negative dynode
+
From mass filter
e-
104 e-
Positive dynode
Ammeter
Mass spectrum
100
90
80
70
60
50
40
30
20
10
0
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
Single ion monitoring (SIM)
m/z
Full scan time = 1.0 sec
(0.002 sec/ion)
0.1s
Time 
Cocaine
H3C
N
O
CH3
O
C17H21NO4
O
MW=303.35
O
82 (base peak)
182 [M-121]+
303 (M+)
121
[M-31]+ 272
Cocaine fragmentation (EI)
H3C
O
N
O
CH3
O
H3C
N
121
182
O
CH3
O
O
H3C
O
O
CH3
N
O
O
O
303
O
82
unstable
H3C
N
O
O
272
O
CH3
O
31
82 (base peak)
182 [M-121]+
303 (M+)
121
[M-31]+ 272
Amphetamine/Methamphetamine
H
N
NH2
CH3
CH3
Amphetamine
CH3
Methamphetamine
44
91
Amphetamine fragmentation
+
NH2
CH3
NH2
44
CH3
+
91
44
91
Methamphetamine fragmentation
H
N
+
CH3
H
N
58
CH3
CH3
+
91
CH3
58
91
TMS derivative of amphetamine
H
N
NH2
Si(CH3)3
BSTFA
CH3
Amphetamine
CH3
TMS-Amphetamine
MW = 207
TMS-amphetamine fragmentation
Si(CH3)3
NH
+
CH3
116
H
N
Si(CH3)3
CH3
+
91
H
N+
Si(CH3)2
CH3
192
Mass spectra of TMS-amphetamine
TMS-methamphetamine fragmentation
Si(CH3)3
N
+
CH3
CH3
Si(CH3)3
130
N
CH3
CH3
+
91
Si(CH3)3
N
+
CH3
206
CH2
Mass spectra of TMSmethamphetamine
Methamphetamine metabolism
H
N
H3C
CH3
H
d-Methamphetamine
NH2
~10%
CH3
H
N
H
CH3
l-Desoxyephedrine
Amphetamine
Cocaine HCl and free base forms
H3C
N
H3C
O
Cl-
NH+
O
CH3
O
CH3
pH<8.6
O
O
O
HCl
O
O
Cocaine metabolism
H3C
N
O
CH3
O
O
O
- C6H5COO
- CH3
- CH3
H3C
N
H3 C
N
H
N
O
O
CH3
OH
O
CH3
O
O
O
O
O
O
OH
Ecgonine methyl ester
Benzoylecgonine
Norcocaine
TMS derivative of benzoylecgonine
H3C
N
H3C
O
N
O
Si(CH3)3
OH
BSTFA
O
O
O
Benzyolecgonine
MW = 289
O
O
TMS-Benzyolecgonine
MW = 361
TMS-benzoylecgonine fragmentation
H3C
O
N
O
Si(CH3)3
O
H3C
N
240
O
Si(CH3)3
O
O
+
122
H3C
O
N
O
361
82
H3C
N
+
Si(CH3)2
O
O
O
O
346
Mass spectra of TMSbenzoylecgonine
Opiates
H3C
H3C
N
N
CH3
H
HO
O
Morphine
H
OH
H3C
O
O
Codeine
OH
Glucuronidation
-D-glucuronic acid
H3C
H3C
COOH
N
O
H
OH
H
H
HO
H
N
OH
H
H
OH
Hepatic glucuronyl transferase
HO
O
Morphine
OH
C6H9O7
O
C6H9O7
Morphine diglucuronide
Morphine hydrolysis
H3C
H3C
N
N
H
H
-glucuronidase

C6H9O7
O
C6H9O7
Morphine diglucuronide
HO
O
Morphine
OH
TMS derivative of codeine
H3C
H3C
N
N
H
H3C
O
O
Codeine
MW = 299
H
BSTFA
OH
H3C
O
O
TMS-Codeine
MW = 371
O
Si(CH3)3
Mass spectra of TMS-codeine
Heroin metabolism
H3C
N
H
- CH3CO
O
H3C
O
O
O
O
Heroin
H3C
N
CH3
H
H 3C
O
N
HO
- CH3CO
O
Morphine
O
6-Monoacetylmorphine
H
HO
O
OH
CH3
9-Tetrahydrocannabinol (THC)
COOH
CH3
OH
OH
Oxidation
H3C
H3C
O
9-THC
H3C
H3C
O
9-THC-COOH
THC-COOH detection
THC-COOH glucuronide (15%)
Hydrolysis
THC-COOH
BSTFA
TMS-THC-COOH