Clinical and Forensic Toxicology
description
Transcript of 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. . .
1. Cleaning Products2. Analgesics3. Cosmetics4. Plants5. Cough/Cold Preparations6. Hydrocarbons7. Bites8. Topicals9. Foreign Bodies10. Chemicals
1. Antidepressants2. Analgesics3. Sedative/Hypnotics4. Street Drugs5. Cardiovascular Drugs6. Alcohols7. Fumes8. Chemicals9. Asthma Medications10. Cleaning Products
Exposures Deaths
PharmacokineticsPl
asm
a dr
ug c
once
ntra
tion
Time
t1/2
Peak plasma concentration
ktt eCC 0
Pharmacokinetics Summary
t0.693=k
0.693=kt=(2)=C0.5
C
kt=CC
-kt=CC
eC=C
1/2
1/20
0
t
0
0
t
-kt0t
lnln
ln
ln
Pharmacokinetics Summary
21
693.0
0
tt
t eCC
C
Pharmacokinetics Summary
C
Vt
0.693 = Vk = Clearance d1/2
d
The Autonomic Nervous System
Sympathetic(Thoracocolumbar)
Norepinephrine
Parasympathetic(Craniosacral)Acetylcholine
21Cardiovascular Smooth muscle
Insulin releaseGluconeogenesis
GIVascular smooth
muscle
Muscarinic 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
Blood alcohol concentration (mg/dL, %)
0 0.250.150.100.05 0.300.20 0.35
CN
S im
pairm
ent
Loss of inhibition
Impaired motor coordination
Slurred speech
Staggering gait
Loss of consciousness
Ethanol distribution
Serum95% H2O
Cells82% H2O
EtOH
Serum (or plasma) ethanol is 5 – 15% higher than whole blood ethanol
Enzymatic Ethanol Methods
• ADH is selective, but not specific for ethanol
• Other enzymes that involve NADH can potentially interfere
CH3CH2OH CH3CHO
NAD+ NADH
ADH
CH3CH2OHADH
NAD+ NADH
CH3CHO
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:
8.2182 BUNGlucNa
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
AlcoholToxicmg/dL MW
Add to serum osmol (mosm/mg/dL)
Ethanol 300 46 0.22
Methanol 50 32 0.31
Isopropanol 200 60 0.17
Ethylene glycol 50 60 0.17
Osmol Gap Mnemonic
MethanolEthanol
Diuretics (glycerol, mannitol, sorbitol)IsopropanolEthylene glycol
Analgesic Poisoning
Acetaminophen64%
Aspirin17%
Ibuprofen19%
Acetaminophen62%
Aspirin34%
Ibuprofen4%
Exposures
Fatalities
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 MetabolismNHCOCH3
OH
NHCOCH3
OH
Glutathione
NHCOCH3
Glucurinide
NCOCH3
O
Hepatic Glucuronyl transferase
Microsomal mixedfunction oxidases
Glutathione conjugation
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
0
1
2
3
4
5
6
7
Perc
ent u
sing
in p
revi
ous 3
0 da
ys
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 NLCP-certified 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 Workplace-compliant 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
Injector
GC Column
Ionizer Mass Filter
Detector
Data System
0
10
20
30
40
50
60
70
80
90
100
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
e-
e-
++ +
(+)
From GC
To MS
Filament
CollectorIon volume(or source)
Focusinglens
(-)
Power supply
The “Right Hand Rule”
Direction of current
Direction ofmagneticfield
Magnetic sector mass spectrometer
To detector
+From ion source
Quadrupole mass spectrometer
+From ion source To detector
Electron multiplier
e-
104 e-+
From mass filter
Positive dynode
Negative dynode
Ammeter
Mass spectrum
0
10
20
30
40
50
60
70
80
90
100
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)
Time
m/
z
Full scan time = 1.0 sec(0.002 sec/ion)
0.1s
Cocaine
N
H3C
O
OCH3
O
OC17H21NO4
MW=303.35
303 (M+)[M-31]+ 272
182 [M-121]+
82 (base peak)
121
Cocaine fragmentation (EI)
N
H3C
O
OCH3
O
O
N
H3C
O
OCH3
O
O
N
H3C
O
OCH3
O
O
N
H3C
O
O
CH3
O
O
272 31
82
121182
303
unstable
303 (M+)[M-31]+ 272
182 [M-121]+
82 (base peak)
121
Amphetamine/Methamphetamine
HN
CH3
Methamphetamine
CH3
CH3
Amphetamine
NH2
44
91
Amphetamine fragmentation
CH3
NH2CH3
NH2
44
91
+
+
44
91
Methamphetamine fragmentation
HN
CH3
CH3
HN
CH3
CH3+
91
+
58
58
91
TMS derivative of amphetamine
CH3
NH2
BSTFA
CH3
HN
Si(CH3)3
Amphetamine TMS-AmphetamineMW = 207
TMS-amphetamine fragmentation
CH3
HN
Si(CH3)3
CH3
HN
Si(CH3)2
CH3
NH
Si(CH3)3
116
91
+
+
192
+
Mass spectra of TMS-amphetamine
TMS-methamphetamine fragmentation
N
CH3
CH3
Si(CH3)3
N
CH3
CH3
Si(CH3)3
N
CH3
CH2
Si(CH3)3
+
91
+
130
206
+
Mass spectra of TMS-methamphetamine
Methamphetamine metabolism
CH3
Amphetamine
NH2
HN
H3C H
d-Methamphetamine
HN
H CH3
l-Desoxyephedrine
CH3
~10%
Cocaine HCl and free base forms
N
H3C
O
OCH3
O
O
NH+
H3C
O
OCH3
O
O
Cl-
pH<8.6
HCl
Cocaine metabolism
N
H3C
O
OCH3
O
O
N
H3C
O
OCH3
OH
N
H3C
OH
O
O
O
HN
O
OCH3
O
O
Ecgonine methyl ester Benzoylecgonine Norcocaine
- C6H5COO
- CH3- CH3
TMS derivative of benzoylecgonine
N
H3C
OH
O
O
O
BSTFA
N
H3C
O
O
O
O
Si(CH3)3
BenzyolecgonineMW = 289
TMS-BenzyolecgonineMW = 361
TMS-benzoylecgonine fragmentation
N
H3C
O
OSi(CH3)3
O
O
N
H3C
O
OSi(CH3)3
N
H3C
O
O
N
H3CO
O
OO
Si(CH3)2
346
82
122240
361
+
+
Mass spectra of TMS-benzoylecgonine
Opiates
OHO OH
N
H3C
H
OO OH
N
H3C
H
H3C
Morphine Codeine
CH3
Glucuronidation
OHO OH
N
H3C
H
OH
HO
OH
HH
OHH
OH
COOH
OC6H9O7 C6H9O7
N
H3C
H
Morphine
Hepatic glucuronyl transferase
Morphine diglucuronide
-D-glucuronic acid
Morphine hydrolysis
OC6H9O7 C6H9O7
N
H3C
H
OHO OH
N
H3C
H
MorphineMorphine diglucuronide
-glucuronidase
TMS derivative of codeine
OO OH
N
H3C
H
H3C
BSTFA
OO O
N
H3C
H
H3C Si(CH3)3
CodeineMW = 299
TMS-CodeineMW = 371
Mass spectra of TMS-codeine
Heroin metabolism
OO O
N
H3C
H
Heroin
OHO OH
N
H3C
H
Morphine
H3C
O
CH3
O
OHO O
N
H3C
H
6-Monoacetylmorphine CH3
O
- CH3CO
- CH3CO
9-Tetrahydrocannabinol (THC)
O
CH3
OH
H3CH3C
O
COOH
OH
H3CH3C
Oxidation
9-THC 9-THC-COOH
THC-COOH detection
THC-COOH glucuronide (15%)
THC-COOH
TMS-THC-COOH
Hydrolysis
BSTFA