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