Download Forensic toxicology

Toxicology
Drugs, poisons, alcohol in forensic
medicine
2012
History
What is forensic toxicology?
Intoxications
Mayer Mátyás
Forensic toxicology
Toxicology
Paracelsus (born Philippus Aureolus
Theophrastus Bombastus von Hohenheim, 1493 –
the father of toxicology,
wrote: Alle Ding' sind Gift, und
nichts ohn' Gift; allein die Dosis
macht, daß ein Ding kein Gift
ist.*
1541),
*All things are poison, and nothing is without poison; only the dose permits something not
to be poisonous
What is a Poison?
All substances are poisons;
there is none that is not a poison.
The right dose differentiates a poison and a remedy.
Dose-Response Relationship
As the dose of a toxicant increases, so does the response.
RESPONSE
Therapeutic range
5
4
therapeutic effects
undesired/unwanted effects
0-1 NOAEL (No Observable Effect Level)
3
2-3 Linear Range
4 Maximum Response
5 Plateau
2
0
1
DOSAGE
What is a lethal Dose?
• LD50 is the idea that is used by pharmacologist
and companies in a statistical sense in animal
experiments.
• LD50 Over a large number of tests, it provides
a toxic level at which half the animals will be
expected to die.
Comparison of LD50
Chemical
Ethyl Alcohol
Sodium Chloride
Ferrous Sulfate
Morphine Sulfate
Strychnine Sulfate
Nicotine
Black Widow
Curare
Rattle Snake
Dioxin (TCDD)
Botulinum toxin
LD50 (mg/kg)
10,000
4,000
1,500
900
150
1
0.55
0.50
0.24
0.001
0.0001
Toxicology as science
Toxicodynamics investigates the effect of xenobiotics to the
human body (dose-response relationship, mechanism of action etc.)
Toxikokinetics investigates the effect of the organism to the
harmful xenobiotics (absorption , distribution, metabolism, routes of
excretion)
• Xenobiotics is derived from the Greek words xenos (foreigner, stranger)
and bios (life)
Toxic substance
• Medicaments
(TCA, neuroleptics, opiates, NSAID, ...)
• Pleasure drugs (heroine, PCP, ethanol, mephedrone,
ethanol+BZD/barbiturates, …)
• Household chemicals (HCL, HOCl, NaOH, ethylene glycol)
• Pesticides
•
Toxins from plants, animals and mushrooms
Pathways of intoxication
• Routes and Sites of Exposure
– Ingestion (Gastrointestinal Tract)
– Inhalation (Lungs)
– Dermal/Topical (Skin)
– Injection
• intravenous, intramuscular, intraperitoneal
Typical Effectiveness of Route of Exposure
iv > inhale > ip > im > ingest > topical
Duration of exposure
Acute
< 24hr
usually 1 exposure
Subacute
1 month
repeated doses
Subchronic
1-3 month
repeated doses
Chronic
> 3 month
repeated doses
Over time, the amount of chemical in the body can build up, it can
redistribute, or it can overwhelm repair and removal mechanisms
Individual Susceptibility
• Genetics-species, strain variation, interindividual
variations (yet still can extrapolate between mammalssimilar biological mechanisms)
• There can be 10-30 fold difference in response to a
toxicant in a population
• Gender (gasoline nephrotoxic in male mice only)
• Nutritional status
• Health conditions
Individual Susceptibility II.
• Age
– young
• underdeveloped excretory mechanisms, biotransformation enzymes and
blood-brain barrier
– old
• changes in excretion rates, metabolism rates and body fat
• Previous or Concurrent Exposures
– additive
– synergistic
– antagonistic
Causes of death
• Respiratory failure
– RCD (opiates, EtOH)
– Airway obstruction (aspiration, swallowing the tongue)
• Circulatory failure
– Cardial
– Vascular
– Hypovolaemia
• Systemic hypoxaemia
– O2 transporrt/use
• Seizures
• Damage of other organs
– Liver, lung
• Accidents
– Behavioral effects (gasoline+whiskey=car crash)
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Mayer Mátyás
Forensic toxicology
Forensic toxicology
Mathieu Joseph Bonaventure
Orfila (1787–1853), the father of
forensic toxicology published the
first comprehensive work on
forensic toxicology in 1813.
Marie LaFarge 1840 (arsenic)
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Mayer Mátyás
Forensic toxicology
• Toxicology
–Experimental Toxicology
–Applied toxicology
• Clinical toxicology
• Forensic toxicology
• Ecotoxicology
• Occupational toxicology
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Mayer Mátyás
Forensic toxicology
Forensic toxicology
• The study of alcohol, drugs and poisons, including their
chemical composition, preparations and identification
• The science of detecting and identifying the presence
of drugs and poisons in body fluids, tissues, and
organs.
• Forensic toxicology is toxicology with medicolegal
applications
Multidisciplinar applied science
includes toxicology, analytical
chemistry, pharmacology and clinical chemistry
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Mayer Mátyás
Forensic toxicology
Objectives
• Postmortem forensic toxicology
is used in death
investigations to establish whether or not drugs were the
cause or contributing factor in death.
• Human performance toxicology
– Alcohol in the body an its effects
– Drugs and driving
– Workplace drug testing
• Forensic drug testing is the evaluation of illegal drug
consumption
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Forensic toxicology
Samples (biological matrices)
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Evidence from the crimescene
Urine
Femoral blood PM (venous blood)*
Blood from the heart PM (arterial blood)*
Liquor
Saliva
Tissues (liver, kidney, etc.)
Nails
Hair
*plasma or serum
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Forensic toxicology
Narcotics
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Pains relievers
Depressing action on CNS and RC
Used legally also
Opium: from poppy bulb
Contains alkaloids:
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Morphine: heroin is synthetised from it (semi-synthetic opiate)
Codeine
Thebaine
Narcotine
Narceine
Opioids: non-peptide components which bind to the opiate receptors
Abuse potential
• Opiates addictions are chronic problems
• Compulsive drug seeking
• Neurochemical and molecular changes in
the brain
• Heroin: tolerance and physical
dependence
Depressants
• Depress the CNS, inhibition of neurotransmitter
receptors
• Side effects: slow reflexes, slow thinking,
poor decision making (influenced driving
behaviour!)
• Alcohol
• Barbiturates
• TCA
Stimulants
Caffeine, Theophylline, Nicotin legal, socially accepted drugs
Amphetamine, Metamphetamine, MDMA
Cathinones
Atropine, Scopolamine
• Exitement of central nervous system
• Create a sense of euphoria
• Long term use: stroke, high blood pressure,
paranoia, heart attack
• Hyperthermia, water poisoning MDMA
Amphetamines
• In contrast to opiates, these drugs created an
excitatory condition characterized by elevated
heart rate
• Extreme feelings of euphoria
• These drugs have some of the highest
incidences of abuse
Cocaine
• Cocaine is a stimulant that resembles amphetamine
in it’s abusive potential.
• Unlike amphetamine, cocaine is a natural stimulant
found in the coca leaf.
• Cocaine can be refined to produce crack cocaine:
intensifies its euphoric effects and cost effectiveness.
• Increase of potency when the drug is smoked, the
large surface area of the lungs provides rapid
absorption into the body
• Cocaine is fully metabolized
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Mayer Mátyás
Forensic toxicology
Hallucinogens
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Changes in thoughts, perception, moods
Marijuana
Ketamine and phencyclidine (PCP): dissociative anesthetics
Ketamine:Fast acting anaesthetic and pain killer used
primarily in veterinary surgery, and human medicine
• Produces vivid dreams of hallucinations
• Makes user feel that the mind is separated from the body
“dissociation”
• Ecstasy is called “love pill” increases
perception of color, sound and sensations
Cannabinoids
• Marijuana is a name that applies to parts of the Cannabis
sativa plant.
• Tetrahydrocannabinol (THC) is the major active agent in
marijuana.
• Typically marijuana has a 2-6 % by volume concentration of
THC, Hashish has higher concentration of THC (12 %).
• The average marijuana cigarette contains about 75 mg of THC
but it is rapidly absorbed into the blood stream reaching peak
concentration around 10 – 20 minutes after smoking.
• Physical dependency is rare
• Long term use: risk of harm (cardiodepression
Marijuana
sinsemilla
Short Term Effects:
• Increase in heart rate, lead
to anxiety and paranoia
• Distorted concept of time
and space
• Decrease in concentration
skills, short-term memory
capacity
• Feeling tired after the
“high” wears off
• Increase in appetite, weight
gain
Long Term Effects:
• Breathing problems
• Lung cancer
• Damage cells and tissues in
the body that fight disease
• Lack of motivation
• Difficulty processing new
information
Phencyclidine
• Commonly known as PCP
• Developed by the Park Davis company and
was intended for use as a surgical anesthetic.
– However it was found to be unsatisfactory because some
patients exhibited manic behavior after usage.
– PCP is measured in blood or urine levels around 25 ng/ml.
Levels of greater than 100 ng/ml have been related to
seizure and some deaths.
„Designer” Drugs
New challenges
Cyanide
• Highly toxic substance found in various places throughout
nature.
• Cyanide is dangerous because it binds with ferric ions in
cytochrom oxidase, which is an enzyme critical for electron
transport.
• Because it interrupts electron transport it stops the bodies
main mechanism for energy production.
• Most labs can test for cyanide in whole blood and its
concentration correlates well with severity of poisoning.
• Levels higher than 1000 nanograms per mil are associated
with stupor and amounts greater than 2500 nanograms per
mil are usually fatal.
Carbon Monoxide CO
• Some studies suggest that CO causes more
deaths than any other toxin.
• This is because CO is present in all fires and
typically is the cause of death in fire.
• CO binds to hemoglobin much more tightly
than O2 does and decreases our blood’s ability
to carry oxygen.
• CO also binds with myoglobin which is also
important in electron transport.
Metal poisoning
• Copper
– Copper-sulphate most frequent
– Ulceration
– Vomiting: first greenish, later bloody
• Lead
– Concentrates in blood, soft tissues, bones
– Causes haemolysis, anemia, encephalopathy
• Cadmium
– Itai-itai disease
• Mercury
– limate (HgCl2), calomel (Hg2Cl2), merury nitrite
– Oral ingestion: metallic taste in the mouth,, pain in the pharynx, bloody
vomiting, oral mucosa is grayish-white, coagulation necrosis, anuria
– Can accumulate in the hair
– the fish and shellfish of Minamata Bay methylmercury
atomic absorption spectroscopy
Kayser–Fleischer ring
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Mayer Mátyás
Forensic toxicology
Lead
dense metaphyseal bands
Dark deposits of lead sulphid on the gum
(lead lines)
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Mayer Mátyás
Forensic toxicology
Mercury
Mercury-Amalgam filling
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Mayer Mátyás
Forensic toxicology
Cadmium
Tobacco accumulates Cd
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Mayer Mátyás
Forensic toxicology
Itai-itai Disease
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weak and brittle bones
spinal and leg pain
bone deformities
complications
• coughing
• anemia
• kidney failure
Acid poisoning
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Coagulative necrosis
Acidosis, haemolysis, hypercapnia
Hydrochloric gas: laryngeal oedema and spasm
Hydrochloric acid: grayish-white scab
Sulphuric acid: grayish-black scab
Nitric acid: yellow colour (xanthoprotein reaction)
Healing occurs with scarring
Alkaline poisoning
• Ammonium or sodium hydroxide: household
substances, mainly children by accident, or
with the intention of suicide
• They cause colliquative necrosis
– Surface becomes softened and slippery
– Necrosis is deep, may cause perforation
– Healing with serious scarring
• Dysphasia, vomitting, gastric pain, salivation,
haematemesis
Corrosive metal salts
• Silver nitrate
– Grayish-white area on buccal mucosa
– Later becomes grayish-black
• Zink chloride
– From batteries
– Burning pain in the mouth, nausea, vomitting, bloody
diarrhoea
– Liver and kindney damage
• Potassium permanganate
– Coagulation necrosis
– Irritated mucosa turns into a purplish-red colour
Alcohols
• Ethanol is beverage alcohol
• Low molecular weight alcohols such as methanol and
isopropyl may also contribute to human injury.
• It takes around 90 minutes for the peak level of
ethanol to enter the blood.
• Gas Chromatography is the preferred method for
testing alcohol in blood and a proper chain of
custody must be observed for criminal cases.
Toxicology of Alcohol
• Alcohol intoxication depends on
– Amount of alcohol consumed
– Time of consumption
– Body weight
– Rate of alcohol absorption
– Gender
Factors that Affect Alcohol Absorption
• Time of consumption
• Type of alcoholic
beverage
• Presence of food in
stomach
Calculation of BAC
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Widmark formula:
C= A/ W x r
C – concentration of alcohol (mg/ 100 ml)
A – amount of alcohol in gramms
– Multiply volume% with 0.8
• W – body weight in kg
• r – Widmark factor: 0.7 males, 0.6 females
• In general as average: 1 dl wine or 3 dl beer is
eliminated completely in an hour (means: 7-8
gramms of pure alcohol)
Effects of alcohol
Physical Effects
• Co-ordination is impaired,
clumsiness, slower reflexes
• High blood pressure,
damage to the heart
• Liver damage
• If drinking when pregnant
• Life threatening when
mixed with other drugs
Mental and Emotional
• Behave in ways that you
normally wouldn’t
• Increase in aggressive and
violent behaviour
• Problems with school and
learning
Fate of Alcohol I.
• Alcohol is absorbed into the bloodstream
• Distributed through-out the body’s water
• And finally eliminated by oxidation and excretion
Note:
A.
Oxidation is the combination of oxygen and alcohol to produce new products by the liver
B.
Elimination is removing alcohol from the body in an unchanged state; normally excreted in breath and
urine
Alcohol and the Law
• 1939-1964: intoxicated
= 0.15% BAC
• 1965: intoxicated =
0.10% BAC
• 2003: intoxicated =
0.08% BAC
In Hungary: zero tolerance
From 0.081 % - crime
Assay Procedure
• Studying the anamnesis
• Screening test for substance group
(immunological tests)
• Confirmational analysis qualitative or
Fourier IR spectroscopy, mass
quantitative (NMR,
spectrometry, UV-VIS spectroscopy)
Mayer Mátyás
Forensic toxicology
Rapid Drug Screen (RDS)
OPI TCA COC THC MTD mAMP AMP BZO PCP BAR
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Mayer Mátyás
Forensic toxicology
Fluorescence Polarization
Immunoassay (FPIA) I.
• Fully automated
immunoassay analyzer
• Fit for medium and high
volume clinical laboratories
• Up to 81 tests analyzed per
hour
• FPIA is a patented
technology used for
measurement of serum and
urine drug concentration
Axsym Abott
11-10-2011
Mayer Mátyás
Forensic toxicology
Fluorescence Polarization
Immunoassay (FPIA) II.
SAMPLE PREPARATION
• Protein precipitation
• Separation (filtration, centrifugation, dialysis, etc.)
Protein precipitation
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„salting out” ((NH4)2SO4)
Organic solvent
Acid (TCA, HClO4)
Heavy metal salt
Heat denaturation
HClO4 + KHCO3
KClO4 + H2O + CO2
Control: MALDI-TOF
EXTRACTION METHODS
• Dialysis
• Supercritical Fluid Extraction
• Liquid-liquid extraction (LLE)
• Solid-phase extraction (SPE)
Mayer Mátyás
2012.10.18.
MICROEXTRACTION METHODS
• Microdialysis (in vivo)
• Micro-diffusion
• Liquid-liquid microextraction
- DLLME, Direct-SDME, LLLME, HS-SDME, HF-LPME
• Solid-phase microextraction
- DI-SPME, HS-SPME, MEPS
Liquid-liquid extraction
(LLE)
Dispersive liquid-liquid microextraction
(DLLME)
Extraction and derivatisation in one step
Extracting and dispersing solvents
Δ-9-THC, cannabinol, cannabidiol
urine
Moradi M, Yamini Y, Baheri T.
J Sep Sci. 2011 Jul;34(14):1722-9.
MA, MDMA, ketamine, heroine (59-81%)
Liang Meng, Bin Wang, Feng Luo, Guijun Shen,
Zhengqiao Wang, Ming Guo
Forensic Science International 209 (2011) 42–47
THC-COOH, A, MA, MDA (65-76%)
Blood, urine, seum
Abdulmumin A.Nuhu, ChanbashaBasheer, BahruddinSaadb
Journal ofChromatographyB (2011)
M. Rezaee, Y. Assadi, M.R. Milani Hosseini, E. Aghaee, F. Ahmadi, S. Berijani, J.
Chromatogr. A 1116, 2006
Mayer Mátyás
2012.10.18.
Single-drop microextraction
(SDME)
• Cheap
• Not labor intensive
• Slow
(A) direct SDME
(B) liquid-liquid-liquid microextraction (LLLME)
(C) headspace SDME (HS-SDME).
A, MA (99-107%)
urine
LLLME 500X
Yi He, Youn-Jung Kang
Journal of Chromatography A, 1133 (2006) 35–40
Francisco Pena-Pereira, Isela Lavilla, Carlos Bendicho
Trends in Analytical Chemistry, Vol. 29, No. 7, 2010
Hollow fibre liquid phase microextraction
(HF-LPME)
• 1990’s
• Very clear extract relatively fast
A, MA (60-70%)
Blood, serum, saliva
Knut Einar Rasmussen, Stig Pedersen-Bjergaard
Trends in Analytical Chemistry, Vol. 23, No. 1, 2004
A, MA, MDMA, MDA (56-77%)
hair
Lorena do Nascimento Pantaleão, Beatriz Aparecida Passos Bismara Paranhos,
Mauricio Yonamine
Journal of Chromatography A, 1254 (2012) 1– 7
Mayer Mátyás
2012.10.18.
Solid-phase extraction (SPE)
Normal Phase SPE procedure
Reversed phase SPE
Ion exchange SPE
– Anion exchange
– Cation Exchange
Vacuummanifold
Mayer Mátyás
Forensic toxicology
Solid Phase Microextraction
(SPME)
• DI-SPME
• HS-SPME
Synthetic cannabinoids
saliva
Mayer Mátyás
2012.10.18.
Melissa Toms
Northern Kentucky University, poster
SPME II.
divinylbenzene/carboxene/ polydimethylsiloxane
Coating: polypyrrole, immuneaffinity,
monolithic, C18…
GHB
Hiroyuki KATAOKA
ANALYTICAL SCIENCES SEPTEMBER 2011, VOL. 27
Kenneth G. Furton, JingWang, Ya-Li Hsu, JohnWalton, José R. Almirall
Journal of Chromatographic Science, Vol. 38, July 2000
GHB
On column derivatization
Jodi E. Meyers, Jos´e R. Almirall
J Forensic Sci, Jan. 2005, Vol. 50, No. 1
A, MA, MDMA, MDA, MDE
urine
Benkő András; Artemis Dona; Kovács Anikó; Maravelias, Costas;
Mikóné Hideg Zsuzsanna; Kerner Ágnes
Acta Pharmaceutica Hungarica - 1998. 68. évf. 5. sz.
A, MA, EPH (89-98%)
urine
Mayer Mátyás
2012.10.18.
Sunanta Wangkarn, Wisitsak Wutiadirek
Mj. Int. J. Sci. Tech. 2007, 01(2), 145-156
MEPS
microextraction by packed sorbent
45µm silica baesed particles with 60Å pore size
C2, C4, C8, C18, Sil, M1 ...
A, MA (98-100%)
haj
Hajime Miyaguchi, Yuko T. Iwata, Tatsuyuki Kanamori, Kenji
Tsujikawa, Kenji Kuwayama, Hiroyuki Inoue
Journal of Chromatography A, 1216 (2009) 4063–4070
Molecular imprinting
A. Prieto, S. Schrader, C. Bauer, M. Möder
Analytica Chimica Acta 685 (2011) 146–152
Mayer Mátyás
2012.10.18.
CAPILLARY ELECTROPHORESIS
• Fast
• Low sample needs
MASS SPECTROMETRY
• M/Z (mass per charge)
• Molecular ion
• Fragmentation
Chromatography I.
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Stationary phase
Mobile phase (eluent)
Elution
Analyte
Retention time
Chromatography II.
• Mobile phase
– Gas chromatography
– Liquid chromatography
• Bed shape
– Column chromatography
– Planar chromatography
• Separation mechanism
– Ion exchange chromatography
– Size-exclusion chromatography
– Normal-/Reversed-phase chromatography
– Chiral chromatography
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Mayer Mátyás
Forensic toxicology
Thin Layer Chromatography
TLC/HP-TLC
Densitogram
• Normal phase
• Multiple samples
• Detection with Densitometer
Mayer Mátyás
Forensic toxicology
Thin Layer Chromatography II.
Gas chromatography
(GC)
Analysing compounds that can be vaporized
without decomposition
Mobile phase is an inert unreactive carrier
gas (helium, nitrogen)
Similar to fractional distillation
High-performance liquid
chromatography
(HPLC)
Pressure: 100-150 bar -> 1000-1200 bar
Time of analysis: 30-60 min. -> 0,5-5 min
Solvent consumption: 40-50 ml -> 0,5-5 ml (green chemistry)
Flow rate: 1-1,5 ml/min-> 200 nl/min-300 µl/min
Solvent reservoirs, Degasser, Gradient valve, Mixing vessel for delivery of the mobile phase, High-pressure pump,Switching valve injector,
Sample injection loop, Pre-column (guard column), Analytical column, Detector, Waste or fraction collector.
Analytical colmn
• Filled column
– Fully porous
– Core shell
• Monolithic
• Silica based
• Synthetic polymer based
FILLED COLUMN
Fully porous vs. Core-shell particles
MONOLITHIC COLUMN
• Polymer: increased pH range (0-14)
STATIONARY PHASES
Detectors (GC, LC)
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Flame ionization detector (FID)
Mass spectrometer (MS)
UV detector
Diode array detector (DAD or PDA)
Fluorescence detector (FLD)
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Mayer Mátyás
Forensic toxicology
AUTOMATIZATION
Thank you for your attention!
11-10-2011
Mayer Mátyás
Forensic toxicology