TOXICOLOGY
III. Toxicological examination
M. Balíková
M. Balíková: Toxicological
examination
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Why to care about
toxicological examination
Understanding drug effects
Differential diagnosis
Correct and effective therapy,
reduction of adverse drug effects
Appropriate subsequent measures –
social or forensic
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Information in a request for
toxicological examination
• Name of a person requiring examination,
date of sampling, date of autopsy
• Circumstances of an incident, estimated
time of drug application/time of death
• Medical history of a person, medical
treatment
• Note of occupation, hobies
• Clinical symptoms/Preliminary pathological
report, if available
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General sample requirements
for toxicology
• Whole blood
a) 10 ml into plain tube
b) 10 ml into 1-2% NaF
• Urine, 50-100 ml
(liver, kidney - can substitute urine
if not available)
• Gastric content, 50 – 100 ml
• Scene material
• Other tissues (brain, lung, vitreous
humour, fat, hair……)
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Drug detection windows
a) sample
b) method
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Different sample types
Specimen
Advantage
Disadvantage Comment
Blood
Urine
Present parent
compounds
Quantitation
Large volume
High concentr.
Easier/longer
detection
Gastric
content
Useful after
drug ingestion
Limited volume
Trace
concentrations
Often not
available
Metabolites
Quantitative
data not useful
Variable sample
Additional May contain
high
tissues
concentrations
Careful
individual
interpretation
Standard
sample for
initial
screening
Analysis may help to interpret
postmortem blood data.
Quantitative data - problems
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Hair sample – to get information about
the life style of a subject in the past
• Drugs are located in the hair in relation
to the time when appeared in blood
• Different hair growth rate at the body
• Cyclus of hair growth, anagen 85% ….
• Ideal sample at the vertex of the head
• Before cutting close to the skin - tie
the strand with the cotton thread
• Correct sampling:
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Toxicological laboratory diagnostics
1)
2)
Nonspecific preliminary methods for screening
Specific methods ( confirmation,
quantification
For pharmaceuticals, illegal drugs:
ad1) Immunochemical screening, colour
reactions
ad 2) Chromatographic systems, mass
spectrometry (MS)
UNKNOWN DRUG – Systematic toxicological analysis – STA
logical sequence of applied methods
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Immunoassays in toxicology
•
•
•
•
•
ADVANTAGES
Technically simple
Sensitive
Rapid
Important initial
information about
sample
On site
performance
•
•
•
•
•
•
DISADVANTAGES
Not all drugs detected
Group detection only
Potentional
interferences
Preliminary results
Confirmation
necessary
Continuous reagent
consumption, supply
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Immunoassays – Principle - 1
Interaction of the target molecule (antigendrug) with a corresponding antibody
To generate measurable signal or visual detection:
Antibody for the drug being assayed
Labelled form of the structurally related drug
or labelled antibody
Competition between the antigen in a sample and labelled
antigen (reagent) for binding to the fixed amount of the
antibody to create the specific immunocomplex:
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Immunoassays – Principle - 2
The proportion of labelled drug molecules bound in the
complex is inversely proportional to the number of
unlabelled drug molecules in the sample
VARIOUS SPECIFICITY of ANTIBODIES
1) for SCREENING – detection of selected groups
(barbiturates)
2) for QUANTITATION of specific individual
(phenobarbitone)
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INITIAL TOXICOLOGICAL
SCREENING by IMMUNOASSSAYS
Adopted cut off values
Cut off need not be LOD
Lower cut off – more FP
High cut off – more FN
Positive detection need to be
confirmed by a specific
method (GC-MS)
Confirmatory method – higher
sensitivity
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Immunoscreening
Cut off values
Selection negative / positive samples
by definition
1) Instrumental methods – flexible, adaptable
2) On site testing – fixed cut off value
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Cut off value and detection windows
Example:
THCOOH
detection
in urine
by three
various
methods –
different
detection
windows
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Various specificity of antibodies
Compound cross reactivities
1) Different reactivities of
related drugs in a selected
group – insufficient
sensitivity for MDMA
detection - FN
Modification of cut off –
method adaptation for
MDMA detection too
2) Adulterants, dilution - FN
3) Reactivities of unwanted
substances – FP
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CONFIRMATION
Basic principle in forensic toxicology :
Verification of preliminary results by another
independent method – more specific, more
sensitive
Identification of specific compounds
Individual compounds differ in
potency/toxicity (codeine – morphine)
Forensic aspects for drug distinguishing
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Drug detection by
chromatography
ADVANTAGES
 Flexible open systems
 Possibility to expand
and update
 Individual drugs
 Uncommon drugs
 More selective
 Less consumables
DISADVANTAGES
 Sample preparation
 Time consuming
 Reference
substances needed
 Experienced
personal
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STA and METHOD COMBINATION
TLC
 No instrument
 Flexible open system
 Reference standards
necessary
 Lower separation
efficiency
 Lower sensitivity-high
drug concentrations
 Larger sample (urine)
GC-MS or LC-MS
Instrument necessary
Flexible open system
Reproducible results
Spectra Data Base
High separation efficiency
High sensitivity-trace
analysis (blood)
 Low sample volume (blood)






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Systematic TLC -1
Various systems developed in the world
System by J. Večerková used overal Czechoslovakia
since 1970
SCREENING – 3 indicators to assess:
1) Extractability – compound acidobasicity
2) Compound mobility related to reference mixture
3) Colour detection by set of reagents
CONFIRMATION >>> Potential drug candidates
to be confirmed in other chromatographic
conditions with specific reference standards
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Systematic TLC -2
Practical application:
Adsorbent : Kieselgel G - Merck
Mobile phase: ETOAC-ETOH-NH3
1 – REFERENCE STANDARDS
2- URINE EXTRACT of BASES
3- URINE EXTRACT after
HYDROLYSIS
Potentional intoxication with
opiates and methamphetamine with
presence of nicotine – subsequent
confirmation in another system TLC
or GC-MS
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FLEXIBLE METHOD APPLICATION
in CONFIRMATION
EXAMPLE:
METHADONE SUBSTITUTION
TARGETED OPIATE CONTROL in URINE
PROCEDURES APPLIED:
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TANDEM GC and MS
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MAS SPECTRA and molecular
structure
 Direct reflection of molecular structure
 Destructive mass detection
 Fragmentation reflects strength of bonds
 Fragmentation rules,
logical mass loses
 Specificity of MS
 Structure modification
to get specific spectra
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Targeted opiates confirmation
by GC-MS after positive IA
Codeine
identification
by
1) RETENTION
2) MASS
SPECTRA
HIGH REPRODUCIBILITY – MASS SPECTRA DATABASES
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Standard requirements for
correct compound identification
by GC-MS
 Acceptable deviations in retention related to
standard (CODEINE RI=2375+/-1%)
 Acceptable deviations in individual fragment mass
abundances when compared with standard
(CODEINE m/z 299100 229261624612423)
 Logical molecular structure fragmentation
 Check for carry over in sample sequence
 Included controls in sample sequence
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GS-MS screening for unknown drug
Many psychoactive substances causing intoxication need
not to be detected by commercial immunoassays, e. g.:
Atropine, scopolamine,DMT….
Synthetic drugs, techno drugs
GHB, ………….
Some of them can be detected by GC-MS screening
system for unknowns
Some of them to be detected require specific sample
preparation, specific chromatography (GHB) – suspicious
cases, not routinely
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Ayahuasca case - 1
Collective session of lucid thinking supported by
some oriental plant extract-more than 35
participants aged 20 – 50 years
40-60 min after ingestion participants started to
be agitated, agressive, with tachycardia,
hyperthermia, dry skin, excessive salivation, some
people fell into coma – medical rescue service called
Urgent clinical need to idetify the toxic agent –
for diferential diagnosis
By laboratory GC-MS screening for uknown drugs
identified in tea extract and in human samples of
blood, urine or gastric content :
atropine,scopolamine, harmine, harmaline, DMT …..
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Ayahuasca case - 2
Subsequently partly determined:
Herbal infusion (free bases):
 atropine 27 mg/L
 harmine 179 mg/L
 scopolamine 515 mg/L
Estimated ingested dose (cup of 150mL):
 atropine 4 mg
 harmine 27 mg
 scopolamine 78 mg
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Ayahuasca case - 3
Clinical impacts:
 All patients recovered
 Some were dismissed from hospitals after a few hrs
 Some requirred intensice care for some days
Forensic impacts:
The organizator taken into detention by police and
investigated – why? Defended himself with no intention
to harm anybody. Legal problem to prove the intention.
Sent for psychiatric examination. Found irresponsible
for his behavior – dissmissed from detention without
any sanction. (However, ???)
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IA missing response for
structuraly related
amphetamines- FN
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DOB Case - important experience
4-bromo-2,5-dimethoxy-amphetamine
 strong hallucinogen, effective dose 1–3 mg
 agonist of serotonin receptors
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DOB Case History - 1
 Two men ingested unknown powdered drug
 Found in open space in coma vomitted with severe
cramps
 Hospitalized in diffent departments
 Biological fluids sent for toxicology for unknowns
 Laboratory applied general procedures without
initial information what happened
 One of men developed strong metabolic acidosis
pH 6.6 – laboratory check for methanol, diols
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DOB Case History - 2
Immunochemical CEDIA for drug groups in urine:
THCOOH COCAINE AMPHETAMINES
M-28
113 kg
M-29
65 kg
positive
positive
negative
positive
negative
negative
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DOB Case HISTORY - 3
GC-MS
screening for
unknowns
Hit from MS
PMW Library
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DOB Case HISTORY - 4
GC-MS
confirmation by
targeted analysis
for amines after
acetylation
Metabolism not
known – research
biotransformation
study
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Hair analysis for drugs
 To get information about the life style
of a subject
 To document chronic drug abuse in a
specific time span
 To explain the pathological autopsy
findings
 Trace analyses – GC-MS or LC-MS
sensitive methods are prerequisite
 More efficient incorporation of bases
than acids
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Hair analyses for
methamphetamine
30 years old man
died shortly after
drug application .
Autopsy revealed
arteriosclerosis
and bleeding into
the cerebellum.
Hair analyses
documented
chronic MA abuse
for approx. 8
months.
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examination
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Hair analyses for opiates
Deceased
24 years
old woman
Documented
chronic
variable
opiates
abuse for
approx. 6
months
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Hair analyses for drugs
Interpretation
Hair – stable sample
Biological variability in hair growth
and drug incorporation
Individual attitude to a case
Standards from sampling to
interpretation expressed by SOHT
(Society for Hair Testing)
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Basic literature:
 R. C. Baselt: Disposition of Toxic Drugs and Chemicals in Man.
Biomedical Publ., 6th ed., 2002 , Foster City, Ca.
 B. Brinkman, B. Madea: Handbuch Gerichtliche Medizin. Springer Vrlg.
2002, Berlin.
 S. Moeschlin: Klinik und Therapie der Vergiftungen. George Thieme
Vrlg. 1986, Stuttgart.
 H. Lüllmann, K. Mohr, M. Wehling: Pharmakologie und Toxikologie,
George Thieme Vrlg. 1999, Stuttgart.
 R. J. Flanagan, A. Taylor, I. D. Watson. R. Whelpton: Fundamental of
Analytical Toxicology, Wiley, 2007, West Sussex.
 S, B. Karch, ed.: Drug Abuse Handbook, CRC Press, 1998, Boca Raton.
 O. H. Drummer, M. Odell: The Forensic Pharmacology of Drugs of
Abuse. Arnold Publ. 2001, London.
 F. Pragst, M. Balíková: State of the art in hair analysis for detection of
drug and alcohol abuse. A review. Clinica Chimica Acta 370 (2006) 17-49.
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