TOXICOLOGY III. Toxicological examination

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TOXICOLOGYIII. Toxicological examination
M. Balíková
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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 windowsa) sampleb) method
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Different sample types
Specimen Advantage Disadvantage Comment
Blood Present parent compounds Quantitation Limited volume Trace concentrations Careful individual interpretation
Urine Large volume High concentr. Easier/longer detection Often not available Metabolites Quantitative data not useful Standard sample for initial screening
Gastric content Useful after drug ingestion Variable sample
Additional tissues May contain high concentrations Analysis may help to interpret postmortem blood data. Quantitative data - problems 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

• 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 (antigen-drug)
  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
valuesSelection 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. Vecerková used overal Czechoslovakia
  since 1970
• SCREENING 3 indicators to assess
• Extractability compound acidobasicity
• Compound mobility related to reference mixture
• Colour detection by set of reagents
• CONFIRMATION gtgtgt 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
SUBSTITUTIONTARGETED 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
• RETENTION
• 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 RI2375/-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 13 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 positive positive negative
M-29 65 kg 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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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.