Toxicology iii toxicological examination
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TOXICOLOGY III. Toxicological examination. M. Balíková. 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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Toxicology iii toxicological examination

TOXICOLOGYIII. Toxicological examination

M. Balíková

M. Balíková: Toxicological examination


Why to care about toxicological examination

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

M. Balíková: Toxicological examination


Information in a request for toxicological examination

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

M. Balíková: Toxicological examination


General sample requirements for toxicology

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……)

M. Balíková: Toxicological examination


Drug detection windows a sample b method

Drug detection windowsa) sampleb) method

M. Balíková: Toxicological examination


Different sample types

Different sample types

M. Balíková: Toxicological examination


Hair sample to get information about the life style of a subject in the past

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:

M. Balíková: Toxicological examination


Toxicological laboratory diagnostics

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

M. Balíková: Toxicological examination


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

Immunoassays in toxicology

M. Balíková: Toxicological examination


Immunoassays principle 1

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:

M. Balíková: Toxicological examination


Immunoassays principle 2

Immunoassays – Principle - 2

The proportionof 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)

M. Balíková: Toxicological examination


Initial toxicological screening by immunoasssays

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

M. Balíková: Toxicological examination


Immunoscreening cut off values selection negative positive samples by definition

Immunoscreening Cut off valuesSelection negative / positive samples by definition

  • Instrumental methods – flexible, adaptable

  • On site testing – fixed cut off value

M. Balíková: Toxicological examination


Cut off value and detection windows

Cut off value and detection windows

Example:

THCOOH detection in urine by three various methods – different detection windows

M. Balíková: Toxicological examination


Various s pecificity of antibodies compound cross reactivities

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

M. Balíková: Toxicological examination


Confirmation

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

M. Balíková: Toxicological examination


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

Drug detection by chromatography

M. Balíková: Toxicological examination


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)

STA and METHOD COMBINATION

M. Balíková: Toxicological examination


Systematic tlc 1

Systematic TLC -1

  • Various systems developed in the world

  • System by J. Večerková 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>>> Potential drug candidates to be confirmed in other chromatographic conditions with specific reference standards

M. Balíková: Toxicological examination


Systematic tlc 2

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

M. Balíková: Toxicological examination


Flexible method application in confirmation

FLEXIBLE METHOD APPLICATION in CONFIRMATION

EXAMPLE: METHADONE SUBSTITUTIONTARGETED OPIATE CONTROL in URINE

PROCEDURES APPLIED:

M. Balíková: Toxicological examination


Toxicology iii toxicological examination

TANDEM GC and MS

M. Balíková: Toxicological examination


Mas spectra and molecular structure

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

M. Balíková: Toxicological examination


Targeted opiates confirmation by gc ms after positive ia

Targeted opiates confirmation by GC-MS after positive IA

  • Codeine identification by

  • RETENTION

  • MASS SPECTRA

HIGH REPRODUCIBILITY – MASS SPECTRA DATABASES

M. Balíková: Toxicological examination


Standard requirements for correct compound identification by gc ms

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

M. Balíková: Toxicological examination


Gs ms screening for unknown drug

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

M. Balíková: Toxicological examination


Ayahuasca case 1

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 – medicalrescue 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 …..

M. Balíková: Toxicological examination


Ayahuasca case 2

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

M. Balíková: Toxicological examination


Ayahuasca case 3

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, ???)

M. Balíková: Toxicological examination


Ia missing response for structuraly related amphetamines fn

IA missing response for structuraly related amphetamines- FN

M. Balíková: Toxicological examination


Dob case important experience

DOB Case - important experience

  • 4-bromo-2,5-dimethoxy-amphetamine

  • strong hallucinogen, effective dose 1–3 mg

  • agonist of serotonin receptors

M. Balíková: Toxicological examination


Dob case history 1

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

M. Balíková: Toxicological examination


Dob case history 2

DOB Case History - 2

Immunochemical CEDIA for drug groups in urine:

M. Balíková: Toxicological examination


Dob case history 3

DOB Case HISTORY - 3

GC-MS screening for unknowns

Hit from MS PMW Library

M. Balíková: Toxicological examination


Dob case history 4

DOB Case HISTORY - 4

GC-MS confirmation by targeted analysis for amines after acetylation

Metabolism not known – research biotransformation study

M. Balíková: Toxicological examination


Hair analysis for drugs

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

M. Balíková: Toxicological examination


Hair analyses for methamphetamine

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.

M. Balíková: Toxicological examination


Hair analyses for opiates

Hair analyses for opiates

Deceased 24 years old woman

Documented chronic variable opiates abuse for approx. 6 months

M. Balíková: Toxicological examination


Hair analyses for drugs interpretation

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)

M. Balíková: Toxicological examination


Basic literature

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.

M. Balíková: Toxicological examination


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