TOXICOLOGY III Toxicological examination M Balkov Toxicological examination

TOXICOLOGY III. Toxicological examination M. Balíková: Toxicological examination

Why to care about toxicological examination Ø Understanding drug effects Ø Differential diagnosis Ø Correct and effective therapy, reduction of adverse drug effects Ø Appropriate subsequent measures – social or forensic M. Balíková: Toxicological examination 2

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 3

General sample requirements for toxicology • Whole blood a) 10 ml into plain tube b) 10 ml into 1 -2% Na. F • 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 4

Drug detection windows a) sample b) method M. Balíková: Toxicological examination

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 Additional tissues Useful after drug ingestion Variable sample May contain high concentrations Analysis may help to interpret postmortem blood data. Quantitative data - problems M. Balíková: Toxicological examination 6

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 7

Toxicological laboratory diagnostics 1) Nonspecific preliminary methods for screening 2) Specific methods ( confirmation, quantification For pharmaceuticals, illegal drugs: ad 1) 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 8

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 M. Balíková: Toxicological examination 9

Immunoassays – Principle - 1 Interaction of the target molecule (antigendrug) with a corresponding antibody To generate measurable signal or visual detection: ØAntibody for the drug being assayed ØLabelled 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 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) M. Balíková: Toxicological examination

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 1) Instrumental methods – flexible, adaptable 2) On site testing – fixed cut off value M. Balíková: Toxicological examination

Cut off value and detection windows Example: THCOOH detection in urine by three various methods – different detection windows M. Balíková: Toxicological examination

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 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 Ø Flexible open systems Ø Possibility to expand update Ø Individual drugs Ø Uncommon drugs Ø More selective Ø Less consumables DISADVANTAGES Ø Sample preparation Ø Time consuming Ø Reference substances needed Ø Experienced personal M. Balíková: Toxicological examination 17

STA and METHOD COMBINATION TLC Ø No instrument Ø Flexible open system Ø Reference standards necessary Ø Lower separation efficiency Ø Lower sensitivity-high drug concentrations Ø Larger sample (urine) GC-MS or LC-MS Ø Instrument necessary Ø Flexible open system Ø Reproducible results Ø Spectra Data Base Ø High separation efficiency Ø High sensitivity-trace analysis (blood) Ø Low sample volume (blood) M. Balíková: Toxicological examination 18

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 M. Balíková: Toxicological examination

Systematic TLC -2 Practical application: Adsorbent : Kieselgel G - Merck Mobile phase: ETOAC-ETOH-NH 3 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 EXAMPLE: METHADONE SUBSTITUTION TARGETED OPIATE CONTROL in URINE PROCEDURES APPLIED: M. Balíková: Toxicological examination

TANDEM GC and MS M. Balíková: Toxicological examination 22

MAS SPECTRA and molecular structure Ø Direct reflection of molecular structure Ø Destructive mass detection Ø Fragmentation reflects strength of bonds Ø Fragmentation rules, logical mass loses Ø Specificity of MS Ø Structure modification to get specific spectra M. Balíková: Toxicological examination 23

Targeted opiates confirmation by GC-MS after positive IA Codeine identification by 1) RETENTION 2) MASS SPECTRA HIGH REPRODUCIBILITY – MASS SPECTRA DATABASES M. Balíková: Toxicological examination

Standard requirements for correct compound identification by GC-MS Ø Acceptable deviations in retention related to standard (CODEINE RI=2375+/-1%) Ø Acceptable deviations in individual fragment mass abundances when compared with standard (CODEINE m/z 299100 229261624612423) Ø Logical molecular structure fragmentation Ø Check for carry over in sample sequence Ø Included controls in sample sequence M. Balíková: Toxicological examination

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 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, harmaline, DMT …. . M. Balíková: Toxicological examination

Ayahuasca case - 2 Subsequently partly determined: Herbal infusion (free bases): Ø atropine 27 mg/L Ø harmine 179 mg/L Ø scopolamine 515 mg/L Estimated ingested dose (cup of 150 m. L): Ø atropine 4 mg Ø harmine 27 mg Ø scopolamine 78 mg M. Balíková: Toxicological examination

Ayahuasca case - 3 Clinical impacts: Ø Ø Ø 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 M. Balíková: Toxicological examination

DOB Case - important experience 4 -bromo-2, 5 -dimethoxy-amphetamine Ø strong hallucinogen, effective dose 1– 3 mg Ø agonist of serotonin receptors M. Balíková: Toxicological examination

DOB Case History - 1 Ø Two men ingested unknown powdered drug Ø Found in open space in coma vomitted with severe cramps Ø Hospitalized in diffent departments Ø Biological fluids sent for toxicology for unknowns Ø Laboratory applied general procedures without initial information what happened Ø One of men developed strong metabolic acidosis p. H 6. 6 – laboratory check for methanol, diols M. Balíková: Toxicological examination

DOB Case History - 2 Immunochemical CEDIA for drug groups in urine: THCOOH COCAIN AMPHETAMINE E S M-28 113 kg M-29 65 kg positive negative M. Balíková: Toxicological examination

DOB Case HISTORY - 3 GC-MS screening for unknowns Hit from MS PMW Library M. Balíková: Toxicological examination

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 Ø To get information about the life style of a subject Ø To document chronic drug abuse in a specific time span Ø To explain the pathological autopsy findings Ø Trace analyses – GC-MS or LC-MS sensitive methods are prerequisite Ø More efficient incorporation of bases than acids M. Balíková: Toxicological examination 36

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 37

Hair analyses for opiates Deceased 24 years old woman Documented chronic variable opiates abuse for approx. 6 months M. Balíková: Toxicological examination 38

Hair analyses for drugs Interpretation Ø Hair – stable sample Ø Biological variability in hair growth and drug incorporation Ø Individual attitude to a case Ø Standards from sampling to interpretation expressed by SOHT (Society for Hair Testing) M. Balíková: Toxicological examination 39

Basic literature: § R. C. Baselt: Disposition of Toxic Drugs and Chemicals in Man. Biomedical Publ. , 6 th 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 40