m. balíková: toxicological examination 1 toxicology iii. toxicological examination m. balíková

M. Balíková: Toxicological examination

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TOXICOLOGYTOXICOLOGY

III. Toxicological examinationIII. Toxicological examination

M. BalíkováM. Balíková


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Why to care about Why to care about toxicological toxicological examinationexamination

Understanding drug effectsUnderstanding drug effectsDifferential diagnosisDifferential diagnosisCorrect and effective therapy, Correct and effective therapy,

reduction of adverse drug reduction of adverse drug effectseffects

Appropriate subsequent Appropriate subsequent measures – social or forensicmeasures – social or forensic


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Information in a request Information in a request for toxicological for toxicological

examinationexamination• Name of a person requiring Name of a person requiring

examination, date of sampling, date of examination, date of sampling, date of autopsyautopsy

• Circumstances of an incident, Circumstances of an incident, estimated time of drug estimated time of drug application/time of deathapplication/time of death

• Medical history of a person, medical Medical history of a person, medical treatmenttreatment

• Note of occupation, hobiesNote of occupation, hobies• Clinical symptoms/Preliminary Clinical symptoms/Preliminary

pathological report, if availablepathological report, if available


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General sample General sample requirements for requirements for

toxicologytoxicology• Whole bloodWhole blood

a) 10 ml into plain tubea) 10 ml into plain tubeb) 10 ml into 1-2% NaFb) 10 ml into 1-2% NaF

• UrineUrine, 50-100 ml, 50-100 ml (liver, kidney - can substitute urine (liver, kidney - can substitute urine

if not available) if not available)• Gastric content, 50 – 100 mlGastric content, 50 – 100 ml• Scene materialScene material• Other tissues (brain, lung, vitreous Other tissues (brain, lung, vitreous

humour, fat, hair……)humour, fat, hair……)


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Drug detection windowsDrug detection windowsa) samplea) sampleb) methodb) method


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Different sample typesDifferent sample typesSpecimeSpecimenn

AdvantageAdvantage DisadvantaDisadvantagege

CommentComment

BloodBlood Present Present parent parent compoundscompounds

QuantitationQuantitation

Limited Limited volumevolume

Trace Trace concentratioconcentrationsns

Careful Careful individual individual interpretatiinterpretationon

UrineUrine Large Large volumevolume

High High concentr.concentr.

Easier/Easier/longer longer detectiondetection

Often not Often not availableavailable

MetabolitesMetabolites

Quantitative Quantitative data not data not usefuluseful

Standard Standard sample for sample for initial initial screeningscreening

Gastric Gastric contentcontent

Useful after Useful after drug drug ingestioningestion

Variable Variable samplesample

AdditionAdditional al tissuestissues

May contain May contain high high concentratioconcentrationsns

Analysis may help to Analysis may help to interpret postmortem interpret postmortem blood data.blood data. Quantitative Quantitative data - problemsdata - problems


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

the pastthe past• Drugs are located in the hair in Drugs are located in the hair in

relation to the time when appeared in relation to the time when appeared in blood blood

• Different hair growth rate at the bodyDifferent hair growth rate at the body• Cyclus of hair growth, anagen 85% ….Cyclus of hair growth, anagen 85% ….• Ideal sample at the vertex of the headIdeal sample at the vertex of the head• Before cutting close to the skin - tie Before cutting close to the skin - tie

the strand with the cotton threadthe strand with the cotton thread• Correct sampling: Correct sampling:


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Toxicological laboratory Toxicological laboratory diagnosticsdiagnostics

1)1) Nonspecific preliminary methods for Nonspecific preliminary methods for screeningscreening

2)2) Specific methods ( confirmation, Specific methods ( confirmation, quantificationquantification

For pharmaceuticals, illegal drugs:For pharmaceuticals, illegal drugs:ad1) Immunochemical screening, colour ad1) Immunochemical screening, colour

reactionsreactionsad 2) Chromatographic systems, mass ad 2) Chromatographic systems, mass

spectrometry (MS)spectrometry (MS)

UNKNOWN DRUG – Systematic toxicological analysis – UNKNOWN DRUG – Systematic toxicological analysis – STA STA logical sequence of applied methodslogical sequence of applied methods


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Immunoassays in Immunoassays in toxicologytoxicology

ADVANTAGESADVANTAGES• Technically Technically

simplesimple• SensitiveSensitive• Rapid Rapid • Important initial Important initial

information information about sampleabout sample

• On site On site performanceperformance

DISADVANTAGESDISADVANTAGES• Not all drugs detectedNot all drugs detected• Group detection onlyGroup detection only• Potentional Potentional

interferencesinterferences• Preliminary resultsPreliminary results• Confirmation Confirmation

necessarynecessary• Continuous reagent Continuous reagent

consumption, supplyconsumption, supply


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Immunoassays – Principle Immunoassays – Principle - 1- 1

Interaction of the target molecule Interaction of the target molecule ((antigen-drugantigen-drug) with a corresponding) with a corresponding antibodyantibody

To generate measurable signal or visual To generate measurable signal or visual detection:detection:

Antibody for the drug being assayedAntibody for the drug being assayed

Labelled form of the structurally related Labelled form of the structurally related drug drug or labelled antibodyor labelled antibody

Competition between the antigen in a sample and Competition between the antigen in a sample and labelled antigen (reagent) for binding to the fixed labelled antigen (reagent) for binding to the fixed amount of the antibody to create the specific amount of the antibody to create the specific immunocompleximmunocomplex::


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Immunoassays – Principle Immunoassays – Principle - 2- 2

The proportionThe proportion of labelled drug molecules of labelled drug molecules bound in the complex is inversely proportional bound in the complex is inversely proportional to the number of unlabelled drug molecules in to the number of unlabelled drug molecules in the samplethe sample

VARIOUS SPECIFICITY of ANTIBODIESVARIOUS SPECIFICITY of ANTIBODIES

1) for 1) for SCREENINGSCREENING – detection of selected – detection of selected groups groups (barbiturates)(barbiturates)

2) for 2) for QUANTITATIONQUANTITATION of specific individual of specific individual (phenobarbitone)(phenobarbitone)


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INITIAL TOXICOLOGICAL INITIAL TOXICOLOGICAL SCREENING by SCREENING by

IMMUNOASSSAYSIMMUNOASSSAYSAdopted cut off valuesAdopted cut off values

Cut off need not be Cut off need not be LOD LOD

Lower cut off – more FPLower cut off – more FP

High cut off – more FNHigh cut off – more FN

Positive detection need to Positive detection need to be confirmed by a specific be confirmed by a specific method (GC-MS)method (GC-MS)

Confirmatory method – Confirmatory method – higher sensitivityhigher sensitivity


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Immunoscreening Immunoscreening Cut off valuesCut off values

Selection negative / positive Selection negative / positive samples by definitionsamples by definition

1)1) Instrumental methods – flexible, Instrumental methods – flexible, adaptableadaptable

2)2) On site testing – fixed cut off valueOn site testing – fixed cut off value


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Cut off value and detection Cut off value and detection windowswindows

Example:Example:

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


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Various sVarious specificity of antibodies pecificity of antibodies Compound cross reactivities Compound cross reactivities

1) 1) Different reactivities of Different reactivities of related drugs in a related drugs in a selected group – selected group – insufficient sensitivity insufficient sensitivity for MDMA detection -for MDMA detection - FNFN

Modification of cut off – Modification of cut off – method adaptation for method adaptation for MDMA detection tooMDMA detection too

2) Adulterants, dilution - FN

3) Reactivities of unwanted substances – FP


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CONFIRMATIONCONFIRMATION

Basic principle in forensic toxicology :Basic principle in forensic toxicology :

Verification of preliminary results by Verification of preliminary results by another independent method – more another independent method – more specific, more sensitivespecific, more sensitive

Identification of specific compoundsIdentification of specific compounds

Individual compounds differ in Individual compounds differ in potency/toxicity (codeine – morphine)potency/toxicity (codeine – morphine)

Forensic aspects for drug Forensic aspects for drug distinguishing distinguishing


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Drug detection by Drug detection by chromatographychromatography

ADVANTAGESADVANTAGES Flexible open Flexible open

systemssystems Possibility to expand Possibility to expand

and updateand update Individual drugsIndividual drugs Uncommon drugsUncommon drugs More selectiveMore selective Less consumablesLess consumables

DISADVANTAGESDISADVANTAGES Sample Sample

preparationpreparation Time consumingTime consuming Reference Reference

substances substances neededneeded

Experienced Experienced personalpersonal


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STA and METHOD COMBINATIONSTA and METHOD COMBINATION

TLCTLC No instrumentNo instrument Flexible open systemFlexible open system Reference standards Reference standards

necessarynecessary Lower separation Lower separation

efficiencyefficiency Lower sensitivity-Lower sensitivity-

high drug high drug concentrationsconcentrations

Larger sample (urine)Larger sample (urine)

GC-MS or LC-MSGC-MS or LC-MS Instrument necessaryInstrument necessary Flexible open systemFlexible open system Reproducible resultsReproducible results Spectra Data BaseSpectra Data Base High separation efficiencyHigh separation efficiency High sensitivity-trace High sensitivity-trace

analysis (blood)analysis (blood) Low sample volume Low sample volume

(blood)(blood)


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Systematic TLC -1Systematic TLC -1Various systems developed in the worldVarious systems developed in the world

System by J. Večerková used overal System by J. Večerková used overal Czechoslovakia since 1970Czechoslovakia since 1970

SCREENINGSCREENING – 3 indicators to assess: – 3 indicators to assess:

1)1) Extractability – compound acidobasicityExtractability – compound acidobasicity

2)2) Compound mobility related to reference Compound mobility related to reference mixturemixture

3)3) Colour detection by set of reagentsColour detection by set of reagents

CONFIRMATIONCONFIRMATION >>> >>> Potential drug Potential drug candidates to be confirmed in other candidates to be confirmed in other chromatographic conditions with specific chromatographic conditions with specific reference standardsreference standards


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Systematic TLC -2Systematic TLC -2Practical application:Practical application:

Adsorbent : Kieselgel G - Adsorbent : Kieselgel G - MerckMerck

Mobile phase: ETOAC-ETOH-Mobile phase: ETOAC-ETOH-NH3NH3

11 – – REFERENCE STANDARDSREFERENCE STANDARDS

22- - URINE EXTRACT of BASESURINE EXTRACT of BASES

33- - URINE EXTRACT after URINE EXTRACT after HYDROLYSISHYDROLYSISPotentional intoxication with Potentional intoxication with

opiates and opiates and methamphetamine with methamphetamine with presence of nicotine – presence of nicotine – subsequent confirmation in subsequent confirmation in another system TLC or GC-MSanother system TLC or GC-MS


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FLEXIBLE METHOD FLEXIBLE METHOD APPLICATION in CONFIRMATIONAPPLICATION in CONFIRMATION

EXAMPLE: METHADONE EXAMPLE: METHADONE SUBSTITUTIONSUBSTITUTIONTARGETED OPIATE CONTROL in URINETARGETED OPIATE CONTROL in URINE

PROCEDURES APPLIED:PROCEDURES APPLIED:


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TANDEM GC and MSTANDEM GC and MS


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MAS SPECTRA and MAS SPECTRA and molecular structuremolecular structure

Direct reflection of molecular structureDirect reflection of molecular structure Destructive mass detectionDestructive mass detection Fragmentation reflects strength of Fragmentation reflects strength of

bondsbonds Fragmentation rules, logical Fragmentation rules, logical

mass losesmass loses Specificity of MSSpecificity of MS Structure modification Structure modification

to get specific spectrato get specific spectra


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Targeted opiates Targeted opiates confirmation by GC-MS confirmation by GC-MS

after positive IAafter positive IA Codeine Codeine

identificatioidentification byn by

1)1) RETENTIONRETENTION

2)2) MASS MASS SPECTRASPECTRA

HIGH REPRODUCIBILITY – MASS SPECTRA DATABASES


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Standard requirements for Standard requirements for correct compound correct compound

identification identification by GC-MSby GC-MS

Acceptable deviations in retention related to Acceptable deviations in retention related to standard standard (CODEINE RI=2375+/-1%)(CODEINE RI=2375+/-1%)

Acceptable deviations in individual fragment Acceptable deviations in individual fragment mass mass abundances abundances when compared when compared with standard with standard (CODEINE m/z 299(CODEINE m/z 299100 100

229229262616216246461241242323))

Logical molecular structure fragmentationLogical molecular structure fragmentation

Check for carry over in sample sequence Check for carry over in sample sequence

Included controls in sample sequenceIncluded controls in sample sequence


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GS-MS screening for GS-MS screening for unknownunknown drugdrug

Many psychoactive substances causing Many psychoactive substances causing intoxication need intoxication need not to be detected by not to be detected by commercial immunoassayscommercial immunoassays, e. g.:, e. g.:

Atropine, scopolamine,DMT…. Atropine, scopolamine,DMT….

Synthetic drugs, techno drugsSynthetic drugs, techno drugs

GHB, ………….GHB, ………….

Some of them can be detected by GC-MS Some of them can be detected by GC-MS screening system for unknownsscreening system for unknowns

Some of them to be detected require specific Some of them to be detected require specific sample preparation, specific chromatography sample preparation, specific chromatography (GHB) – suspicious cases, not routinely(GHB) – suspicious cases, not routinely


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Ayahuasca case - 1 case - 1Collective session of lucid thinking Collective session of lucid thinking supported by some oriental plant extract-supported by some oriental plant extract-more than 35 participants aged 20 – 50 yearsmore than 35 participants aged 20 – 50 years

40-60 min after ingestion participants 40-60 min after ingestion participants started to be agitated, agressive, with started to be agitated, agressive, with tachycardia, hyperthermia, dry skin, tachycardia, hyperthermia, dry skin, excessive salivation, some people fell into excessive salivation, some people fell into coma – coma – medicalmedical rescue service called rescue service called Urgent Urgent clinical need to clinical need to idetify the idetify the toxic agent – toxic agent –

for diferential diagnosis for diferential diagnosis

By laboratory GC-MS screening for uknown By laboratory GC-MS screening for uknown drugs identified in tea extract and in human drugs identified in tea extract and in human samples of blood, urine or gastric content : samples of blood, urine or gastric content : atropine,scopolamine, harmine, harmaline, atropine,scopolamine, harmine, harmaline, DMT DMT …..…..


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Ayahuasca case - 2 case - 2Subsequently partly determined: Subsequently partly determined:

Herbal infusion (free bases):Herbal infusion (free bases): atropine 27 mg/Latropine 27 mg/L harmine 179 mg/Lharmine 179 mg/L scopolamine 515 mg/Lscopolamine 515 mg/L

Estimated ingested dose (cup of Estimated ingested dose (cup of 150mL):150mL): atropine 4 mgatropine 4 mg harmine 27 mgharmine 27 mg scopolamine 78 mgscopolamine 78 mg


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Ayahuasca case - 3 case - 3Clinical impacts:Clinical impacts: All patients recoveredAll patients recovered Some were dismissed from hospitals after a Some were dismissed from hospitals after a few hrsfew hrs Some requirred intensice care for some daysSome requirred intensice care for some days

Forensic impacts:Forensic impacts:

The organizator taken into detention by police The organizator taken into detention by police and investigated – why? Defended himself with and investigated – why? Defended himself with no intention to harmno intention to harm anybody. Legal problem to anybody. Legal problem to prove the intention. Sent for psychiatric prove the intention. Sent for psychiatric examination. Found irresponsible for his examination. Found irresponsible for his behavior – dissmissed from detention without behavior – dissmissed from detention without any sanction. (However, ???)any sanction. (However, ???)


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IA missing response for IA missing response for structuraly related structuraly related amphetamines- FNamphetamines- FN


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DOB Case - important experience

4-bromo-2,5-dimethoxy-amphetamine4-bromo-2,5-dimethoxy-amphetamine

strong hallucinogen, effective dose strong hallucinogen, effective dose 1–3 mg1–3 mg

agonist of serotonin receptorsagonist of serotonin receptors


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DOB Case History - 1DOB Case History - 1

Two men ingested unknown powdered drug Two men ingested unknown powdered drug

Found in open space in coma vomitted with Found in open space in coma vomitted with severe severe crampscramps

Hospitalized in diffent departmentsHospitalized in diffent departments

Biological fluids sent for toxicology for Biological fluids sent for toxicology for unknownsunknowns

Laboratory applied general procedures Laboratory applied general procedures without without initial information what happenedinitial information what happened

One of men developed strong metabolic One of men developed strong metabolic acidosis acidosis pH 6.6 – laboratory check for pH 6.6 – laboratory check for methanol, diolsmethanol, diols


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DOB Case History - 2DOB Case History - 2

Immunochemical CEDIA for drug groups in Immunochemical CEDIA for drug groups in urine:urine:

THCOOTHCOOHH

COCAINCOCAINEE

AMPHETAMINEAMPHETAMINESS

M-28113 kg

positivepositive positivepositive negativenegative

M-2965 kg

positivepositive negativenegative negativenegative


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DOB Case HISTORY - 3DOB Case HISTORY - 3GC-MS GC-MS

screening screening for for

unknownsunknowns

Hit from Hit from MS PMW MS PMW LibraryLibrary


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DOB Case HISTORY - 4DOB Case HISTORY - 4

GC-MS GC-MS confirmation by confirmation by targeted targeted analysis for analysis for amines after amines after acetylationacetylation

Metabolism not Metabolism not known – known – research research biotransformatibiotransformation studyon study


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Hair analysis for drugsHair analysis for drugs

To get information about the life To get information about the life style of a subjectstyle of a subject

To document chronic drug abuse in To document chronic drug abuse in a specific time spana specific time span

To explain the pathological autopsy To explain the pathological autopsy findingsfindings

Trace analyses – GC-MS or LC-MS Trace analyses – GC-MS or LC-MS sensitive methods are prerequisitesensitive methods are prerequisite

More efficient incorporation of More efficient incorporation of bases than acidsbases than acids


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Hair analyses for Hair analyses for methamphetaminemethamphetamine

30 years old 30 years old man died man died shortly after shortly after drug drug application . application . Autopsy Autopsy revealed revealed arteriosclerosis arteriosclerosis and bleeding and bleeding into the into the cerebellum. cerebellum. Hair analyses Hair analyses documented documented chronic MA chronic MA abuse for abuse for approx. 8 approx. 8 months.months.


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Hair analyses for opiatesHair analyses for opiates

Deceased Deceased 24 years 24 years old old womanwoman

DocumentDocumented chronic ed chronic variable variable opiates opiates abuse for abuse for approx. 6 approx. 6 monthsmonths


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Hair analyses for drugs Hair analyses for drugs InterpretationInterpretation

Hair – stable sampleHair – stable sampleBiological variability in hair Biological variability in hair

growth growth and drug and drug incorporationincorporation

Individual attitude to a caseIndividual attitude to a caseStandards from sampling to Standards from sampling to

interpretation expressed by interpretation expressed by SOHT (Society for Hair Testing)SOHT (Society for Hair Testing)


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Basic literature:Basic literature: R. C. Baselt: Disposition of Toxic Drugs and Chemicals in Man. R. C. Baselt: Disposition of Toxic Drugs and Chemicals in Man.

Biomedical Publ., 6th ed., 2002 , Foster City, Ca. Biomedical Publ., 6th ed., 2002 , Foster City, Ca. B. Brinkman, B. Madea: Handbuch Gerichtliche Medizin. Springer B. Brinkman, B. Madea: Handbuch Gerichtliche Medizin. Springer

Vrlg. 2002, Berlin.Vrlg. 2002, Berlin. S. Moeschlin: Klinik und Therapie der Vergiftungen. George Thieme S. Moeschlin: Klinik und Therapie der Vergiftungen. George Thieme

Vrlg. 1986, Stuttgart.Vrlg. 1986, Stuttgart. H. Lüllmann, K. Mohr, M. Wehling: Pharmakologie und Toxikologie, H. Lüllmann, K. Mohr, M. Wehling: Pharmakologie und Toxikologie,

George Thieme Vrlg. 1999, Stuttgart.George Thieme Vrlg. 1999, Stuttgart. R. J. Flanagan, A. Taylor, I. D. Watson. R. Whelpton: Fundamental of R. J. Flanagan, A. Taylor, I. D. Watson. R. Whelpton: Fundamental of

Analytical Toxicology, Wiley, 2007, West Sussex.Analytical Toxicology, Wiley, 2007, West Sussex. S, B. Karch, ed.: Drug Abuse Handbook, CRC Press, 1998, Boca Raton.S, B. Karch, ed.: Drug Abuse Handbook, CRC Press, 1998, Boca Raton. O. H. Drummer, M. Odell: The Forensic Pharmacology of Drugs of O. H. Drummer, M. Odell: The Forensic Pharmacology of Drugs of

Abuse. Arnold Publ. 2001, London.Abuse. Arnold Publ. 2001, London. F. Pragst, M. Balíková: State of the art in hair analysis for detection of 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-drug and alcohol abuse. A review. Clinica Chimica Acta 370 (2006) 17-49.49.

m. balíková: toxicological examination 1 toxicology iii. toxicological examination m. balíková

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