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Post Mortem Forensic Toxicology

Jeffery Hackett

MSc PhD CSci CChem MRSC

jhackett@unitedchem.com

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What is it?

Post-Mortem Forensic Toxicology: determines the absence or presence of drugs and their metabolites, chemicals such as ethanol and other volatile substances, carbon monoxide and other gases, metals, and other toxic chemicals in human fluids and tissues, and evaluates their role as a determinant or contributory factor in the cause and manner of death

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Where do I find out about it?

Journals:Forensic Science InternationalJ.Forensic ScienceJ. Anal. ToxicologyJ.Chromatogr. B (and A)

Books:R.C. Baselt: Disposition of Toxic Drugs and

Chemicals in ManClarke’s Analysis of Drugs and PoisonsJ.Garriot: Medico-legal Aspects of Alcohol

Interesting cases

• Dr. Crippen: Hyocine

• Georgi Markov: Ricin

• Justin Devilliers: Fentanyl• Ivan Litvanenko: Polonium

• Mrs Cahill: Cyanide• Stacy Castor: Ethylene Glycol

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What’s it all really about

1. How drugs get into people

2. How we get the drugs out

3. How drugs are tested for

4. What does it all mean??

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1. How do drugs get into people??

Intoxicants: Ethanol (Other Alcohols)

Oral administration

Butane (Other gases)

Toluene(also Solvents)

Via Inhalation

Marilyn Monroe?

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Illicit Drugs:

Cannabinoids: Smoked

Amphetamines: Oral

Cocaine/ Heroin :Smoked, Snorted, Injected

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Prescription Drugs:Acetaminophen

PropoxypheneFentanylOxycodoneZolpidemTemazepamMethadone

Mainly oral administration, some have been abused in other ways

Weird and Wonderful

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Spice (JWH-018)Mephedrone MCat

Salvia Divinorum

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Route of Drugs:Oral administration

A.D.M.E.

Drugs will leave from the stomach

Pass through the Liver

Enter into Blood stream/ CSF

Leave through Liver/Kidneys

Pass into Urine

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What does this mean for sampling?

Blood is always important (Site?)

Liver will sequester higher levels of drugs

Lungs are excellent for solvents

Urine is the last point for drugs

Hair has problems all of its own

Vitreous Humor: Alcohol Data

Brain??: Oral Fluid

THC in Blood (LC-MSMS)1 ng per mL

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TIC: from Sample 5 (0.1) of 0112.wiff (Turbo Spray) Max. 1.9e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.030 60 89 119 147 177 206 236

Time, min

0.0

1.0e5

1.9e5

I.

..

2.55

2.06

Printing Time: 3:30:22 PMPrinting Date: Monday, March 09, 2009

Polarity/Scan Type: Negative MRM, ...Analyst Version: 1.4.2

*POST PM BY RHH *API 3200 QTRAP S/N AF011810604

XIC of +MRM (4 pairs): Period 2, 318.3/196.2 amu from Sample 5 (0.1) of 0112.wiff (Turbo Spray) Max. 1.1e5 cps.

2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.46 12 18 24 30 36 42 48 53 59 65 71 77 83 89 95 101 107 112 118 124

Time, min

0.00

1.00e5

I.

..

2.55

Printing Time: 3:30:22 PMPrinting Date: Monday, March 09, 2009

Polarity/Scan Type: Negative MRM, ...Analyst Version: 1.4.2

*POST PM BY RHH *API 3200 QTRAP S/N AF011810604

XIC of +MRM (4 pairs): Period 2, 315.2/193.2 amu from Sample 5 (0.1) of 0112.wiff (Turbo Spray) Max. 1.2e4 cps.

2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.46 12 18 24 30 36 42 48 53 59 65 71 77 83 89 95 101 107 112 118 124

Time, min

0.00

1.00e4

I.

..

2.57

Printing Time: 3:30:22 PMPrinting Date: Monday, March 09, 2009

Polarity/Scan Type: Negative MRM, ...Analyst Version: 1.4.2

*POST PM BY RHH *API 3200 QTRAP S/N AF011810604

XIC of -MRM (4 pairs): Period 1, 346.1/302.3 amu from Sample 5 (0.1) of 0112.wiff (Turbo Spray) Max. 1.9e4 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.213 25 36 48 60 72 83 95 107 119 130

Time, min

0.0

1.0e4

1.9e4

I.

..

2.06

Printing Time: 3:30:22 PMPrinting Date: Monday, March 09, 2009

Polarity/Scan Type: Negative MRM, ...Analyst Version: 1.4.2

*POST PM BY RHH *API 3200 QTRAP S/N AF011810604

XIC of -MRM (4 pairs): Period 1, 343.1/299.3 amu from Sample 5 (0.1) of 0112.wiff (Turbo Spray) Max. 1780.0 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.213 25 36 48 60 72 83 95 107 119 130

Time, min

0

1000

1780

I.

..

2.06

Printing Time: 3:30:22 PMPrinting Date: Monday, March 09, 2009

Polarity/Scan Type: Negative MRM, ...Analyst Version: 1.4.2

*POST PM BY RHH *API 3200 QTRAP S/N AF011810604

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2. Getting the drugs out:

Volatile/Semivolatile: Headspace

Acid drugs : Solvent extraction

+

Basic drugs : Solid phase (micro) extraction

Metals/Metalloids: Digestion

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Headspace Analysis:

• Samples are warmed to create an atmosphere

• Aliquots are taken of the vapour

• This is free of contamination/ interferences

Low boiling:Propane

Medium : Ethanol

High : Toluene

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Solvent extraction:

pH Modification

Addition of acid/ base

COOH COO- + H+

H+ + R-COO- COOH

Neutral Species are more soluble in organic solvents

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Solid phase extraction: Samples are pH modified

Filtered through porous sorbents

Drugs are collected onto modified surfaces

Interferences are washed off

Compounds of interest are eluted off

**pH= pKa +2**

pH=pKa +log [ ionised/unionised]

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Digestion:Dry digestion

Samples are: heated, ignited and ashedResidue is dissolved in dilute acid

Good for Copper, Lead, Zinc etcWet digestion

Samples are heated in strong acid solutionSolution is reduced in volume, neutralized and

diluted Good for volatile metals: Mercury

Thallium

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Drug Testing:

Immunoassay:

ELISA/EMIT/FPIA/RIA

Chromatography:

Gas Chromatography (FID, ECD, MS)

Liquid Chromatography (PDA, FLD, MS/MS)

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ELISA Instrumentation

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Method:

*Immunoassays are Presumptive Tests

*All operators are competency trained.

Calibrators are matrix matched

Set at Negative, Cut off and Positive values

Good for Blood, Urine and Tissue

Wide range of analytes available

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ELISA Analysis:Analyte Negative Cut-Off Positive  

Amphetamine 2.816 1.023 0.181 0.992 2.562

Barbiturate 1.609 0.504 0.196 1.266 1.555

Benzodiazepine 2.151 0.413 0.113 2.136 0.155

Cannabinoids 1.238 0.658 0.398 1.326 0.497

Cocaine 1.070 0.739 0.430 1.045 0.114

Methadone 1.240 0.668 0.156 1.426 1.529

Methamphetamine 2.170 0.799 0.310 1.658 2.069

Opiate 1.393 0.331 0.145 1.504 0.104

Oxycodone 1.704 1.266 0.488 1.612 1.039

Phencylcidine 2.195 1.210 0.793 2.237 2.332

 

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Gas-ChromatographyInstrumentation

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Example of Gas-Chromatography

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Gas-Chromatography Mass Spectrometry

3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

Time-->

Abundance

TIC: 0401005.D

3.74 4.22 4.29

5.18

5.37

5.52

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Mass Spectrometry

50 100 150 200 250 300 350 400 4500

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

m/z-->

Abundance

Scan 299 (4.820 min): 0201003.D312

286

266

238

183

109 21075 144

51163

353 376 421 462397

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Liquid ChromatographyInstrumentation

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Example of Liquid Chromatography: (xanthines: Theophylline/ Theobrmine/ Caffeine/ 8-

Chlorotheophylline)

TWC of DAD Spectral Data: from Sample 8 (600) of dec112007d.wiff Max. 4.4e4 mAU.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23Time, min

-2000.0

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

Ab

so

rba

nc

e, m

AU

14.28

7.549.43

5.81 17.80

6.24

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Different Detectors (LC)UV and Fluorescence

min5 6 7 8 9 10 11

mAU

0

1

2

3

4

DAD1 E, Sig=280,5 Ref=360,100 (062506JA\044-0501.D)

6.27

3

9.35

4

min5 6 7 8 9 10 11

LU

2.6

2.8

3

3.2

3.4

3.6

3.8

FLD1 A, Ex=280, Em=390 (062506JA\044-0501.D)nm200 225 250 275 300 325 350 375

mAU

0

2

4

6

8

10

12

14

16

DAD1, 6.349 (17.3 mAU,Bln) of 044-0501.D

LC-MSMS

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Chromatographic Methods

• Use Validated/Peer Reviewed ones• Internal Standards (IS) must match analytes• Best IS are: deuterated (MS)

: Substituted Analogues (FID,NPD)

: Non-drug materials

extraction involves : Volatile Solvents (GC)

: Miscible Solvents (LC)

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Chromatographic Analysis Involves:

Calibration curves with known standards and controls

Derivitization with recognized modifiers (BSTFA, PFPA)

Recognized detection methods(SIM, MRM, PDA)

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Calibration Curve:THC and Metabolite

Untitled 5 (THC 1): "Linear" Regression ("1 / x" weighting): y = 0.114 x + -0.00295 (r = 0.9996)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50Analyte Conc. / IS Conc.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

5.8

An

aly

te

A

re

a / IS

A

re

a

Untitled 5 (THC-COOH 1): "Linear" Regression ("1 / x" weighting): y = 0.0899 x + -0.0213 (r = 0.9968)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50Analyte Conc. / IS Conc.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

4.6

An

aly

te

A

re

a / IS

A

re

a

THC (0.25 to 50 ng/ mL)

Carboxy-THC (0.25 to 50 ng/ mL)

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4. What does it all mean: Toxicology Report

RESULTS: Blood (Iliac): Ethyl Alcohol – Not Detected5

Benzoylecgonine – 0.05 mg/L1,2,4 Sertraline – 0.11 mg/L2,4 Desmethylsertraline – 0.01 mg/L2,4 Quetiapine – 0.50 mg/L Negative for Acetaminophen, Amitriptyline, Barbiturates, Chlordiazepoxide, Citalopram, Clomipramine, Cocaethylene, Cocaine, Desipramine, Diazepam, Doxylamine, Ibuprofen, Imipramine, Meperidine, Methadone, Methamphetamine, N-desmethyltramadol, Norpropoxyphene, Pentazocine, Phenytoin, Procainamide, Procaine, Strychnine, Thioridazine, Tramadol, Trazodone, Trimipramine, Venlafaxine, and Verapamil.2,4

For example use only………Not a real report.

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Interpretation of Drugs:THC Cocaine Heroin Amphetamine

1st

Carboxy-THC

1st BE

2nd EME

1st 6AM

2nd Morph

3rd M3G/6G

Various:

MDMA to

MDA

Time in blood :Hrs

Time in blood :Hrs Time in blood :Min*

Time in blood :Hrs

Time in Urine :days/wks

Time in Urine : 1day

Time in Urine :Hrs

Time in Urine :days

Psilocybin/ Psilocin Question

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PB IS PC

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Interpretation of Alcohol:Clinical Values

BAC/g% Effect0.02 Relaxed

0.05 Tranquil0.10 Coordination problems

0.20 Intoxication Obvious0.30 Passing Out0.40 Comatose0.50 Death

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Post Mortem Alcohol:A Point of View

Widmark:BAC= 100* A

Wt*Wf

Back-Extrapolation: *Post Absorptive Period Linear Elimination No Alcohol Consumption

**Should not be applied because of the number variables**

New Issue (EtG/ EtS)

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Ethylglucuronide molecule

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My Thanks:

Albert Elian

Bob Osiwiecz (Erie Co. Med Examiner Lab)

J. Spencer

Mike Telepchak