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Post Mortem Forensic Toxicology. Jeffery Hackett MSc PhD CSci CChem MRSC [email protected]. What is it?. - PowerPoint PPT Presentation
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sufw2010
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