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 International
J.Forensic Science
J. Anal. Toxicology
J.Chromatogr. B (and A)
Books:
R.C. Baselt: Disposition of Toxic Drugs and
Chemicals in Man
Clarke’s Analysis of Drugs and Poisons
J.Garriot: Medico-legal Aspects of Alcohol
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Interesting cases
• Dr. Crippen:
Hyocine
• Georgi Markov:
Ricin
• Justin Devilliers:
• Ivan Litvanenko:
Fentanyl
Polonium
• Mrs Cahill:
• Stacy Castor:
Cyanide
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
Propoxyphene
Fentanyl
Oxycodone
Zolpidem
Temazepam
Methadone
Mainly oral administration, some have been abused in other ways
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Weird and Wonderful
Mephedrone
MCat
Spice (JWH-018)
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
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THC in Blood (LC-MSMS)
1 ng per mL
Polarity/Scan Type: Negative MRM, ...
Analyst Version: 1.4.2
.
Printing Time: 3:30:22 PM
Printing Date: Monday, March 09, 2009
Max. 1.9e5 cps.
.
TIC: from Sample 5 (0.1) of 0112.wiff (Turbo Spray)
2.55
.
1.9e5
1.0e5
I
2.06
0.0
0.5
30
1.0
60
1.5
89
2.0
119
2.5
147
3.0
177
3.5
206
4.0
236
.
Time, min
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.
.
I
1.00e5
2.55
0.00
2.5
12
2.6
18
2.7
24
2.8
30
2.9
36
3.0
42
3.1
48
3.2
53
3.3
59
3.4
65
Time, min
XIC of +MRM (4 pairs): Period 2, 315.2/193.2 amu from Sample 5 (0.1) of 0112.wiff (Turbo Spray)
3.5
71
3.6
77
3.7
83
3.8
89
3.9
95
4.0
101
4.1
107
4.2
112
4.3
118
4.4
124
.
2.4
6
.
Max. 1.2e4 cps.
I
.
2.57
1.00e4
0.00
2.5
12
2.6
18
2.7
24
2.8
30
2.9
36
3.0
42
3.1
48
3.2
53
3.3
59
3.4
65
Time, min
XIC of -MRM (4 pairs): Period 1, 346.1/302.3 amu from Sample 5 (0.1) of 0112.wiff (Turbo Spray)
3.5
71
3.6
77
3.7
83
3.8
89
3.9
95
4.0
101
4.1
107
4.2
112
4.3
118
4.4
124
.
2.4
6
.
Max. 1.9e4 cps.
2.06
.
1.9e4
I
1.0e4
0.0
0.4
25
0.6
36
0.8
48
1.0
60
1.2
72
Time, min
XIC of -MRM (4 pairs): Period 1, 343.1/299.3 amu from Sample 5 (0.1) of 0112.wiff (Turbo Spray)
1.4
83
1.6
95
1.8
107
2.0
119
2.2
130
.
0.2
13
.
Max. 1780.0 cps.
.
1780
2.06
I
1000
0
*POST PM BY RHH
0.2
13
0.4
25
0.6
36
0.8
48
1.0
60
1.2
72
Time, min
*API 3200 QTRAP S/N AF011810604
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1.4
83
1.6
95
1.8
107
2.0
119
2.2
130
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 ashed
Residue is dissolved in dilute acid
Good for Copper, Lead, Zinc etc
Wet digestion
Samples are heated in strong acid solution
Solution 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-Chromatography
Instrumentation
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Example of GasChromatography
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Gas-Chromatography
Mass Spectrometry
Abundance
T IC: 0 4 0 1 0 0 5 .D
5.18
2600000
2400000
2200000
2000000
1800000
1600000
1400000
1200000
5.37
1000000
5.52
800000
600000
400000
3.74
4.29
4.22
200000
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
Time-->
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7.50
8.00
8.50
9.00
9.50
Mass Spectrometry
Abundanc e
Sc an
299
(4 . 8 2 0
m in ): 0 2 0 1 0 0 3 . D
312
1400000
286
1300000
1200000
1100000
1000000
900000
800000
700000
266
600000
238
500000
400000
183
300000
200000
75
100000
109
51
144
210
163
3 5 33 7 63 9 74 2 1
0
50
100
150
200
250
m / z -->
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300
350
400
462
450
Liquid Chromatography
Instrumentation
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Example of Liquid Chromatography: (xanthines:
Theophylline/ Theobrmine/ Caffeine/ 8Chlorotheophylline)
TWC of DAD Spectral Data: from Sample 8 (600) of dec112007d.wiff
Max. 4.4e4 mAU.
14.28
4.4e4
4.2e4
4.0e4
3.8e4
3.6e4
3.4e4
7.54
9.43
3.2e4
A b s o rb a n c e , m A U
3.0e4
2.8e4
2.6e4
2.4e4
2.2e4
2.0e4
1.8e4
1.6e4
1.4e4
1.2e4
1.0e4
8000.0
5.81
17.80
6000.0
4000.0
2000.0
6.24
0.0
-2000.0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Time, min
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14
15
16
17
18
19
20
21
22
23
Different Detectors (LC)
UV and Fluorescence
DAD1, 6.349 (17.3 mAU,Bln) of 044-0501.D
9.354
6.273
DAD1 E, Sig=280,5 Ref=360,100 (062506JA\044-0501.D)
mAU
mAU
16
4
14
12
3
10
8
2
6
1
4
2
0
0
5
6
FLD1 A, Ex=280, Em=390 (062506JA\044-0501.D)
7
8
9
10
11
min
7
8
9
10
11
min
LU
3.8
3.6
3.4
3.2
3
2.8
2.6
5
6
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200
225
250
275
300
325
350
375
nm
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)
5.8
5.5
5.0
A n a ly te A re a / IS A re a
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
Analyte Conc. / IS Conc.
30
32
34
36
38
40
42
44
46
48
50
44
46
48
50
THC (0.25 to 50 ng/ mL)
Untitled 5 (THC-COOH 1): "Linear" Regression ("1 / x" weighting): y = 0.0899 x + -0.0213 (r = 0.9968)
4.6
4.4
4.2
4.0
3.8
3.6
A n a ly te A re a / IS A re a
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
Analyte Conc. / IS Conc.
30
32
34
36
38
40
42
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
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Psilocybin/ Psilocin Question
PB
IS PC
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Interpretation of Alcohol:
Clinical Values
BAC/g%
0.02
0.05
0.10
0.20
0.30
0.40
0.50
Effect
Relaxed
Tranquil
Coordination problems
Intoxication Obvious
Passing Out
Comatose
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**
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New Issue (EtG/ EtS)
Ethylglucuronide molecule
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My Thanks:
Albert Elian
Bob Osiwiecz (Erie Co. Med Examiner Lab)
J. Spencer
Mike Telepchak
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