shawn p. vorce technical director chesapeake toxicology
TRANSCRIPT
Shawn P. Vorce
Technical Director
Chesapeake Toxicology Resources
"The opinions or assertions presented hereafter are the private views of Shawn P. Vorce and should not be construed as official or as reflecting the views of the Department of Defense, its branches, the US Army Medical Research and Materiel Command or the Armed Forces Medical Examiner System.“
Forensic Toxicology is defined as the application of toxicology for the purposes of the law
Postmortem Toxicology Death investigations Cause and manner
Human Performance Toxicology DUI/DWI DFSA
Forensic Drug Testing Military Criminal Justice System Workplace drug testing Athletic programs
Accessioning
Screening
negative positive Confirmation/Quantitation
Issue Report
Consultation/Testimony
Special Testing
Method Development
Screening◦ Immunoassay (IA)
◦ Thin Layer Chromatography (TLC)◦ Headspace (GC/FID)
◦ Gas Chromatography (NPD/FID)◦ Gas Chromatography/Mass Spectrometry (GC/MS)
Confirmation/Quantitation◦ Gas Chromatography/Mass Spectrometry (GC/MS)
Full Scan
Selected Ion Monitoring (SIM)
◦ Liquid Chromatography/Mass Spectrometry (LC/MS)◦ Liquid Chromatography Tandem Mass Spectrometry
(LC/MS/MS)
◦ Time of Flight Mass Spectrometry (TOF)
Immunoassay Screening
◦ EMIT enzyme multiplied immunoassay technique
◦ CEDIA cloned enzyme donor immunoassay
◦ ELISA enzyme-linked immunosorbent assay
◦ FPIA fluorescence polarization Immunoassay
◦ KIMS kinetic interaction of micro-particles in solution
◦ DRI Diagnostic Reagent Inc
Immunoassay Screening◦ EMIT
Sample, Antibody, Enzyme-drug complex, Substrate
Drug
Sample
Drug
Enzyme-drug complex
Drug
substrate
Absorbs light λ= 340 nm
Sample
substrate
Does NOT Absorb light
Immunoassay Screening
Negative Result
Positive Result
IA Cross Reactivity
Benzodiazepines (CEDIA)
◦ Oxazepam antibodies
◦ 200 ng/mL cut-off
◦ Package insert list interfering compounds and levels
IA Cross Reactivity
Amphetamines (CEDIA)
◦ Amphetamine antibodies
◦ 500 ng/mL cut-off
◦ Package insert list interfering compounds and levels
Compound 500 ng/mL Cut-off
Bupropion 13000
Fenfluramine 1200
Phenmetrazine 1500
Phenothiazines 10000
Phentermine 17500
Pseudoephedrine 75000
DMAA 2500
Methamphetamine 500
MDA 1400
MDMA 800
Headspace-GC/FID
BAC
◦ Headspace Sample and ISTD are
transferred to a headspace vial
Vial is heating to a constant Temp (90oC).
Volatile component diffuse into gas phase forming equilibrium between liquid and headspace.
Sample is taken from the Headspace and analyzed. H
eadspace
BAC◦ Headspace
http://blog.restek.com/wp-content/uploads/2014/05/HS-GC-Instrument-Diagram2.jpg
BAC◦ Headspace (GC/FID)
Acetaldehyde
Ethanol
2-Propanol
ISTD
Acetone
0.0 0.02 0.05 0.08 0.10 0.12 0.15
Ethanol Concentration
Eth
anol Response/
ISTD
Response
0.0
0
.50 1.0
0 1
.50 2
.00 2
.50 3
.00
= 0.088 BAC
Inhalants◦ Headspace (GC/MS)
min0.5 1 1.5 2 2.5 3 3.5
50
100
150
200
0.8
23
1.1
16
1.6
15
1.7
42
1.8
57
2.9
86
Standard Volatile Profile
0.5 1 1.5 2 2.5 3 3.5
40
80
120
160
0.8
23
0.8
83
2.9
85
Unidentified Peak
Inhalants◦ Headspace (GC/MS)
(mainlib) Ethane, 1,1-difluoro-
0 10 20 30 40 50 60 70 800
50
100
2
15 2627
3133 43
45
46
47
51
64
65
67
FF
0.5 1 1.5 2 2.5 3 3.5
40
80
120
160
0.8
23
0.8
83
2.9
85
Unidentified Peak
Screening◦ Thin Layer Chromatography (TLC)
Spray
Spot
•sample
•standards develop
Dry plates in an oven
Gas/Liquid Chromatography
Gas/Liquid Chromatography
min1.0 2.0 3.0 4.0 5.0 6.0 7.0
50
100
150
200
2.2
16 3.7
56
5.9
91
3.5
11
Chromatogram
DetectorInject Sample Fused Silica Capillary Column Temperature Programmable Oven
Screening◦ Gas Chromatography (GC/FID or GC/NPD)
Positive Identification
RT Library
Automated Search
Criteria:RT ±2% of STD
Mass Spectrometry
a-methyltryptamine
H
N
C
CF2CF3
O
N
N
F3CF2CCO
N
F3CF2CCO
N
HN
F3CF2CCO
CCF2CF3
O
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460
9500
0
50010001500200025003000350040004500500055006000650070007500800085009000
100001050011000115001200012500
m/z-->
Abundance276
303
129
190
69 102
156 466
51256
Screening/Confirmation◦ Gas Chromatography/Mass Spectrometry (GC/MS)
5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.000
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
Time-->
Abundance
AB
C
D
E
FG
H
I
J
K
L
M
N
O
P
QR
S
T U
V
W
Figure 7 Full scan EI total ion chromatogram of 27 designer phenethylamines and tryptamines. (A) 4-FA (B) 4-FMA (C) 4-MA (D) 4-MTA (E) DOM (F) DOET (G) 3,4,5-trimethoxyamphetamine (H) 2C-C (I) DOC (J) 5-IT (K) AMT (L) DMT (M) 2C-B (N) DOB (O) 2C-T (P) 2C-I, DOI (Q) 2C-T-2 (R) 5-MeO-AMT (S) 5-MeO-DMT (T) DiPT (U) 5-MeO-MiPT (V) 5-MeO-DiPT (W) 5-MeO-DALT.
Screening/Confirmation◦ Gas Chromatography/Mass Spectrometry (GC/MS)◦ Full Scan MS
Positive Identification
Mass Spectral Library
Automated Search
Criteria:RT ±2% of STD
Match of >70% screenMatch of >90% confirm
No extraneous ions
Urine Sample
Standard Solution
MS Full Scan Library Match
Confirmation/Quantitation◦ Gas Chromatography/Mass Spectrometry (GC/MS)
Full ScanSpectrum at 11.25 min
Library Match
7 CyclesCycle:Scan 40-550 m/z
Confirmation/Quantitation◦ Gas Chromatography/Mass Spectrometry (GC/MS)
Selected Ion Monitoring (SIM)
16 Cycles
Improves peak shape, sensitivity, quantitations
SIM Cycle:303, 182, 82 m/z
Confirmation/Quantitation◦ Liquid Chromatography/Mass Spectrometry (LC/MS)
Confirmation/Quantitation◦ Liquid Chromatography Tandem Mass Spectrometry
(LC/MS/MS)
LC/MS/MS
MRM Transition342.0 / 155.1
Product Ion Scan (Full Scan)
MRM Scan
Full Scan
Monitor specific Compound at Expected RT 750 compounds in 12.0 min
Waters Xevo G2 QTOF
AB Sciex 5600+ TripleTOF
10-7 Torr
1
1
MCP Detector
1 Torr 10 mTorr 10-5 Torr
10 mTorr
Collision Gas (Ar)
Fragmenter
Octupole 1 Quad Mass Filter (Q1)
Collision Cell
(CC or Q2)
DC Quad
(Shaping Quad
or QDC)
Puller
Pusher
Ion Mirror
Accelerating
Column
Time of Flight (TOF)
Turbo Pump B
Turbo Pump ARough Pump
Shield (“Liner”)
Octupole 2
Exit Pulse
Skimmer 1
Lens 1 and 2
Slicer
Ion Focus Upper SlitShroud
Shroud
Lower Slit
Shroud
DC DC1 DC2
Version 20040304
2
2
Measure Current for
Beam Alignment
a b Time of Flight = m/z
Nominal mass 300
Exact (calculated) mass 300.1594
CODEINE [M+H]+
H+
300.1595
Exact Mass Libraries◦ 700-1400 compounds pre-loaded
High resolution – (4-5 decimals)◦ Same NOMINAL mass but different EXACT mass
Elucidate unknown peaks◦ Propose chemical formula
“All data” acquired◦ Reprocess archived data for new compounds
Quantitation
ISTD Conc
6-acetylmorphine
Drug ISTD Drug Conc ISTD Conc x y Calc Value % Diff
0 0 0 0 0 0 0 0
386 2097 5.0 25.0 0.2 0.1841 5.38 7.61% Unknown Drug Response ISTD Response
2123 2325 25.0 25.0 1.0 0.9131 26.69 6.76% 5.29 575 3166 25.0
4312 2220 50.0 25.0 2.0 1.9423 56.77 13.55%
10732 3123 100.0 25.0 4.0 3.4364 100.45 0.45% y-int m (slope)
23461 3466 200.0 25.0 8.0 6.7689 197.85 -1.07% 0 0.8553
y = 0.8553xR² = 0.9977
0.0000
1.0000
2.0000
3.0000
4.0000
5.0000
6.0000
7.0000
8.0000
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Series1
Linear (Series1)
Confirmation/Quantitation
Positive Identification
Criteria:RT ±2% of avg Calibrators
2 Ions (ISTD)
3 Ions (Compound)
Ion Ratios ±20% of avgCalibrators
Gaussian shaped peaks
Peak asymmetry
Confirmation/QuantitationFull scan EI mass spectra of 5-IT and AMT
RT
5-IT 9.10 min
AMT 9.30 min
Ion Ratios
5-IT 131/130 m/z 1.75
AMT 131/130 m/z 1.14
40 50 60 70 80 90 100 110 120 130 140 150 160 170 1800
5000
10000
15000
20000
25000
30000
35000
m/z -->
Abundance 131
44
77 103 17415451 65 11589
40 50 60 70 80 90 100 110 120 130 140 150 160 170 1800
2000
6000
10000
14000
18000
22000
26000
30000
m/z-->
Abundance131
44
77103
17463 1178952
159143
NH
NH2
C11H14N2
mw 174.25
5-IT
NH2
NH
C11H14N2
mw 174.25
AMT
Chain of Custody Case (list of item received) Each aliquot tested
Standard Operating Procedure (SOPs) Administrative, Accessioning, Safety, Testing Methods, etc.
Quality Assurance/Quality Control Controls
Screening/Confirmation/Quantitations Methods Method Validations
Specificity, selectivity, accuracy, precision, carryover, stability, variation, robustness, etc.
Instrumentations Tuning Calibration System Suitability Controls
Criteria for Positives/Confirmation◦ Retention Times (RT)
GC ±2% of avg Calibrators LC ±3% of avg Calibrators
◦ Full Scan MS >70% screen
>90% confirmation
◦ SIM 2 Ions (ISTD) / 3 Ions (Compound of Interest) Ion ratios ±20% of avg Calibrators
◦ MRM Ion Transitions 2 MRMs (ISTD or Compound of interest) MRM Ratios ±20% of avg Calibrators
◦ ToF LC ±3% of avg Calibrators
Exact mass ±5 ppm
◦ Peak asymmetry 0.5 – 2.0