Shawn P. Vorce Technical Director Chesapeake Toxicology … · 2016-08-11 · Screening/Confirmation Gas Chromatography/Mass Spectrometry (GC/MS) 5.00 6.00 7.00 8.00 9.00 10.00 11.00

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


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


Full ScanSpectrum at 11.25 min

Library Match

7 CyclesCycle:Scan 40-550 m/z


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


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

Documents

Shawn P. Vorce Technical Director Chesapeake Toxicology … · 2016-08-11 · Screening/Confirmation Gas Chromatography/Mass Spectrometry (GC/MS) 5.00 6.00 7.00 8.00 9.00 10.00 11.00