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 Special Testing

Screening Method Development negative positive Confirmation/Quantitation

Issue Report

Consultation/Testimony  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

substrate

Sample Does NOT Absorb light

Drug

Drug Drug substrate

Sample Absorbs Enzyme-drug complex light λ= 340 nm  Immunoassay Screening

Positive Result

Negative Result  IA Cross Reactivity

 Benzodiazepines (CEDIA)

◦ Oxazepam antibodies

◦ 200 ng/mL cut-off

◦ Package insert list interfering compounds and levels  IA Cross Reactivity

Compound 500 ng/mL  Amphetamines (CEDIA) Cut-off Bupropion 13000 ◦ Amphetamine antibodies Fenfluramine 1200 Phenmetrazine 1500 ◦ 500 ng/mL cut-off Phenothiazines 10000 ◦ Package insert list interfering Phentermine 17500 compounds and levels 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. Headspace  BAC ◦ Headspace

http://blog.restek.com/wp-content/uploads/2014/05/HS-GC-Instrument-Diagram2.jpg  BAC ◦ Headspace (GC/FID)

Ethanol

2-Propanol

Acetaldehyde

Acetone

ISTD Ethanol Response/ ISTD Response Response ISTD Response/ Ethanol

0.0 0.50 1.00 1.50 2.00 2.50 3.00 2.50 2.00 1.50 1.00 0.50 0.0 0.0 0.02 0.05 0.08 0.10 0.12 0.15

Ethanol Concentration

= 0.088 BAC  Inhalants

◦ Headspace (GC/MS)

0.883 1.615 200 Standard 160 Volatile Unidentified 1.857 Profile Peak

150 120

1.116 2.985

100 1.742

2.986 80

50

40

0.823 0.823

0.5 1 1.5 2 2.5 3 3.5 min 0.5 1 1.5 2 2.5 3 3.5  Inhalants

◦ Headspace (GC/MS) 0.883

51 160 100 Unidentified Peak

120 2.985 F F 50 65 80

47 45 40 27 15 26 31 46 64 0.823 2 33 43 67 0 0 10 20 30 40 50 60 70 80 0.5 1 1.5 2 2.5 3 3.5 (mainlib) Ethane, 1,1-difluoro-  Screening ◦ Thin Layer Chromatography (TLC) Spot •sample •standards develop

Dry plates in an oven

Spray  Gas/Liquid Chromatography  Gas/Liquid Chromatography

Inject Sample Fused Silica Capillary Column Temperature Programmable Oven Detector

Chromatogram

200 3.756

150 2.216 3.511 100 5.991

50

1.0 2.0 3.0 4.0 5.0 6.0 7.0 min  Screening ◦ Gas Chromatography (GC/FID or GC/NPD)

Positive Identification

RT Library

Automated Search

Criteria: RT ±2% of STD  Mass Spectrometry a-methyltryptamine

N

F3CF2CC Abundance O 276 12500 12000 11500

11000 N 10500 F3CF2CC 10000 O 9500 303 9000

8500 N H 8000 N O 7500 C 129 CF2CF3 7000 6500 6000 190 5500 5000 H O N C 4500 CF2CF3 4000 3500 N F3CF2CC 3000 O 2500 69 102 2000 1500 156 466 1000 51 500 256 0 m/z--> 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460  Screening/Confirmation ◦ Gas Chromatography/Mass Spectrometry (GC/MS)

Abundance L

360000 340000 320000 300000 280000 260000 240000 220000 N 200000 D 180000 V 160000 M 140000 H K 120000 C 100000 O 80000 R Q T U I 60000 F 40000 B G J S W A E P 20000 0 Time--> 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

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

Urine Sample Positive Identification

Mass Spectral Library

Automated Search

Criteria: RT ±2% of STD Standard Solution

Match of >70% screen Match of >90% confirm

No extraneous ions  MS Full Scan Library Match  Confirmation/Quantitation ◦ Gas Chromatography/Mass Spectrometry (GC/MS)  Full Scan

Spectrum at 11.25 min

Cycle: 7 Cycles Scan 40-550 Library Match m/z  Confirmation/Quantitation ◦ Gas Chromatography/Mass Spectrometry (GC/MS)  Selected Ion Monitoring (SIM)

SIM Cycle: 16 Cycles 303, 182, 82 m/z Improves peak shape, sensitivity, quantitations  Confirmation/Quantitation ◦ Liquid Chromatography/Mass Spectrometry (LC/MS)  Confirmation/Quantitation ◦ Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS)  LC/MS/MS

MRM Transition 342.0 / 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 Ion Mirror

Shield (“Liner”)

10-7 Torr

Turbo Pump B

Rough Pump Turbo Pump A

1 Torr 10 mTorr 10-5 Torr

Shroud Accelerating Shroud 10 mTorr MCP Detector Ion Focus Upper Slit Column Slicer Shroud 1 2 Fragmenter Puller DC DC1 DC2 Pusher Skimmer 1 1 2

a b Lower Slit Collision Cell Exit Pulse Time of Flight = m/z Lens 1 and 2 (CC or Q2)

Measure Current for Beam Alignment Octupole 1 Quad Mass Filter (Q1) Octupole 2 DC Quad Time of Flight (TOF) Collision Gas (Ar) (Shaping Quad or QDC)

Version 20040304  Nominal mass 300  Exact (calculated) mass 300.1594 H+ CODEINE [M+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 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 ISTD Conc 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

8.0000 y = 0.8553x R² = 0.9977 7.0000

6.0000

5.0000

4.0000 Series1 Linear (Series1) 3.0000

2.0000

1.0000

0.0000 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0  Confirmation/Quantitation

Positive Identification

Criteria: RT ±2% of avg Calibrators

2 Ions (ISTD)

3 Ions (Compound)

Ion Ratios ±20% of avg Calibrators

Gaussian shaped peaks

Peak asymmetry  Confirmation/Quantitation

Abundance 131 Full scan EI mass spectra of 5-IT NH2 44 35000 and AMT

30000 N H 25000 5-IT C11H14N2 20000 mw 174.25

15000 10000 RT 5000 77 103 174 51 65 89 115 154 0 5-IT 9.10 min m/z --> 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

131 Abundance AMT 9.30 min 30000 44 NH2 26000

22000 N Ion Ratios 18000 H AMT 14000 C11H14N2 mw 174.25 5-IT 131/130 m/z 1.75 10000

6000 77 AMT 131/130 m/z 1.14 103 52 2000 63 89 117 143 159 174 m/z--> 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180  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