Shawn P. Vorce Technical Director Chesapeake Toxicology Resources

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 Enzyme-drug complex Absorbs light λ= 340 nm

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 Bupropion 13000 Fenfluramine 1200 Phenmetrazine 1500 Phenothiazines 10000 Phentermine 17500 Pseudoephedrine 75000 DMAA 2500 Methamphetamine 500 MDA 1400 MDMA 800 500 ng/ml Cut-off

Headspace-GC/FID

Headspace BAC Headspace Sample and ISTD are transferred to a headspace vial Vial is heating to a constant Temp (90 o C). Volatile component diffuse into gas phase forming equilibrium between liquid and headspace. Sample is taken from the Headspace and analyzed.

BAC Headspace http://blog.restek.com/wp-content/uploads/2014/05/hs-gc-instrument-diagram2.jpg

Ethanol Response/ ISTD Response 0.0 0.50 1.00 1.50 2.00 2.50 3.00 BAC Headspace (GC/FID) Ethanol 2-Propanol Acetaldehyde Acetone ISTD 0.0 0.02 0.05 0.08 0.10 0.12 0.15 Ethanol Concentration = 0.088 BAC

0.823 1.742 2.986 2.985 1.116 1.857 1.615 Inhalants Headspace (GC/MS) 200 150 100 50 Standard Volatile Profile 160 120 80 40 0.823 0.883 Unidentified Peak 0.5 1 1.5 2 2.5 3 3.5 min 0.5 1 1.5 2 2.5 3 3.5

2.985 Inhalants Headspace (GC/MS) 100 50 0 2 F 27 15 26 F 31 33 0 10 20 30 40 50 60 70 80 43 47 45 46 51 (mainlib) Ethane, 1,1-difluoro- 65 64 67 160 120 80 40 0.823 0.883 Unidentified Peak 0.5 1 1.5 2 2.5 3 3.5

Screening Thin Layer Chromatography (TLC) Spot sample standards develop Dry plates in an oven Spray

Gas/Liquid Chromatography

3.511 5.991 2.216 3.756 Gas/Liquid Chromatography Inject Sample Fused Silica Capillary Column Temperature Programmable Oven Detector Chromatogram 200 150 100 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 Abundance m/z--> 12500 12000 11500 11000 10500 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 51 69 102 N 129 H N 190 O C CF 2 CF 3 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 F 3 CF 2 CC 276 O N F 3 CF 2 CC 303 O N F3CF2CC 156 466 256 O H N O C CF2CF3

Screening/Confirmation Gas Chromatography/Mass Spectrometry (GC/MS) Abundance 360000 340000 320000 300000 280000 260000 240000 220000 200000 180000 160000 140000 120000 100000 80000 60000 40000 20000 0 Time--> L N D V M H K C O Q R T U I F A B G E J S W P 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 Positive Identification Urine Sample 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: Scan 40-550 m/z 7 Cycles Library Match

Confirmation/Quantitation Gas Chromatography/Mass Spectrometry (GC/MS) Selected Ion Monitoring (SIM) SIM Cycle: 303, 182, 82 m/z 16 Cycles 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 Shroud 10 mtorr Ion Focus Upper Slit Accelerating Column MCP Detector ragmenter Shroud 1 2 Slicer Puller Skimmer 1 Lens 1 and 2 DC DC1 DC2 1 2 Pusher a b Lower Slit Collision Cell Exit Pulse Time of Flight = m/z (CC or Q2) Measure Current for Beam Alignment Octupole 1 Quad Mass Filter (Q1) Collision Gas (Ar) Octupole 2 DC Quad (Shaping Quad or QDC) Time of Flight (TOF) Version 20040304

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 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 7.0000 y = 0.8553x R² = 0.9977 6.0000 5.0000 4.0000 3.0000 Ser Line 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 44 35000 30000 25000 20000 N H 5-IT C 11 H 14 N 2 mw 174.25 NH 2 Full scan EI mass spectra of 5-IT and AMT 15000 10000 5000 0 m/z --> Abundance 30000 26000 51 65 77 103 89 115 154 174 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 131 44 NH 2 RT 5-IT AMT 9.10 min 9.30 min 22000 18000 14000 10000 6000 2000 m/z--> 0 77 103 52 63 89 117 143 159 174 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 N H AMT C 11 H 14 N 2 mw 174.25 Ion Ratios 5-IT 131/130 m/z 1.75 AMT 131/130 m/z 1.14

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