Shawn P. Vorce Technical Director Chesapeake Toxicology Resources

Transcription

1 Shawn P. Vorce Technical Director Chesapeake Toxicology Resources

2 "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.

3 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

4 Accessioning Special Testing Screening Method Development negative positive Confirmation/Quantitation Issue Report Consultation/Testimony

5 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)

6 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

7 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

8 Immunoassay Screening Negative Result Positive Result

9 IA Cross Reactivity Benzodiazepines (CEDIA) Oxazepam antibodies 200 ng/ml cut-off Package insert list interfering compounds and levels

10 IA Cross Reactivity Amphetamines (CEDIA) Amphetamine antibodies 500 ng/ml cut-off Package insert list interfering compounds and levels Compound Bupropion Fenfluramine 1200 Phenmetrazine 1500 Phenothiazines Phentermine Pseudoephedrine DMAA 2500 Methamphetamine 500 MDA 1400 MDMA ng/ml Cut-off

11 Headspace-GC/FID

12 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.

13 BAC Headspace

14 Ethanol Response/ ISTD Response BAC Headspace (GC/FID) Ethanol 2-Propanol Acetaldehyde Acetone ISTD Ethanol Concentration = BAC

15 Inhalants Headspace (GC/MS) Standard Volatile Profile Unidentified Peak min

16 2.985 Inhalants Headspace (GC/MS) F F (mainlib) Ethane, 1,1-difluoro Unidentified Peak

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

18 Gas/Liquid Chromatography

19 Gas/Liquid Chromatography Inject Sample Fused Silica Capillary Column Temperature Programmable Oven Detector Chromatogram min

20 Screening Gas Chromatography (GC/FID or GC/NPD) Positive Identification RT Library Automated Search Criteria: RT ±2% of STD

21 Mass Spectrometry

22 a-methyltryptamine N Abundance m/z--> N 129 H N 190 O C CF 2 CF F 3 CF 2 CC 276 O N F 3 CF 2 CC 303 O N F3CF2CC O H N O C CF2CF3

23 Screening/Confirmation Gas Chromatography/Mass Spectrometry (GC/MS) Abundance Time--> L N D V M H K C O Q R T U I F A B G E J S W P 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.

24 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

25 MS Full Scan Library Match

26 Confirmation/Quantitation Gas Chromatography/Mass Spectrometry (GC/MS) Full Scan Spectrum at min Cycle: Scan m/z 7 Cycles Library Match

27 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

28 Confirmation/Quantitation Liquid Chromatography/Mass Spectrometry (LC/MS)

29 Confirmation/Quantitation Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS)

30 LC/MS/MS MRM Transition / Product Ion Scan (Full Scan)

31 MRM Scan Full Scan

32 Monitor specific Compound at Expected RT 750 compounds in 12.0 min

33 Waters Xevo G2 QTOF AB Sciex TripleTOF

34 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

35 Nominal mass 300 Exact (calculated) mass CODEINE [M+H] + H

36 Exact Mass Libraries 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

37 Quantitation 6-acetylmorphine Drug ISTD Drug Conc ISTD Conc x y Calc Value % Diff % Unknown Drug Response ISTD Response ISTD Conc % % % y-int m (slope) % y = x R² = Ser Line

38 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

39 Confirmation/Quantitation Abundance N H 5-IT C 11 H 14 N 2 mw NH 2 Full scan EI mass spectra of 5-IT and AMT m/z --> Abundance NH 2 RT 5-IT AMT 9.10 min 9.30 min m/z--> N H AMT C 11 H 14 N 2 mw Ion Ratios 5-IT 131/130 m/z 1.75 AMT 131/130 m/z 1.14

40 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

41 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