1. Technology Transition Workshop RTI International is a trade name of Research Triangle Institute Evaluation AccuTOF DART for Postmortem Toxicology Screening Peter R. Stout

2. Technology Transition Workshop NIJ Project Grant No. 2006-DN-BX-K014 Opinions are those of the authors and not necessarily the U.S. Department of Justice

3. Technology Transition Workshop Objective Evaluate the Jeol Ltd. AccuTOF DART system as a novel application for use in postmortem toxicology laboratories –Qualitative –Screening methodology Specifically postmortem –Urine –Blood and tissue

4. Technology Transition Workshop Presentation goals Equipment set up Discuss standards run Urine based testing Blood/tissue testing Summary of experience with the instrument Future work

5. Technology Transition Workshop RTI set up JEOL TOF DART LEAP Technologies CombiPal autosampler Mass Center software More recently trying the Schraeder software

6. Technology Transition Workshop AccuTOF DARTSnorkel LEAP Autosampler Ion Source DART

7. Technology Transition Workshop How Autosampler Works

8. Technology Transition Workshop Sample Introduction

9. Technology Transition Workshop AccuTOF- DART Strengths Minimal sample preparation Broad range of sensitivity Rapid analysis Simultaneous determination of multi-drug analytes Sufficient mass accuracy for formula determination

10. Technology Transition Workshop Reference materials Examined 112 compounds –drugs and metabolites Methanolic standard materials Directly introduced

11. Technology Transition Workshop Isomers DrugM+H Pentobarbital/Amobarbital227.138 Codeine/Hydrocodone300.159 2-Oxo-3-hydroxy LSD/3-Oxo-3-hydroxy LAMPA356.196 Morphine/Hydromorphone/Norcodeine286.143 Benzoylecgonine/Norcocaine290.138 PMA/Ephedrine166.122 Cocaine/Scopolamine304.154 Talbutal/Butalbutal225.123 Phentermine/methamphetamine150.120  9-THC/  6-THC 315.224 Imiprimine/Despropyl Fentanyl281.193

12. Technology Transition Workshop Near Isomers Near isomersM+H Phentermine/triethanolamine150.120/150.105 Methamphetamine/triethanolamine150.120/150.105 Amitriptyline/venlafaxine278.183/278.204 Tramadol/nortriptyline264.188/264.167 Ranatidine/Clomipramine315.141/315.154 Diphenhydramine/Tripelennamine 255.162/255.173

13. Technology Transition Workshop Urine Method Drug standards diluted in blank human urine Glass probe dipped in sample Analyzed with DART in positive mode DART temperature @ 300°C Mass calibrated using polyethylene glycol Orifice 1 voltage set at 20V Archived previously confirmed postmortem samples analyzed

14. Technology Transition Workshop Urine Components Urea+H Creatinine+H Creatinine Dimer +H

15. Technology Transition Workshop Cocaine 100 µg/mL M + H = 304.1548

16. Technology Transition Workshop Methamphetamine 100 µg/mL M+H = 150.1278

17. Technology Transition Workshop Triazolam 100 µg/mL M + H = 343.0508

18. Technology Transition Workshop LOD for Drug Analytes in Urine (µg/mL) DrugsLODDrugLOD Amitriptyline1.5Heroin25 Amphetamine3.1Hydrocodone3.1 Benzoylecgonine12.5Methadone 3.1 Caffeine15Methamphetamine6.2 Cocaethylene12.5Morphine3.1 Cocaine1.2Nicotine1.5 Codeine12.5Oxycodone3.1 Dextromethorphan3.1Oxazepam(25) Diphenhydramine3.1PCP0.78 Diazepam3.1Propoxyphene1.5 Ecgonine Methyl Ester1.5  9-THC 3.1 Fentanyl0.31Trazodone50 Fluoxetine12.5Triazolam3.1

19. Technology Transition Workshop Creatinine Issues Oxazepam (10 µg/mL) in DI water (M+H 287.0578) Oxazepam (10 µg/mL) with 10 µg/mL creatinine

20. Technology Transition Workshop Oxazepam (10 µg/mL) with 100 µg/mL creatinine Creatinine Issues Oxazepam (10 µg/mL) with 200 µg/mL creatinine

21. Technology Transition Workshop Archived Case Example Methadone=310.216 EDDP=278.190 310.21327 278.20765 -3mmu +17mmu

22. Technology Transition Workshop Evaluation of Postmortem Blood and Tissue Samples

23. Technology Transition Workshop “Minimal” Blood/Tissue Method 2 mL of ACN added to 1 mL of sample Mixture was shaken and vortexed Spun in centrifuge at 2500 rpm for 5 minutes ~100uL of ACN saved for DART analyses Remainder poured off and dried down at 45°C Reconstituted in 100 uL of ACN and analyzed

24. Technology Transition Workshop Cases used SpecimenCases Analyzed Blood28 Liver11 Kidney2 Brain4

25. Technology Transition Workshop Blood method Acetonitrile added to blood

26. Technology Transition Workshop Blood Method Vortexed

27. Technology Transition Workshop Blood method Centrifuged

28. Technology Transition Workshop Blood method Decanted off supernatant

29. Technology Transition Workshop Blood method Dry down under N 2

30. Technology Transition Workshop Blood method Reconstitute

31. Technology Transition Workshop SPE method

32. Technology Transition Workshop Human Blood Cholestadiene Monosaccharide Urea Plasticizer

33. Technology Transition Workshop Methadone 100 ug/mL Methadone in Blank Blood Unextracted 100 ug/mL Methadone in Blank Blood Extracted in 1:3 ACN 310.21838 M + H =310.216

34. Technology Transition Workshop Methadone 100 ug/mL Methadone in Blank Blood Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL 310. 215 1 ug/mL Methadone in Blank Blood Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN

35. Technology Transition Workshop Postmortem Aorta Blood Specimen Un-extracted Extracted in 1:3 ACN Previously reported at 2.8  g/mL Nondetected

36. Technology Transition Workshop Postmortem Aorta Blood Specimen Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN

37. Technology Transition Workshop Postmortem Liver Specimen Previously reported at 6.6 ug/mL Un-extracted Extracted in 1:3 ACN

38. Technology Transition Workshop Postmortem Liver Specimen Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL

39. Technology Transition Workshop Cocaine 1 ug/mL Cocaine in blank blood extracted in 1:3 ACN 1 ug/mL Cocaine in Blank Blood Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN M+H=304.154 304.15962

40. Technology Transition Workshop Cocaine 0.1 ug/mL Cocaine in Blank Blood Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN

41. Technology Transition Workshop Postmortem Aorta Blood Specimen Extracted in 1:3 ACN Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN Previously reported at 0.52  g/mL 304.16913

42. Technology Transition Workshop ? BE=290.138 Propoxyphene=340.226Norpropoxyphene=326.211 All drugs not found Could not see without concentrating SPE Extraction Postmortem Cocaine Case

43. Technology Transition Workshop Postmortem case by LC/MS and SPE BE BE-d3 cocaine cocaine-d3 cocaethylene cocaethylene- d3 BE 1253 ng/mL, Cocaine 8.8 ng/mL, CE 2.7 ng/mL

44. Technology Transition Workshop Original case reports Case 1 mg/L Cocaine0.03 EME0.22 BE1.76 Oxycodone0.08 Propoxyphene0.45 Norpropoxyphene0.91 AlprazolamPositive

45. Technology Transition Workshop Original case reports Case 2 mg/L Methadone1.1 EDDPPositive Sertraline0.14 Desmesthylsertraline0.76 Doxepin0.74 Desmethyldoxipin3.78

46. Technology Transition Workshop Comparison to “Traditional” MS platforms Case 1Case 2 PlatformCOCBECEMethadone Original Quant30ng/ml1,760ng/mlND1,100 ng/ml LC/MS8.812532.71156 LC/Trap*Detected LC/TOF12.516326.4754** LC/QTOF26.1153910.4898 LC/QQQ1.114480.11134 DART-TOFND * No quantitation attempted on the trap ** Source was saturated, resulting in poor quantitation

47. Technology Transition Workshop Table of Drugs Analyzed in PM Specimens Acetaminophen Alprazolam Amphetamine Atropine Benzoylecgonine Caffeine Carbamazepine Carisoprodol Chlorpheniramine Citalopram Clonazepam Cocaine Codeine Cotinine Cyclobenzaprine Desmethylcitalopram Desmethyldoxepin Dextromethorphan Diazepam Diphenhydramine EME Fluoxetine Fluvoxamine Gabapentin Hydrocodone Ibuprophen Levorphanol MDMA Methamp Methadone Methylphenidate Meprobamate Metoprolol Mirtazapine Morphine Nicotine Nortriptyline Olanzapine Oxcarbazepine Oxycodone Oxymorphone Paroxetine PCP Promethazine Propoxyphene Quetiapine Temazepam THC Topiramate Trazadone                  

48. Technology Transition Workshop Conclusions Reasonable sensitivity for some drugs Greater sensitivity for some drugs over others Creatinine appears to interfere with ionization of some drugs Requirement for some sample preparation to mitigate interference from matrix Auto sampler helps with more consistent sample introduction

49. Technology Transition Workshop Conclusions At least minimal drug extraction appears necessary to achieve best sensitivity Extensive (solid phase extraction), does not necessarily improve things

50. Technology Transition Workshop Conclusions Drug detected in spiked blood have better sensitivity than in postmortem specimens at the same or greater concentrations Not ideal for detecting therapeutic drug levels –Caveat the post mortem samples used are old and compound degradation may have occurred

51. Technology Transition Workshop Conclusions Strong potential for the instrument Issues for implementation: –Sensitivity –Interferences –Software (Mass Center) Mass calibration Stability Retrieval of spectra

52. Technology Transition Workshop Conclusions Sensitivity –GIST/Vapur addition may improve sensitivity Software –Schraeder software addresses many issues Mass calibration Retrieval of spectra –Still acquire using Mass Center Potential stability issues Be proactive about disk management

53. Technology Transition Workshop Acknowledgement Jeri Ropero-Miller Nichole Bynum National Institutes of Justice – Award #2006-DN-BX-K014 LEAP Technologies Maricopa County, Washington State Police and OCME-NC SPEWare