DART Workshop – Ames, IA August 2008

1. DART Workshop – Ames, IAAugust 2008 Use of the AccuTOF-DART system for the forensic analysis of drugs of abuse Bob Steiner Virginia Department of Forensic Science Central Laboratory Richmond, VA 804-786-4707 x22347 robert.steiner@dfs.virginia.gov

2. GLOSSARY OF TERMS: • AccuTOF – Accurate mass Time Of Flight; mass spectrometer • DART – Direct Analysis in Real Time; atmospheric pressure ion source • Orifice 1 – inlet to the AccuTOF; raising voltage causes fragmentation of ions via collision induced dissociation • Profile Spectrum – multiple data points to describe the mass peaks of a spectrum; have “chromatogram” appearance • Centroid – to find the center of a mass peak collected in profile mode; gives resulting histogram spectrum as a mass vs. abundance pair • PEG600 – polyethylene glycol polymer with an average MW of 600 Da; used for internal mass calibration • Internal mass calibration – PEG600 spectrum run within a data file; allows to accurately set the mass axis for all spectra in that file

3. GLOSSARY OF TERMS: • Function switching – method that allows changing the orifice 1 voltage rapidly during data collection; ori 1 voltage changes every 0.25 sec • Protonated or deprontonated molecule – the addition or subtraction of a hydrogen from the neutral molecule, depending on polarity of DART • Millimass unit (mmu) – accuracy of TOF spectra measured to thousandths of a Dalton; acceptable spectra are < 5 mmu from calculated mass • JEOL-DX file – text file of data points in a mass spectrum; can be easily exported to other software for analysis

4. SearchFromList – library searching program; searches by empirical formula and matching user-library spectra • Drugs_Neutral_Masses – library of empirical formulae of drugs • Drug Prep Library_ori20 – library spectra of pharmaceuticals at orifice 1 voltage of 20 V; also libraries at ori30, 60 and 90 V • Drug Std Library_ori20 – library spectra of primary standards at orifice 1 voltage of 20 V; also libraries at ori30, 60 and 90 V • Mirror spectrum – display of unknown (positive) and known (negative) spectra in SearchFromList program GLOSSARY OF TERMS:

5. DART METHODOLOGY • How do I use the AccuTOF-DART for drug samples? • Types of samples • Best sampling methods • Function Switching • Calibration considerations

6. HEADSPACE SAMPLING 1. Start acquisition 2. Open container near DART gas stream 3. Remove container Can’t get simpler than that!!!!!!

7. SAMPLING OF SOLIDS Hold solid material in DART gas stream using tweezers Drawbacks: 1. Hard to hold 2. MESSY 3. Non-homogeneous samples

8. MORE SOLID SAMPLING DRAWBACKS Coated tablets! GAS STREAM IS HOT!!! 4-chloro-2,5-dimethoxyamphetamine

9. “DRIED ON TUBE” METHOD Deposit sample onto tubes with syringe Can measure how much sample is deposited Turned out not so good!

10. “DRIED ON TUBE” METHOD Loading cap tubes with sample Even 1 uL “runs down” tube Inconsistent results obtained

11. Injecting sample into gas stream with syringe Draw up samples from ALS vials Can measure amount injected Turned out not so good!

12. LIQUID SAMPLING Dissolve sample into suitable solvent Dip capillary tube in liquid/hold “wand” in DART gas stream Advantages: 1. EASY!!! 2. Can control concentration of sample 3. Homogeneous sample Preferred method at DFS!!!

13. DART sampling by hand is an ART! Consistency from one person to the next is difficult to obtain!!! Buy an AutoDART???

14. FUNCTION SWITCHING In general, ionization of molecules with DART produces hydride addition or subtraction products of the molecular ion. Methyl stearate spectrum showing [M+H]+ ion at 299.2931 Da. No ‘diagnostic’ ions

15. Collision-Induced Dissociation • Definition*: “An ion/neutral species interaction wherein the projectile ion is dissociated as a result of interaction with a target species. This is brought about by conversion of part of the translation energy of the ion to internal energy in the ion during collision.” • High vacuum prevents this in EI • Basis behind triple stage quadrupole systems • Commonly done in LCMS systems to promote fragmentation • Performed in the AccuTOF by raising orifice 1 voltage *http://mass-spec.lsu.edu/msterms/index.php/Collision-Induced_Dissociation

16. EFFECT OF RAISING ORIFICE 1 VOLTAGE Orifice 1 = 15V Orifice 1 = 30V

17. EFFECT OF RAISING ORIFICE 1 VOLTAGE Orifice 1 = 45V Orifice 1 = 60V

18. Function Switching allows collection of data at SEVERAL orifice 1 voltages AT ONCE!

19. Noscapine Orifice 1 = 20 Function Switching results showing fragmentation at various orifice 1 voltages Orifice 1 = 30 Orifice 1 = 60 Orifice 1 = 90

20. RUNNING A SAMPLE ON THE AccuTOF-DART Lock and chk stds More PEG PEG Sample

21. Mass calibrations Calibration at acquisition – PEG+H Set at tune (after cleaning) “Global” mass calibration Internal mass calibration – temp_PEG Within your data file “Fine tunes” your calibration

22. “Run PEG before AND/OR after samples within your datafile! Intensity matters!!” Peggy the Dimetrodon PEG PEG

23. GOOD PEG vs BAD PEG

24. Where to average your peak for best PEG intensity? TIC profile 30V data centroid

25. TIC Less intensity! profile centroid

26. Centroiding peaks = difficult with low intensity ions Interfering peak or noise “tailing” due to kinetic energy spread in TOF

27. LOCK MASS AND CHECK STANDARDS • Mix of cocaine, methamphetamine and nefazodone • MWs span the mass range • Cocaine is used as drift compensation “lock” at 304.1549 Da • Methamphetamine (150.1283 Da) and Nefazodone (470.2323 Da) cover the low and high ends of mass range • Adds another level of calibration applied to sample data

28. LOCK MASS AND CHECK STANDARDS Cocaine 304.1549 Meth 150.1283 Nefazodone 470.2323

29. SAMPLE DATA Sample signal is averaged, centroided and calibrations are applied for EACH orifice voltage Spectra are saved in “JEOL-DX” format

30. SearchFromList Program – by Dr. Robert “Chip” Cody

31. Match spectra search Load data file Load empirical formula library Adjust these to suit your search Empirical formula search Add or subtract from empirical formula

32. SearchFromList empirical formula search result Lot number of std used to create lib spectrum Spectrum file name From empirical formula library must be < 5 mmu

33. SearchFromList empirical formula search result Search result label Spectrum imported from Mass Center

34. Choose library to match orifice 1 voltage for the spectrum you are searching Match spectra search DOUBLE CLICK on library entry to load all spectra! Search!

35. Match spectra search results Blue = original spectrum Click on entries to view comparison spectra Red = comparison spectrum

36. Suggested Data for casefiles • Print out: TIC[2] from Chromatogram view • Print out spectrum of cocaine lock mass and check stds • Save spectra as averaged, background subtracted, centroided, calibrated JEOL-DX files • Print out: SearchFromList search result for 20V spectrum • Print out: Other spectra or search results at other voltages – enough to characterize the spectrum

37. FUNCTION SWITCHING - ADVANTAGES • FOUR spectra collected every second • Higher orifice 1 voltages result in more fragmentation • Can lead to greater confidence for identification • Except for mixtures, spectra are reproducible

38. FUNCTION SWITCHING - DISADVANTAGES • Mixture spectra – no prior chromatography (can be good OR bad!!) • Less sensitivity – splitting ionization between four functions • Finite database for searching – library databases need to be developed!

39. Application of the DART to DX • Currently being used as a screening tool for DX casework. • Validation took over one year to complete. • Approved by DFS Scientific Advisory Board and full VA Forensic Science Board, May 6-8, 2008.

40. AccuTOF-DART Validation at DFS • DART screening method - LLOD determined for 7 drugs; daily calibration data to show stability of instrument • DART sampling methods – why we use methanol and MP tube ‘wands’ • Comparison of DART to GCMS – new technology vs. established technology; 553 samples run on each – ALL match in highest Scheduled drug found; DART typically showed more cmpds! • Selectivity study – can you tell the difference in DART spectra of drugs with the SAME empirical formula?? • “Rugustness” – same result on different days with different people??

41. LLOD DETERMINATION and INSTRUMENT STABILITY

42. COMPARISON OF DART RESULTS WITH ANALYTICAL SCHEME – 553 SAMPLES! DART result GCMS confirmation 1161 heroin 1161heroin, papaverine, 6-MAM, quetiapine 1665 cocaine, diltiazem, naproxen 1665 cocaine 1910 MDMA, caffeine, procaine 1910 MDMA, caffeine, procaine, dimethylsulfone 1976 cocaine, caffeine, benzocaine 1976 cocaine, caffeine

43. SELECTIVITY STUDY Ori1 30V 119.0872 Easily distinguish methamphetamine from phentermine!

44. SELECTIVITY STUDY Cocaine and scopolamine ori1 30V

45. SELECTIVITY STUDY Cocaine and scopolamine ori1 90V

46. SELECTIVITY STUDY LSD and LAMPA ori1 90V

47. GHB SCREENING “Detection of GHB in various drink matrices via AccuTOF-DART” Bennett MJ, Steiner RR. J. Forensic Sci., in press* Spiked 50 beverages with GHB at “impairment levels” to determine if DART could detect GHB easily seen in all drink matrices Gave better results than using GHB color test! Done in NEGATIVE ion mode * slated for publication Jan 2009

48. GHB SCREENING Ocean Spray Cranberry Juice - blank blank Ocean Spray Cranberry Juice – 2 mg/mL spike with GHB [GHB-H]-

49. DART Spectral Interpretation

50. Molecular Weight Information Electron Impact Ionization DART Ionization • [M+H]+ or [M-H]- • Very stable • Very little fragmentation (without help!) • Proper terms: Protonated or deprotonated molecule • Loss of stable neutrals • Use SearchFromList to find • M+. • Unstable – leads to extensive fragmentation • Usually the peak @ highest mass, excluding isotope peaks • Look for “logical neutral losses” • Sometimes hard to find!