1. Mass Spectrometry in Forensic Science Erin Shonsey March 16, 2011

2. Overview Introduction to forensic sciences Uses of mass spectrometry in forensic sciences Typical instrumentation in forensic sciences Applications of new instrumentation

3. Introduction to Forensic Sciences Forensic Sciences is defined as: the application of a broad spectrum of sciences to answer questions of interest to the legal system.sciences

4. Introduction to Forensic Sciences Typical analytical sections within a forensic science laboratory: Drug Chemistry – Analysis of pills, powders, liquids, plant materials, and other suspicious items for illegal drug content Toxicology – Analysis of biological samples for alcohol, prescription medication, drugs of abuse, and other chemicals that are not naturally occurring in the body DNA – Extraction and amplification of DNA from biological fluids for identification Firearms – Bullet pattern recognition and analysis of gun powder Fire Debris -- Identification of ignitable liquids used in arsons

5. Alabama –Frye standard: the court must decide if the questioned procedure, technique, and principles are “generally accepted” by a relevant community –Federal Rule 702: a witness qualified as an expert may testify in the form of an opinion Federal –Daubert: Has it been tested? Has it been published and peer reviewed? Potential rate of error Existence and maintenance of standards controlling the techniques operation Accepted in the relevant scientific community Standards for Accepting the Scientific Validity of a Procedure, Technique, and Principle

6. Mass Spectrometry in Forensic Science A gas chromatograph with a mass spec detector is the final tool used in the analysis of drug chemistry and toxicology samples for identification and confirmation.

7. Typical forms of Mass Spectrometry in Every Forensic Science Lab Gas Chromatography-Mass Spectrometry (GC-MS) http://www.chem.arizona.edu/massspec/intro_html/intro.html

8. Typical forms of Mass Spectrometry in Every Forensic Science Lab Gas Chromatography-Mass Spectrometry (GC-MS) http://www.microbialcellfactories.com/content/figures/1475-2859-6-6-4-l.jpg

9. Typical forms of Mass Spectrometry in Every Forensic Science Lab

10. Gas Chromatography-Mass Spectrometry (GC-MS) Spectrum Spectra are searched against a library of known compounds in an effort to identify every peak in the TIC A standard is analyzed on the instrument to generate a known retention time and spectrum of the compound for that instrument

11. Problems Encountered with the GC/MS Lose the parent ion of the compound upon ionization in the instrument Example: Methadone

12. Problems Encountered with the GC/MS Derivatize the compound for analysis with GC/MS which decreases detection of low level compounds Example: THC

13. Problems Encountered with the GC/MS Heat labile compound will be identified as a related compound, but not the actual compound Example: Clorazepate to Nordiazepam

14. New Technology Four new instruments have been brought into the department in October 2008 –AccuTOF-DART mass spectrometer –3200 QTRAP mass spectrometer with LC –3200 QTRAP mass spectrometer with DART –HS-GC-MSD

15. Different forms of Mass Spectrometry Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC-ESI-MS)

16. Different forms of Mass Spectrometry Direct Analysis in Real Time with Time of Flight Mass Spectrometry N 2 Electrostatic reflector TOF detector

17. HS-GC-MSD This instrument provides opportunity for qualitative and quantitative identification of volatile compounds

18. TIC of Volatiles Mix

19. Isopropanol Spectrum of Peak at 1.44 min

20. Acetone Spectrum of Peak at 1.61 min

21. 1-propanol (IS) Spectrum of Peak at 1.70 min

22. TIC of Ethanol Standard

23. Ethanol Spectrum of Peak at 1.29 min

24. 1-propanol (IS) Spectrum of Peak at 1.70 min

25. Summary Method development is underway with the HS-GC-MSD –Good separation and spectra from the volatiles mix and ethanol standard Ready to start validation –Developing method for commonly abused inhalants –Developing a screening for other volatile compounds Example: GHB

26. AccuTOF-DART MS The DART is the first open air, ambient ion source for a mass spectrometer Coupled to a time of flight instrument exact mass measurements can be used in the putative identification of compounds

27. 3200 QTRAP-DART MS Coupled to a hybrid triple quadrupole/Trap instrument molecular ions can be individually fragmented for identification of sample components

28. DART Ionization Penning ionization: energy is transferred from metastable ions (M*) Positive ions: He* ionizes water which transfers a proton to the sample Negative ions: Penning electrons are rapidly thermalized and captured by oxygen which ionizes the sample http://www.jeolusa.com/PRODUCTS/AnalyticalInstruments/MassSpectrometers/AccuTOFDART/AccuTOFDARTIonizati onMechanisms/tabid/450/Default.aspx

29. He* He He* He H2OH2O H2OH2O H2OH2O H2OH2O He* [(H 2 O + ) n H] + M M M M MH + H2OH2O H2OH2O H2OH2O H2OH2O He*MH + DART Ionization

30. Time of Flight Detector Accelerating pulse TOF detector t = (d/√(2U))((√m/z)) t = time d = flight tube distance U = accelerating voltage m = mass z = charge

31. N 2 Electrostatic reflector TOF detector AccuTOF Mass Spectrometer The reflector doubles the length of the flight tube Orthogonal acceleration time of flight Repelling plate

32. Q1 Q2Q3 Detector Collision gas N2N2 Vacuum Different forms of Mass Spectrometry DART Ionization Tandem Mass Spectrometry

33. mass scanning mode m1 m3 m4 m2 m3 m1 m4 m2 single mass transmission mode m3 m1 m4 Quadrupoles have variable ion transmission modes m2

34. Q1 Q2Q3 Detector N2N2 Vacuum Molecular Ion Scanning

35. Q1 Q2Q3 Detector Collision gas N2N2 Vacuum N2N2 N2N2 N2N2 N2N2 N2N2 Product Ion Scanning

36. Q1 Q2Q3 Detector Collision gas N2N2 Vacuum N2N2 N2N2 N2N2 N2N2 N2N2 Multiple Reaction Monitoring (MRM)

37. Sample Introduction with the AccuTOF-DART MS Liquid samples are introduced with a glass capillary tube closed at one end Solid samples are introduced into the stream with tweezers

38. Sample Introduction with the AccuTOF-DART MS Every sampling device is analyzed for contamination prior to use 00.20.40.60.81.01.21.41.6 Time[min] 500 1000 1500 Intensity(1696474)x10 3 1.46 1.58 0.85 0.401.15 1.03 0.30 1.37 1.36 0.18 1.17

39. Types of Samples Analyzed with the AccuTOF-DART MS http://www.ecstasy2.com/img/ecstasy_pill_collage1.jpg

40. White Powder Analyzed with the AccuTOF-DART MS Name Neutral comp. Meas. Calc. Diff(u) Abund. Methamphetamine C10H15N 150.1302 150.1283 0.0020 100.0000 Phentermine C10H15N 150.1302 150.1283 0.0020 100.0000 Amantadine C10H17N 152.1460 152.1439 0.0020 21.3243 Phendimetrazine C12H17NO 192.1422 192.1388 0.0034 21.6783

41. Q1 of White Powder m/z, Da 406080100120140160180200220240260280300320340360380400 150.3 342.5 210.3 192.3 121.2 152.3 166.3 234.3 284.3 299.4 252.4 315.4 263.4 168.2 312.4 180.3 154.3 212.3 256.2 292.4 194.2 226.3 317.3 334.4 78.1 279.4 232.3 131.4 366.4 382.4 354.4 138.2 386.4 117.3 0.0 2.0e5 4.0e5 6.0e5 8.0e5 1.0e6 1.2e6 1.4e6 1.6e6 1.8e6 2.0e6 2.2e6 2.4e6 2.6e6 2.8e6 Intensity, cps Possible Methamphetamine Molecular Ion Mass Spectrum

42. NameFitRevFitPurityCE METHAMPHETAMINE91.46896.18890.5845 BZP86.34185.98174.23730 PHENTERMINE53.92688.36953.51625 METH-TPC13.57320.92913.44110 METH-TPC27.90611.8367.835 TFMPP1.73227.981.65330 Metanephrine1.54450.8181.54225 Product Ion Mass Spectrum

43. METHAMPHETAMINE 1METHAMPHETAMINE 2RATIO% OF STANDARD STANDARD2780007880002.834532 CASE4950127002.56565790.51% Total Analysis Time = 5 min

44. 50100150200250300350400450500 m/z 0 100 Intensity(110649)x10 3 315.23 316.23 205.20 Plant Material Analyzed with the AccuTOF-DART MS Name Neutral comp.Meas. Calc. Diff(u) Abund. Cannabidiol C21H30O2 315.2355 315.2324 0.0031 100.0000 Tetrahydrocannibinols C21H30O2 315.2355 315.2324 0.0031 100.0000

45. NameFitRevFitPurityCE THC92.06290.78187.47540 MDMA358.0944.1463.98623 BZP57.5561.3071.2530 MDMA256.8344.0473.923 MDMA47.2795.885.19723 TFMPP39.2186.2844.72830 Phenobarbitol37.6023.9981.852-30 PSEUDOEPHEDRINE36.0441.9041.47425 METHAMPHETAMINE26.5781.6941.4695 Epinephrine24.31223.6710.2925 Normetanephrine20.67612.5968.32725 Metanephrine17.59716.1388.37525 Epinephrine1.9062.9660.70110 Metanephrine1.0253.1170.0585

46. THC 1THC 2RATIO% OF STANDARD STANDARD2.49E+058.31E+040.33 CASE1.11E+053.35E+040.3090.43% Total Analysis Time = 5 min

47. Name Neutral comp. Meas. Calc. Diff(u) Abund. Benzylpiperazine C11H16N2 177.1409 177.1392 0.0017 100.0000 Caffeine C8H10N4O2 195.0891 195.0882 0.0009 33.0937 TFMPP C11H13F3N2 231.1119 231.1109 0.0010 69.6525 Clandestine Tablet Analyzed with the AccuTOF-DART MS 50100150200250300350400450500 m/z 0 100 Intensity(174735)x10 3 177.14 231.11 195.09

48. NameFitRevFitPurityCE BZP99.904 30 METHAMPHETAMINE80.0184.70767.7745 PHENTERMINE50.67884.70742.92825 METH-TPC2.19684.7071.8610

49. BZP 1BZP 2RATIO% OF STANDARD STANDARD1.51E+061.19E+040.01 CASE Item #14.66E+053.91E+030.01106.47% CASE Item #26.21E+055.27E+030.01107.68% Total Analysis Time for 2 Items = 8 min Analyst Time for 2 Items = 3 hrs

50. Name Neutral comp. Meas. Calc. Diff(u)Abund. Phentermine C10H15N 150.1277 150.1283-0.000568.2766 Methamphetamine C10H15N 150.1277 150.1283-0.0005 68.2766 Benzylpiperazine C11H16N2 177.1400 177.1392 0.0008100.00 Butamben C11H15NO2 194.1187 194.1181 0.000621.7282 MDMA C11H15NO2 194.1187 194.1181 0.000621.7282 TFMPP C11H13F3N2 231.1117 231.1109 0.000812.8296 Clandestine Tablet Analyzed with the AccuTOF-DART MS

51. NameFitRevFitPurityCE MDMA92.94387.45885.9223 METHAMPHETAMINE80.010.5310.4255 BZP38.0981.6491.57730 PHENTERMINE25.3197.3964.87525 Warfarin16.99243.5512.89625 PSEUDOEPHEDRINE8.3319.0291.60425 Epinephrine5.47328.112.24525 THC3.9339.9992.88540 Normetanephrine3.40927.071.39825 METH-TPC2.1960.5310.01210 Lorazepam1.39458.4550.91830 Milrinone1.14711.6640.134-29 Epinephrine0.40519.9930.11710 NameFitRevFitPurityCE METHAMPHETAMINE91.94295.96690.3755 BZP86.34175.23864.96130 PHENTERMINE54.63590.21654.63525 METH-TPC22.54734.5122.16310 METH-TPC215.4422.94415.1775 TFMPP2.09531.0691.83630 Metanephrine2.01667.3282.01625 NameFitRevFitPurityCE BZP95.65594.53393.78630 METHAMPHETAMINE80.0193.5374.8335 PHENTERMINE50.67893.5347.39925 METH-TPC2.19693.532.05410

52. METHAMPHETAMINE 1METHAMPHETAMINE 2RATIO% OF STANDARD STANDARD26800018000006.716418 CASE12500878007.024104.58% MDMA 1MDMA 2RATIO% OF STANDARD STANDARD12500001880000.1504 CASE56907290.1281285.19% BZP 1BZP 2RATIO% OF STANDARD STANDARD1510000119000.007881 CASE198001870.009444119.84% Analysis Time = 10 min

53. 50100150200250300350400450500 m/z 0 20 Intensity(33499x10 3 150.13 166.12 391.29 50100150200250300350400450500 m/z 0 5000 Intensity(8355 150.13 282.28 166.12 Methamphetamine (Pseudo)ephedrine Clandestine Laboratory Samples Analyzed with the AccuTOF- DART MS Pen tube Aluminum Foil

54. 50100150200250300350400450500 m/z 0 20 40 Intensity(50970)x10 3 166.12 167.13 50100150200250300350400450500 m/z 0 100 Intensity(182939)x10 3 166.13 167.13 148.11 50100150200250300350400450500 m/z 0 50 Intensity(72800)x10 3 150.13 151.13 Blender Jar White Powder Aluminum Foil Methamphetamine (Pseudo)ephedrine

55. 50100150200250300350400450500 m/z 0 20 40 Intensity(57470)x10 3 391.30 150.13 392.30 50100150200250300350400450500 m/z 0 50 Intensity(98301)x10 3 121.0861.06 Methamphetamine (Pseudo)ephedrine Rubber Tubing White Powder

56. NameFitRevFitPurityCE PSEUDOEPHEDRINE90.80590.62886.36325 METHAMPHETAMINE80.011.1070.8865 BZP67.8671.2411.08130 Metanephrine25.12452.89821.7925 Epinephrine24.3742.55321.95125 Normetanephrine23.86214.94213.37710 Epinephrine15.52316.08312.04910 PHENTERMINE14.56712.5748.78725 Normetanephrine14.23726.8311.46225 MDMA11.15714.11.57323 TFMPP6.28954.9894.16430 Milrinone6.1714.9271.113-29 Phenobarbitol5.61311.620.665-30 Warfarin1.04213.6760.288-35 Warfarin0.89611.2590.68925 METH-TPC0.8854.4620.1110 Metanephrine0.53857.8420.3695

57. PSEUDO 1PSEUDO 2RATIO STANDARD5.75E+051.10E+050.19 White Powder5.17E+051.00E+050.19101.11% White Powder4.08E+046.97E+030.1789.30% Pen Tube5.75E+041.18E+040.21107.27% Blender5.94E+051.14E+050.19100.32% Clandestine Laboratory Samples Analyzed with the DART- QTRAP Total Analysis Time for 7 Items = 1 hr Analyst Time for 7 Items = 2 days METHAMPHETAMINE 1METHAMPHETAMINE 2RATIO% OF STANDARD STANDARD2.78E+057.88E+052.83 White Powder1.51E+043.97E+042.6392.75% Pen Tube6.85E+041.88E+052.7496.82% Aluminum Foil9.25E+032.31E+042.5088.10% Aluminum Foil3.02E+047.99E+042.6593.34% Rubber Tubing7.89E+042.13E+052.7095.24%

58. Efficient screening instrument –Soft ionization keeps the molecular ion intact Mass accuracy allows matches within 5 mmu of the theoretical mass of a compound –No extraction is required for sample analysis Raw samples the preferred sample –High-throughput Typical analysis time for a sample is 1-2 min Real Time Sample Analysis with the AccuTOF- DART MS

59. Compound fragmentation is possible without extraction CID fragmentation allows retention of molecular ion in fragmentation spectrum –These can be searched against an in house library for identification MRM analysis gives ion ratios for a second level of compound identification in comparison to a standard Complex mixtures do not present a problem for analysis –The instrument has the ability to isolate a single compound for fragmentation Real Time Sample Analysis with the DART- QTRAP MS

60. LC-MRM-MS assay for Drug Detection and Quantitation

61. LC-ESI-MS/MS Spectrum of Methadone

62. LC-MRM-MS analysis for Drug Quantitation Q1Q2Q3 Collision gas 310 265

63. Q1Q2Q3 Collision gas 310265 LC-ESI-MRM-MS Compound Molecular Weight Parent ion Product ion Dwell Time (msec) Declustering Potential (DP) Collision Energy (CE) Retention TimepKa Alprazolam308.0829309.12052560508.032.4 Amitriptyline277.183278.2912545427.89.4 Cocaethylene317.37318.21962540394.32-- Cocaine303.1471304.1822530403.68.6 Fentanyl336.2202337.21882555435.188.4 Imipramine280.1939281.2862535327.419.5 Mepivacaine246.1732247.2982542283.327.6 Methadone309.2093310.12652530357.568.6 Methamphetamine149.1204150.1912534272.968.6 Oxycodone315.1471316.12412550402.68.5 THC314.2246315.119325373414.9110.6 Trazodone371.1513372.21762560424.876.1

64. 1234567891011121314151617181920 Time, min TIC of Check Mix Retention Order Oxycodone Methamphetamine Mepivacaine Cocaine Cocaethylene Trazodone Fentanyl Imipramine Methadone Amitriptyline Alprazolam THC

65. Survey Scan (1) Second Dependent Scan (s) IDA Criteria Level 1 Survey Scan (2) Dependent Scan (s) Enhanced Resolution Add to Exclusion List Survey scan be EMS, EMC, Neutral Loss, Precursor Scan, MRM or EPI (combinations of 2 surveys) Improve Resolution/Accuracy Inclusion/Exclusion List Second Level Dependant Scan (MS 3 ) IDA Criteria Level 2 Dependent Scan (s) Second Dependent Scan (s) Two levels of criteria Multiple dependant scans (EPI, Product Ion and MS3) Information Dependent Acquisition (IDA) Courtesy of Ricky Ciner

66. IDA Analysis of Check Mix TIC XIC MRM EPI

67. Library Search of Fragment Spectrum

68. Summary LC-ESI-MS can be used in the qualitative and quantitative analysis of drugs in toxicological specimens –The instrumentation is advantageous in that chemicals do not have to be derivatized –The soft ionization aids detection of the parent ion of the compound

69. Overall Summary Mass spectrometry is a powerful tool in a forensic science lab New instrumentation is expanding the sample analysis possibilities beyond current limitations No one technique is robust enough for everything, therefore a combination of techniques is ideal for screening and confirmation of drug and toxicology samples

70. Acknowledgements UAB Dr. Stephen Barnes Marion Kirk Ray Moore Dr. Matthew Renfrow Landon Wilson ADFS Dr. Dale Carpenter Andrea Headrick Dr. Jack Kalin Gary Wallace