Mass Spectrometry in Forensic Science Erin Shonsey March

Mass Spectrometry in Forensic Science Erin Shonsey March 16, 2011

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

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

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

Standards for Accepting the Scientific Validity of a Procedure, Technique, and Principle • 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

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.

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

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

Typical forms of Mass Spectrometry in Every Forensic Science Lab

Typical forms of Mass Spectrometry in Every Forensic Science Lab 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

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

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

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

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

Different forms of Mass Spectrometry Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC-ESI-MS) N 2 Sample solution Gas - -+ -+- - -5 KV Atmospheric pressure - Collision gas - Q 1 Q 2 Vacuum Q 3 Detector

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

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

TIC of Volatiles Mix

Spectrum of Peak at 1. 44 min Isopropanol

Spectrum of Peak at 1. 61 min Acetone

Spectrum of Peak at 1. 70 min 1 -propanol (IS)

TIC of Ethanol Standard

Spectrum of Peak at 1. 29 min Ethanol

Spectrum of Peak at 1. 70 min 1 -propanol (IS)

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

Accu. TOF-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

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

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/Analytical. Instruments/Mass. Spectrometers/Accu. TOFDARTIonizati on. Mechanisms/tabid/450/Default. aspx

DART Ionization [(H 2 O+)n. H]+ He He* He* He He He*He* He* H 2 O He* [(H 2 O+)n. H]+ H H 2 O He*2 O [(H 2 O+)n. H]+ HO 2 MH+ H 2 O + MH H 2 O MH+

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

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

Different forms of Mass Spectrometry DART Ionization Tandem Mass Spectrometry N 2 Collision gas Q 1 Q 2 Vacuum Q 3 Detector

Quadrupoles have variable ion transmission modes m 2 m 4 m 1 m 4 m 3 m 2 m 3 mass scanning mode m 2 m 4 m 1 m 3 m 2 m 2 single mass transmission mode m 1

Molecular Ion Scanning N 2 Detector Q 1 Q 2 Vacuum Q 3

Product Ion Scanning N 2 Collision gas Detector Q 1 Q 2 Vacuum Q 3

Multiple Reaction Monitoring (MRM) N 2 Collision gas Detector Q 1 Q 2 Vacuum Q 3

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

Sample Introduction with the Accu. TOF-DART MS 3 x 10 Every sampling device is analyzed for contamination prior to use Intensity 1696474 ( ) 1. 46 1500 1. 58 0. 85 0. 40 1000 1. 03 1. 15 0. 30 1. 37 500 1. 36 0. 18 0 0. 2 1. 17 0. 4 0. 6 0. 8 Time ] [min 1. 0 1. 2 1. 4 1. 6

Types of Samples Analyzed with the Accu. TOF-DART MS http: //www. ecstasy 2. com/img/ecstasy_pill_collage 1. jpg

White Powder Analyzed with the Accu. TOF-DART MS 3 0 Intensity 16873 ( ) 150. 13 10 152. 15 192. 14 0 50 100 150 200 250 300 350 400 m /z Name Methamphetamine Phentermine Amantadine Phendimetrazine Neutral comp. Meas. Calc. Diff(u) Abund. C 10 H 15 N 150. 1302 150. 1283 0. 0020 100. 0000 C 10 H 17 N 152. 1460 152. 1439 0. 0020 21. 3243 C 12 H 17 NO 192. 1422 192. 1388 0. 0034 21. 6783 450 500

Molecular Ion Mass Spectrum 150. 3 2. 8 e 6 Possible Methamphetamine 2. 6 e 6 2. 4 e 6 2. 2 e 6 Q 1 of White Powder 2. 0 e 6 1. 8 e 6 Intensity, cps 1. 6 e 6 1. 4 e 6 1. 2 e 6 1. 0 e 6 210. 3 8. 0 e 5 121. 2 152. 3 166. 3 192. 3 6. 0 e 5 234. 3 4. 0 e 5 2. 0 e 5 0. 0 78. 1 40 60 80 284. 3 299. 4 366. 4 252. 4 315. 4 263. 4 168. 2 154. 3 180. 3 256. 2 292. 4 312. 4 194. 2 212. 3 226. 3 317. 3334. 4342. 5 279. 4 232. 3 354. 4 382. 4 138. 2 131. 4 386. 4 117. 3 100 120 140 160 180 200 220 m/z, Da 240 260 280 300 320 340 360 380 400

91. 0 2. 1 e 5 2. 0 e 5 Product Ion Mass Spectrum 1. 9 e 5 1. 8 e 5 1. 7 e 5 1. 6 e 5 Name Fit Rev. Fit Purity CE 1. 4 e 5 METHAMPHETAMINE 91. 468 96. 188 90. 584 5 1. 3 e 5 Intensity, cps 1. 5 e 5 BZP 86. 341 85. 981 74. 237 30 1. 2 e 5 PHENTERMINE 53. 926 88. 369 53. 516 25 METH-TPC 13. 573 20. 929 13. 441 10 METH-TPC 2 7. 906 11. 836 7. 83 5 TFMPP 1. 732 27. 98 1. 653 30 Metanephrine 1. 544 50. 818 1. 542 25 1. 1 e 5 1. 0 e 5 9. 0 e 4 8. 0 e 4 7. 0 e 4 6. 0 e 4 5. 0 e 4 4. 0 e 4 3. 0 e 4 2. 0 e 4 119. 1 1. 0 e 4 0. 0 150. 1 50 60 70 80 90 100 110 120 130 m/z, Da 140 150 160 170 180 190 200

5500 5000 Total Analysis Time = 5 min 4500 4000 3500 Intensity, cps STANDARD 3000 CASE METHAMPHETAMINE 1 METHAMPHETAMINE 2 RATIO % OF STANDARD 278000 788000 2. 834532 4950 12700 2. 565657 90. 51% 2500 2000 1500 1000 500 0 0. 05 0. 10 0. 15 0. 20 0. 25 Time, min 0. 30 0. 35 0. 40 0. 45 0. 50

Plant Material Analyzed with the Accu. TOF-DART MS 3 x 10 Intensity(110649) 315. 23 100 316. 23 205. 20 0 50 100 150 200 250 300 350 400 450 m/z Name Cannabidiol Tetrahydrocannibinols Neutral comp. C 21 H 30 O 2 Meas. 315. 2355 Calc. Diff(u) 315. 2324 0. 0031 Abund. 100. 0000 500

193. 2 4. 8 e 5 4. 6 e 5 4. 4 e 5 4. 2 e 5 Name THC 123. 1 MDMA 3 BZP MDMA 2 MDMA TFMPP Phenobarbitol PSEUDOEPHEDRINE 81. 1 METHAMPHETAMINE Epinephrine 93. 1 Normetanephrine Metanephrine Epinephrine 107. 1 Metanephrine 135. 2 69. 1 4. 0 e 5 3. 8 e 5 3. 6 e 5 3. 4 e 5 3. 2 e 5 3. 0 e 5 Intensity, cps 2. 8 e 5 2. 6 e 5 2. 4 e 5 2. 2 e 5 2. 0 e 5 1. 8 e 5 1. 6 e 5 1. 4 e 5 Fit 92. 062 58. 094 57. 556 56. 834 47. 279 39. 218 37. 602 36. 044 26. 578 24. 312 20. 676 17. 597 1. 906 1. 025 Rev. Fit 90. 781 4. 146 1. 307 4. 047 5. 88 6. 284 3. 998 1. 904 1. 694 23. 67 12. 596 16. 138 2. 966 3. 117 CE 40 23 30 23 23 30 -30 25 5 25 25 25 109. 2 1. 2 e 5 1. 0 e 5 259. 2 8. 0 e 4 6. 0 e 4 43. 1 231. 3 217. 1 177. 2 67. 179. 091. 1 207. 2 235. 2 165. 1 105. 1 111. 1 151. 1 221. 4 247. 1 55. 1 161. 2 191. 3 189. 2 96. 8 4. 0 e 4 2. 0 e 4 0. 0 Purity 87. 475 3. 986 1. 25 3. 9 5. 197 4. 728 1. 852 1. 474 1. 469 10. 29 8. 327 8. 375 0. 701 0. 058 40 121. 1137. 1 60 80 100 120 140 160 180 200 220 240 m/z, Da 260 315. 3 280 300 320 340 360 380 400

0. 33 6. 3 e 4 6. 0 e 4 Total Analysis Time = 5 min 5. 5 e 4 5. 0 e 4 0. 21 4. 5 e 4 4. 0 e 4 Intensity, cps 3. 5 e 4 STANDARD CASE 3. 0 e 4 THC 1 THC 2 2. 49 E+05 8. 31 E+04 1. 11 E+05 3. 35 E+04 RATIO 0. 33 0. 30 % OF STANDARD 90. 43% 2. 5 e 4 2. 0 e 4 1. 5 e 4 1. 0 e 4 5000. 05 0. 10 0. 15 0. 20 0. 25 Time, min 0. 30 0. 35 0. 40 0. 45 0. 50

Clandestine Tablet Analyzed with the Accu. TOF-DART MS 3 x 10 Intensity ( 174735 ) 177. 14 231. 11 100 195. 09 0 50 100 150 200 250 300 350 400 450 m /z Name Benzylpiperazine Caffeine TFMPP Neutral comp. C 11 H 16 N 2 C 8 H 10 N 4 O 2 C 11 H 13 F 3 N 2 Meas. 177. 1409 195. 0891 231. 1119 Calc. 177. 1392 195. 0882 231. 1109 Diff(u) 0. 0017 0. 0009 0. 0010 Abund. 100. 0000 33. 0937 69. 6525 500

91. 1 2. 0 e 5 1. 9 e 5 1. 8 e 5 1. 7 e 5 1. 6 e 5 1. 5 e 5 1. 4 e 5 Name BZP METHAMPHETAMINE PHENTERMINE METH-TPC 1. 3 e 5 Intensity, cps 1. 2 e 5 1. 1 e 5 1. 0 e 5 Fit 99. 904 80. 01 50. 678 2. 196 Rev. Fit 99. 904 84. 707 Purity 99. 904 67. 774 42. 928 1. 86 CE 30 5 25 10 9. 0 e 4 8. 0 e 4 7. 0 e 4 6. 0 e 4 5. 0 e 4 4. 0 e 4 3. 0 e 4 2. 0 e 4 0. 0 177. 2 85. 0 1. 0 e 4 56. 0 50 60 70 80 90 100 110 120 130 m/z, Da 140 150 160 170 180 190 200

7. 8 e 4 7. 5 e 4 7. 0 e 4 Total Analysis Time for 2 Items = 8 min Analyst Time for 2 Items = 3 hrs 6. 5 e 4 6. 0 e 4 5. 5 e 4 5. 0 e 4 Intensity, cps 4. 5 e 4 4. 0 e 4 3. 5 e 4 BZP 1 1. 51 E+06 4. 66 E+05 6. 21 E+05 STANDARD CASE Item #1 CASE Item #2 BZP 2 1. 19 E+04 3. 91 E+03 5. 27 E+03 RATIO % OF STANDARD 0. 01 106. 47% 0. 01 107. 68% 3. 0 e 4 2. 5 e 4 2. 0 e 4 1. 5 e 4 1. 0 e 4 5000. 05 0. 10 0. 15 0. 20 0. 25 Time, min 0. 30 0. 35 0. 40 0. 45

Clandestine Tablet Analyzed with the Accu. TOF-DART MS 3 x 10 Intensity 31002 ( ) 30 147. 11 177. 14 150. 13 20 10 257. 25 194. 12 231. 11 116. 14 285. 28 0 50 100 150 200 250 300 350 400 m /z Name Phentermine Methamphetamine Benzylpiperazine Butamben MDMA TFMPP Neutral comp. C 10 H 15 N C 11 H 16 N 2 C 11 H 15 NO 2 C 11 H 13 F 3 N 2 Meas. 150. 1277 177. 1400 194. 1187 231. 1117 Calc. Diff(u) 150. 1283 -0. 0005 177. 1392 0. 0008 194. 1181 0. 0006 231. 1109 0. 0008 Abund. 68. 2766 100. 00 21. 7282 12. 8296 450 500

91. 1 91. 0 5. 2 e 4 5. 0 e 4 1. 10 e 5 5. 0 e 4 4. 8 e 4 1. 05 e 5 4. 8 e 4 4. 6 e 4 1. 00 e 5 4. 6 e 4 4. 4 e 4 9. 50 e 4 4. 2 e 4 9. 00 e 4 4. 0 e 4 8. 50 e 4 3. 8 e 4 8. 00 e 4 3. 6 e 4 7. 50 e 4 3. 4 e 4 3. 2 e 4 7. 00 e 4 3. 2 e 4 Name MDMA METHAMPHETAMINE BZP PHENTERMINE Warfarin PSEUDOEPHEDRINE Epinephrine THC Normetanephrine METH-TPC Lorazepam Milrinone Epinephrine Name METHAMPHETAMINE BZP PHENTERMINE METH-TPC 2 TFMPP Metanephrine Intensity, cps 3. 0 e 4 6. 50 e 4 3. 0 e 4 2. 8 e 4 6. 00 e 4 2. 6 e 4 5. 50 e 4 2. 4 e 4 5. 00 e 4 2. 2 e 4 4. 50 e 4 2. 0 e 4 4. 00 e 4 1. 8 e 4 1. 6 e 4 3. 50 e 4 1. 6 e 4 1. 4 e 4 3. 00 e 4 1. 2 e 4 2. 50 e 4 1. 0 e 4 2. 00 e 4 8000. 0 1. 50 e 4 6000. 0 1. 00 e 4 4000. 0 5000. 00 2000. 0 0. 0 50 40 50 57. 9 Fit 92. 943 80. 01 38. 098 25. 319 16. 992 8. 33 5. 473 3. 93 3. 409 2. 196 1. 394 1. 147 0. 405 Fit 91. 942 86. 341 54. 635 22. 547 15. 44 2. 095 2. 016 Name BZP METHAMPHETAMINE PHENTERMINE 104. 9 METH-TPC Fit Rev. Fit 133. 3 95. 655 94. 533 80. 01119. 1 93. 53 50. 678 93. 53 2. 196 93. 53 90. 9 85. 190. 1 72. 1 50 60 60 60 70 70 7080 80 Rev. Fit 87. 458 0. 531 1. 649 7. 396 43. 55 19. 029 28. 11 39. 999 27. 07 0. 531 58. 455 11. 664 19. 993 Rev. Fit 95. 966 75. 238 90. 216 34. 51 22. 944 134. 9 31. 069 67. 328 163. 1 Purity 85. 92 0. 425 1. 577 4. 875 12. 896 1. 604 2. 245 2. 885 1. 398 0. 012 0. 918 0. 134 0. 117 Purity 90. 375 64. 961 54. 635 22. 163 15. 177 1. 836 2. 016 Purity 93. 786 74. 833 47. 399 2. 054 150. 2 162. 5 CE 23 5 30 25 25 40 25 10 30 -29 10 CE 5 30 25 10 5 30 25 CE 30 5 25 10 194. 1 177. 2 80 90 90100 100110 110120 130130 140 90 100 110 120 130 140 150 150 160 160 170 170 m/z, Da 180 190 200

1800 1700 Analysis Time = 10 min 1600 1500 1400 1300 1200 1100 Intensity, cps 1000 900 800 700 600 METHAMPHETAMINE 1 METHAMPHETAMINE 2 STANDARD CASE RATIO % OF STANDARD 268000 12500 104. 58% MDMA 2 RATIO % OF STANDARD 1250000 5690 188000 729 0. 1504 0. 12812 85. 19% BZP 1 BZP 2 RATIO % OF STANDARD 1510000 19800 STANDARD CASE 6. 716418 7. 024 MDMA 1 STANDARD CASE 1800000 87800 11900 187 0. 007881 0. 009444 119. 84% 500 400 300 200 100 0 0. 05 0. 10 0. 15 0. 20 0. 25 Time, min 0. 30 0. 35 0. 40 0. 45 0. 50

Clandestine Laboratory Samples Analyzed with the Accu. TOFDART MS 3 x 10 Intensity ( Methamphetamine 33499 Pen tube 150. 13 (Pseudo)ephedrine 20 166. 2 1 391. 29 0 50 100 150 200 250 300 350 400 450 500 m /z Methamphetamine Intensity 8355 ( Aluminum Foil 150. 13 (Pseudo)ephedrine 5000 282. 28 166. 12 0 50 100 150 200 250 300 m /z 350 400 450 500

3 x 10 (Pseudo)ephedrine Intensity (50970) 166. 12 40 Blender Jar 20 167. 13 0 50 100 150 200 250 300 350 400 450 500 m/z (Pseudo)ephedrine 3 x 10 Intensity (182939) White Powder 166. 13 100 167. 13 148. 11 0 50 100 150 200 250 300 350 400 450 500 m/z 3 x 10 Methamphetamine Intensity (72800) Aluminum Foil 150. 13 50 151. 13 0 50 100 150 200 250 300 m/z 350 400 450 500

3 x 10 Intensity (57470) 391. 30 Methamphetamine Rubber Tubing 40 150. 13 20 392. 30 0 50 100 150 200 250 300 350 400 450 500 m/z 3 x 10 Intensity (98301) 61. 06 121. 08 White Powder 50 Methamphetamine (Pseudo)ephedrine 0 50 100 150 200 250 300 m/z 350 400 450 500

148. 1 6. 8 e 4 6. 5 e 4 6. 0 e 4 Name PSEUDOEPHEDRINE METHAMPHETAMINE BZP Metanephrine Epinephrine Normetanephrine Epinephrine PHENTERMINE Normetanephrine MDMA TFMPP Milrinone Phenobarbitol Warfarin METH-TPC Metanephrine 5. 5 e 4 5. 0 e 4 4. 5 e 4 Intensity, cps 4. 0 e 4 3. 5 e 4 3. 0 e 4 2. 5 e 4 2. 0 e 4 1. 5 e 4 Fit 90. 805 80. 01 67. 867 25. 124 24. 37 23. 862 15. 523 14. 567 14. 237 11. 157 6. 289 6. 17 5. 613 1. 042 0. 896 0. 885 0. 538 117. 1 Rev. Fit 90. 628 1. 107 1. 241 52. 898 42. 553 14. 942 16. 083 12. 574 26. 83 14. 1 54. 989 14. 927 11. 62 13. 676 11. 259 4. 462 57. 842 Purity 86. 363 0. 886 1. 081 21. 79 21. 951 13. 377 12. 049 8. 787 11. 462 1. 573 4. 164 1. 113 0. 665 0. 288 166. 2 0. 689 0. 11 0. 369 133. 2 1. 0 e 4 5000. 0 115. 3 121. 2 91. 0 50 60 70 80 90 100 110 147. 0 148. 8 132. 1 120 130 m/z, Da 140 150 CE 25 5 30 25 25 10 10 25 25 23 30 -29 -30 -35 25 10 5 165. 7 160 170 180 190 200

Clandestine Laboratory Samples Analyzed with the DARTQTRAP STANDARD White Powder Pen Tube Aluminum Foil Rubber Tubing METHAMPHETAMINE 1 METHAMPHETAMINE 2 RATIO % OF STANDARD 2. 78 E+05 7. 88 E+05 2. 83 1. 51 E+04 3. 97 E+04 2. 63 92. 75% 6. 85 E+04 1. 88 E+05 2. 74 96. 82% 9. 25 E+03 2. 31 E+04 2. 50 88. 10% 3. 02 E+04 7. 99 E+04 2. 65 93. 34% 7. 89 E+04 2. 13 E+05 2. 70 95. 24% STANDARD White Powder Pen Tube Blender PSEUDO 1 5. 75 E+05 5. 17 E+05 4. 08 E+04 5. 75 E+04 5. 94 E+05 PSEUDO 2 1. 10 E+05 1. 00 E+05 6. 97 E+03 1. 18 E+04 1. 14 E+05 RATIO 0. 19 0. 17 0. 21 0. 19 Total Analysis Time for 7 Items = 1 hr Analyst Time for 7 Items = 2 days 101. 11% 89. 30% 107. 27% 100. 32%

Real Time Sample Analysis with the Accu. TOFDART MS • Efficient screening instrument – Soft ionization keeps the molecular ion intact • Mass accuracy allows matches within 5 mmu of 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 DARTQTRAP MS • 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

LC-MRM-MS assay for Drug Detection and Quantitation N 2 Sample solution Gas - -+ -+- - -5 KV Atmospheric pressure - Collision gas - Q 1 Q 2 Vacuum Q 3 Detector

LC-ESI-MS/MS Spectrum of Methadone 105. 1 265. 3 57. 1 91. 1 117. 2 69. 1 60 77. 1 80 219. 3 223. 2 195. 3 204. 2 159. 3 129. 2 282. 2 310. 3 247. 3 167. 2 187. 3 145. 1 217. 5 275. 2 169. 2 107. 2 221. 3 100 120 140 160 180 200 220 240 260 280 300 320 m/z, amu 340 360 380 400

LC-MRM-MS analysis for Drug Quantitation Collision gas 265 310 Q 1 Q 2 Q 3

LC-ESI-MRM-MS 310 Collision gas Q 1 265 Q 2 Q 3 Compound Molecular Weight Parent ion Product ion Dwell Time (msec) Declustering Potential (DP) Collision Energy (CE) Retention Time p. Ka Alprazolam 308. 0829 309. 1 205 25 60 50 8. 03 2. 4 Amitriptyline 277. 183 278. 2 91 25 45 42 7. 8 9. 4 Cocaethylene 317. 37 318. 2 196 25 40 39 4. 32 -- Cocaine 303. 1471 304. 1 82 25 30 40 3. 6 8. 6 Fentanyl 336. 2202 337. 2 188 25 55 43 5. 18 8. 4 Imipramine 280. 1939 281. 2 86 25 35 32 7. 41 9. 5 Mepivacaine 246. 1732 247. 2 98 25 42 28 3. 32 7. 6 Methadone 309. 2093 310. 1 265 25 30 35 7. 56 8. 6 Methamphetamine 149. 1204 150. 1 91 25 34 27 2. 96 8. 6 Oxycodone 315. 1471 316. 1 241 25 50 40 2. 6 8. 5 THC 314. 2246 315. 1 193 25 37 34 14. 91 10. 6 Trazodone 371. 1513 372. 2 176 25 60 42 4. 87 6. 1

TIC of Check Mix Retention Order Oxycodone Methamphetamine Mepivacaine Cocaethylene Trazodone Fentanyl Imipramine Methadone Amitriptyline Alprazolam THC 1 2 3 4 5 6 7 8 9 10 11 12 Time, min 13 14 15 16 17 18 19 20

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

IDA Analysis of Check Mix TIC XIC MRM EPI

Library Search of Fragment Spectrum

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

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

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