Forensic Drug Testing Part 2: GC/MS Confirmation

1. Forensic Drug TestingPart 2: GC/MS Confirmation Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology

2. Low cost Fast Semi-quantitative High sensitivity Low specificity High cost Slow Quantitative High sensitivity High specificity Screening vs. Confirmation

3. A confirmatory method should . . . • Utilize the most accurate (specific) testing method available • Have sensitivity equal to or better than the screening method • Be economically feasible • Be simple enough to standardize across many laboratories • Produce results that are legally defensible

4. Chromatography • Separation of components based on their. . . • Solubility in mobile and stationary phases • Terminology: • Gas/liquid • Liquid/liquid • Ion exchange • Partition

5. Chromatographic separations Mobile Phase Stationary Phase

6. Chromatographic separations A B Soluble in stationary phase Long retention time Soluble in mobile phase Short retention time

7. A B Chromatographic separations The resolution of a chromatographic separation is defined as: t/mean peak width Detector signal Time

8. Vr w(A) w(B) Chromatographic resolution

9. Chromatographic resolution • The resolution, Rs, is a unitless quantity since it is the ratio of two measures of retention (time, volume, or distance). • In general, in order to satisfactorily separate equal amounts of compounds A and B, the Rs must be greater than 0.8—baseline separation requires an Rs greater than 1.25.

10. 4 Column efficiency

11. Peak broadening Time 

12. The van Deemter equation • = Flow rate A = Eddy diffusion component B = Longitudinal diffusion component C = Mass transfer term

13. The van Deemter equation

14. 1-2 m 15-60 m Packed vs. capillary GC column

15. The van Deemter equation • = Flow rate A = Eddy diffusion component B = Longitudinal diffusion component C = Mass transfer term

16. Gas chromatography stationary phases

17. GC injection techniques • Split injections • Splitless (Gröb) injections • On-column injections

18. Split injections Septum Purge/carrier gas inlet Injector body Split valve Purge gas exit (90 – 99%) To GC column (1 – 10%)

19. Splitless injections Septum Purge/carrier gas inlet Injector body Split valve (shut) Purge gas exit To GC column (>95%)

20. Other inlet systems • Solid probe • Liquid chromatograph • Mass spectrometer?

21. Focusing lens From GC + + To MS + Ion volume (or source) (-) Electron impact ionization Power supply Filament e- e- Collector (+)

22. Other ionization methods • Chemical ionization • Thermospray • Electrospray • Fast atom bombardment (FAB) • Matrix-assisted Laser Desorption (MALDI)

23. Direction of current Direction of magnetic field The “Right Hand Rule”

24. From ion source + To detector Magnetic sector mass spectrometer

25. From ion source + To detector Quadrupole mass spectrometer

26. Other types of mass filters • Ion trap • Ion cyclotron • Time of flight

27. From mass filter e- 104 e- + Ammeter Electron multiplier Negative dynode Positive dynode

28. Mass spectrum

29. Full scan time = 1.0 sec (0.002 sec/ion) 0.1s Single ion monitoring (SIM) m/z Time 

30. Cocaine C17H21NO4 MW=303.35

31. 82 (base peak) 182 [M-121]+ 303 (M+) 121 [M-31]+ 272

32. Cocaine fragmentation (EI)

33. 82 (base peak) 182 [M-121]+ 303 (M+) 121 [M-31]+ 272

34. Amphetamine/Methamphetamine

35. 44 91

36. Amphetamine fragmentation

37. 44 91

38. Methamphetamine fragmentation

39. 58 91

40. TMS derivative of amphetamine

41. TMS-amphetamine fragmentation

42. Mass spectra of TMS-amphetamine

43. TMS-methamphetamine fragmentation

44. Mass spectra of TMS-methamphetamine

45. Methamphetamine metabolism

46. Cocaine HCl and free base forms

47. Cocaine metabolism

48. TMS derivative of benzoylecgonine

49. TMS-benzoylecgonine fragmentation

50. Mass spectra of TMS-benzoylecgonine