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

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

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 separationsB
Soluble in stationary phase
Long retention time
Soluble in mobile phase
Short retention time

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

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

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. Column efficiency 4

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
If we. . . A B C
Increase the temperature + -
Increase the flow rate
+/-
Decrease specimen volume

14. 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
Polarity
100% dimethyl-polysiloxane
Non-polar
50% diphenyl-50% dimethyl-polysiloxane
Intermediate
Polyethylene glycol (Carbowax®)
Polar

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

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

19. Splitless injections Purge/carrier gas inlet Injector body
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. Electron impact ionization
Power supply
Focusing
lens
(-)
Filament
From GC e- +
To MS e-
Ion volume
(or source)
Collector
(+)

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

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

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

25. Quadrupole mass spectrometer+
From ion source
To detector

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

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

28. Mass spectrum

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

51. Opiates

52. Glucuronidation

53. Morphine hydrolysis

54. TMS derivative of codeine

55. Mass spectra of TMS-codeine

56. Heroin metabolism

57. 9-Tetrahydrocannabinol (THC)

58. THC-COOH detection THC-COOH glucuronide (15%) Hydrolysis THC-COOH
TMS-THC-COOH
Hydrolysis
BSTFA

59. Thank You!
Questions?