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

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Presentation on theme: "Forensic Drug Testing Part 2: GC/MS Confirmation Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology Roger."— Presentation transcript:

1 Forensic Drug Testing Part 2: GC/MS Confirmation Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology 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 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 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 Separation of components based on their... –Solubility in mobile and stationary phases Terminology: –Gas/liquid –Liquid/liquid –Ion exchange –Partition

5 Chromatographic separations Stationary Phase Mobile Phase

6 Chromatographic separations AB Soluble in stationary phase Long retention time Soluble in mobile phase Short retention time

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

8 Chromatographic resolution w(A)w(B) V r

9 Chromatographic resolution The resolution, R s, 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 R s must be greater than 0.8baseline separation requires an R s greater than The resolution, R s, 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 R s must be greater than 0.8baseline separation requires an R s 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 = Flow rate A = Eddy diffusion component B = Longitudinal diffusion component C = Mass transfer term

13 The van Deemter equation If we...ABC Increase the temperature++- Increase the flow rate+/--+ Decrease specimen volume+/- -

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

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

16 Gas chromatography stationary phases Stationary phasePolarity 100% dimethyl-polysiloxaneNon-polar 50% diphenyl-50% dimethyl-polysiloxaneIntermediate Polyethylene glycol (Carbowax®)Polar

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

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

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

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

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

22 Other ionization methods Chemical ionization Thermospray Electrospray Fast atom bombardment (FAB) Matrix-assisted Laser Desorption (MALDI) 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 To detector + From ion source

25 Quadrupole mass spectrometer + From ion source To detector

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

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

28 Mass spectrum

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

30 Cocaine C 17 H 21 NO 4 MW=303.35

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

32 Cocaine fragmentation (EI)

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

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%) THC-COOH TMS-THC-COOH Hydrolysis BSTFA

59 Thank You! Questions?


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