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Forensic Drug TestingPart 2: GC/MS Confirmation
Forensic Drug TestingPart 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
Screening vs. ConfirmationScreening 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
• High cost• Slow• Quantitative• High sensitivity• High specificity
A confirmatory method should . . .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
ChromatographyChromatography
• 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
Chromatographic separationsChromatographic separations
Stationary Phase
Mobile Phase
Chromatographic separationsChromatographic separations
A B
Soluble in stationary phaseLong retention time
Soluble in mobile phaseShort retention time
Chromatographic separationsChromatographic separationsD
etec
tor
sign
al
Time
B
A The resolution of a chromatographicseparation is defined as:
t/mean peak width
Chromatographic resolutionChromatographic resolution
w(A) w(B)
Vr
2
)()()()(
BwAwAVBV
R rrs
Chromatographic resolutionChromatographic 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.
• 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.
Column efficiencyColumn efficiency
4
2
)(
)(
A
AVN r
N
LHETP
Peak broadeningPeak broadening
Time
The van Deemter equationThe van Deemter equation
= Flow rateA = Eddy diffusion componentB = Longitudinal diffusion componentC = Mass transfer term
= Flow rateA = Eddy diffusion componentB = Longitudinal diffusion componentC = Mass transfer term
CB
AHETP
The van Deemter equationThe van Deemter equation
CB
AHETP
If we. . . A B C
Increase the temperature + + -
Increase the flow rate +/- - +
Decrease specimen volume +/- +/- -
Packed vs. capillary GC columnPacked vs. capillary GC column1-2 m
15-60 m
The van Deemter equationThe van Deemter equation
= Flow rateA = Eddy diffusion componentB = Longitudinal diffusion componentC = Mass transfer term
= Flow rateA = Eddy diffusion componentB = Longitudinal diffusion componentC = Mass transfer term
CB
AHETP
Gas chromatography stationary phases
Gas chromatography stationary phases
Stationary phase Polarity
100% dimethyl-polysiloxane Non-polar
50% diphenyl-50% dimethyl-polysiloxane Intermediate
Polyethylene glycol (Carbowax®) Polar
GC injection techniquesGC injection techniques
• Split injections
• Splitless (Gröb) injections
• On-column injections
• Split injections
• Splitless (Gröb) injections
• On-column injections
Split injectionsSplit injections
Purge/carrier gas inlet
Purge gas exit (90 – 99%)Split valve
Septum
To GC column (1 – 10%)
Injectorbody
Splitless injectionsSplitless injections
Purge/carrier gas inlet
Purge gas exit
Split valve (shut)
Septum
To GC column (>95%)
Injectorbody
Other inlet systemsOther inlet systems
• Solid probe
• Liquid chromatograph
• Mass spectrometer?
• Solid probe
• Liquid chromatograph
• Mass spectrometer?
Electron impact ionizationElectron impact ionization
e-
e-
++ +
(+)
From GC
To MS
Filament
CollectorIon volume(or source)
Focusinglens
(-)
Power supply
Other ionization methodsOther 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)
The “Right Hand Rule”The “Right Hand Rule”
Direction of current
Direction ofmagneticfield
Magnetic sector mass spectrometerMagnetic sector mass spectrometer
To detector
+
From ion source
Quadrupole mass spectrometerQuadrupole mass spectrometer
+
From ion source To detector
Other types of mass filtersOther types of mass filters
• Ion trap
• Ion cyclotron
• Time of flight
• Ion trap
• Ion cyclotron
• Time of flight
Electron multiplierElectron multiplier
e-
104 e-+
From mass filter
Positive dynode
Negative dynode
Ammeter
Mass spectrumMass spectrum
0
10
20
30
40
50
60
70
80
90
100
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
Single ion monitoring (SIM)Single ion monitoring (SIM)
Time
m/
z
Full scan time = 1.0 sec(0.002 sec/ion)
0.1s
CocaineCocaine
N
H3C
O
O
CH3
O
O
C17H21NO4
MW=303.35
303 (M+)
[M-31]+ 272
182 [M-121]+
82 (base peak)
121
Cocaine fragmentation (EI)Cocaine fragmentation (EI)
N
H3C
O
OCH3
O
O
N
H3C
O
OCH3
O
O
N
H3C
O
OCH3
O
O
N
H3C
O
O
CH3
O
O
272 31
82
121182
303
unstable
303 (M+)
[M-31]+ 272
182 [M-121]+
82 (base peak)
121
Amphetamine/MethamphetamineAmphetamine/Methamphetamine
HN
CH3
Methamphetamine
CH3
CH3
Amphetamine
NH2
44
91
Amphetamine fragmentationAmphetamine fragmentation
CH3
NH2CH3
NH2
44
91
+
+
44
91
Methamphetamine fragmentationMethamphetamine fragmentation
HN
CH3
CH3
HN
CH3
CH3+
91
+
58
58
91
TMS derivative of amphetamineTMS derivative of amphetamine
CH3
NH2
BSTFA
CH3
HN
Si(CH3)3
Amphetamine TMS-AmphetamineMW = 207
TMS-amphetamine fragmentationTMS-amphetamine fragmentation
CH3
HN
Si(CH3)3
CH3
HN
Si(CH3)2
CH3
NH
Si(CH3)3
116
91
+
+
192
+
Mass spectra of TMS-amphetamineMass spectra of TMS-amphetamine
TMS-methamphetamine fragmentationTMS-methamphetamine fragmentation
N
CH3
CH3
Si(CH3)3
N
CH3
CH3
Si(CH3)3
N
CH3
CH2
Si(CH3)3
+
91
+
130
206
+
Mass spectra of TMS-methamphetamine
Mass spectra of TMS-methamphetamine
Methamphetamine metabolismMethamphetamine metabolism
CH3
Amphetamine
NH2
HN
H3C H
d-Methamphetamine
HN
H CH3
l-Desoxyephedrine
CH3
~10%
Cocaine HCl and free base formsCocaine HCl and free base forms
N
H3C
O
O
CH3
O
O
NH+
H3C
O
O
CH3
O
O
Cl-
pH<8.6
HCl
Cocaine metabolismCocaine metabolism
N
H3C
O
O
CH3
O
O
N
H3C
O
O
CH3
OH
N
H3C
OH
O
O
O
HN
O
O
CH3
O
O
Ecgonine methyl ester Benzoylecgonine Norcocaine
- C6H5COO
- CH3
- CH3
TMS derivative of benzoylecgonineTMS derivative of benzoylecgonine
N
H3C
OH
O
O
O
BSTFA
N
H3C
O
O
O
O
Si(CH3)3
BenzyolecgonineMW = 289
TMS-BenzyolecgonineMW = 361
TMS-benzoylecgonine fragmentationTMS-benzoylecgonine fragmentation
N
H3C
O
OSi(CH3)3
O
O
N
H3C
O
OSi(CH3)3
N
H3C
O
O
N
H3CO
O
OO
Si(CH3)2
346
82
122240
361
+
+
Mass spectra of TMS-benzoylecgonine
Mass spectra of TMS-benzoylecgonine
OpiatesOpiates
OHO OH
N
H3C
H
OO OH
N
H3C
H
H3C
Morphine Codeine
CH3
GlucuronidationGlucuronidation
OHO OH
N
H3C
H
OH
HO
OH
HH
OHH
OH
COOH
OC6H9O7 C6H9O7
N
H3C
H
Morphine
Hepatic glucuronyl transferase
Morphine diglucuronide
-D-glucuronic acid
Morphine hydrolysisMorphine hydrolysis
OC6H9O7 C6H9O7
N
H3C
H
OHO OH
N
H3C
H
MorphineMorphine diglucuronide
-glucuronidase
TMS derivative of codeineTMS derivative of codeine
OO OH
N
H3C
H
H3C
BSTFA
OO O
N
H3C
H
H3C Si(CH3)3
CodeineMW = 299
TMS-CodeineMW = 371
Mass spectra of TMS-codeineMass spectra of TMS-codeine
Heroin metabolismHeroin metabolism
OO O
N
H3C
H
Heroin
OHO OH
N
H3C
H
Morphine
H3C
O
CH3
O
OHO O
N
H3C
H
6-Monoacetylmorphine CH3
O
- CH3CO
- CH3CO
9-Tetrahydrocannabinol (THC)9-Tetrahydrocannabinol (THC)
O
CH3
OH
H3C
H3CO
COOH
OH
H3C
H3C
Oxidation
9-THC 9-THC-COOH
THC-COOH detectionTHC-COOH detection
THC-COOH glucuronide (15%)
THC-COOH
TMS-THC-COOH
Hydrolysis
BSTFA
Thank You!
Questions?
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