Transcript Forensic Drug Testing Part 1: Screening

Forensic Drug Testing
Part 2: GC/MS Confirmation
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
Chief of Clinical Chemistry & Toxicology
Screening vs. Confirmation
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Low cost
Fast
Semi-quantitative
High sensitivity
Low specificity
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High cost
Slow
Quantitative
High sensitivity
High specificity
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
Chromatography
• Separation of components based on their. . .
– Solubility in mobile and stationary phases
• Terminology:
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–
–
–
Gas/liquid
Liquid/liquid
Ion exchange
Partition
Chromatographic separations
Mobile Phase
Stationary Phase
Chromatographic separations
A
B
Soluble in stationary phase
Long retention time
Soluble in mobile phase
Short retention time
Detector signal
Chromatographic separations
A
The resolution of a chromatographic
separation is defined as:
B
t/mean peak width
Time 
Chromatographic resolution
Vr ( B)  Vr ( A)
Rs 
w( A)  w( B)
2
Vr
w(A)
w(B)
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.
Column efficiency
Vr ( A) 
N 

  ( A) 
L
HETP 
N
4
2
Peak broadening
Time 
The van Deemter equation
HETP  A 
B

 C
 = Flow rate
A = Eddy diffusion component
B = Longitudinal diffusion component
C = Mass transfer term
The van Deemter equation
HETP  A 
B

 C
If we. . .
A
B
C
Increase the temperature
+
+
-
Increase the flow rate
+/-
-
+
Decrease specimen volume
+/-
+/-
-
Packed vs. capillary GC column
1-2 m
15-60 m
The van Deemter equation
HETP  A 
B

 C
 = Flow rate
A = Eddy diffusion component
B = Longitudinal diffusion component
C = Mass transfer term
Gas chromatography stationary
phases
Stationary phase
100% dimethyl-polysiloxane
50% diphenyl-50% dimethyl-polysiloxane
Polyethylene glycol (Carbowax®)
Polarity
Non-polar
Intermediate
Polar
GC injection techniques
• Split injections
• Splitless (Gröb) injections
• On-column injections
Split injections
Septum
Purge/carrier gas inlet
Injector
body
Split valve
Purge gas exit (90 – 99%)
To GC column (1 – 10%)
Splitless injections
Septum
Purge/carrier gas inlet
Injector
body
Split valve (shut)
Purge gas exit
To GC column (>95%)
Other inlet systems
• Solid probe
• Liquid chromatograph
• Mass spectrometer?
Electron impact ionization
Power supply
Filament
From GC
Focusing
lens
e+ +
To MS
+
eIon volume
(or source)
(-)
Collector
(+)
Other ionization methods
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Chemical ionization
Thermospray
Electrospray
Fast atom bombardment (FAB)
Matrix-assisted Laser Desorption (MALDI)
The “Right Hand Rule”
Direction of current
Direction of
magnetic
field
Magnetic sector mass spectrometer
+
From ion source
To detector
Quadrupole mass spectrometer
+
From ion source
To detector
Other types of mass filters
• Ion trap
• Ion cyclotron
• Time of flight
Electron multiplier
Negative dynode
+
From mass filter
e-
104 e-
Positive dynode
Ammeter
Mass spectrum
100
90
80
70
60
50
40
30
20
10
0
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)
m/z
Full scan time = 1.0 sec
(0.002 sec/ion)
0.1s
Time 
Cocaine
H3C
N
O
CH3
O
C17H21NO4
O
MW=303.35
O
82 (base peak)
182 [M-121]+
303 (M+)
121
[M-31]+ 272
Cocaine fragmentation (EI)
H3C
O
N
O
CH3
O
H3C
N
121
182
O
CH3
O
O
H3C
O
O
CH3
N
O
O
O
303
O
82
unstable
H3C
N
O
O
272
O
CH3
O
31
82 (base peak)
182 [M-121]+
303 (M+)
121
[M-31]+ 272
Amphetamine/Methamphetamine
H
N
NH2
CH3
CH3
Amphetamine
CH3
Methamphetamine
44
91
Amphetamine fragmentation
+
NH2
CH3
NH2
44
CH3
+
91
44
91
Methamphetamine fragmentation
H
N
+
CH3
H
N
58
CH3
CH3
+
91
CH3
58
91
TMS derivative of amphetamine
H
N
NH2
Si(CH3)3
BSTFA
CH3
Amphetamine
CH3
TMS-Amphetamine
MW = 207
TMS-amphetamine fragmentation
Si(CH3)3
NH
+
CH3
116
H
N
Si(CH3)3
CH3
+
91
H
N+
Si(CH3)2
CH3
192
Mass spectra of TMS-amphetamine
TMS-methamphetamine fragmentation
Si(CH3)3
N
+
CH3
CH3
Si(CH3)3
130
N
CH3
CH3
+
91
Si(CH3)3
N
+
CH3
206
CH2
Mass spectra of TMSmethamphetamine
Methamphetamine metabolism
H
N
H3C
CH3
H
d-Methamphetamine
NH2
~10%
CH3
H
N
H
CH3
l-Desoxyephedrine
Amphetamine
Cocaine HCl and free base forms
H3C
N
H3C
O
Cl-
NH+
O
CH3
O
CH3
pH<8.6
O
O
O
HCl
O
O
Cocaine metabolism
H3C
N
O
CH3
O
O
O
- C6H5COO
- CH3
- CH3
H3C
N
H3 C
N
H
N
O
O
CH3
OH
O
CH3
O
O
O
O
O
O
OH
Ecgonine methyl ester
Benzoylecgonine
Norcocaine
TMS derivative of benzoylecgonine
H3C
N
H3C
O
N
O
Si(CH3)3
OH
BSTFA
O
O
O
Benzyolecgonine
MW = 289
O
O
TMS-Benzyolecgonine
MW = 361
TMS-benzoylecgonine fragmentation
H3C
O
N
O
Si(CH3)3
O
H3C
N
240
O
Si(CH3)3
O
O
+
122
H3C
O
N
O
361
82
H3C
N
+
Si(CH3)2
O
O
O
O
346
Mass spectra of TMSbenzoylecgonine
Opiates
H3C
H3C
N
N
CH3
H
HO
O
Morphine
H
OH
H3C
O
O
Codeine
OH
Glucuronidation
-D-glucuronic acid
H3C
H3C
COOH
N
O
H
OH
H
H
HO
H
N
OH
H
H
OH
Hepatic glucuronyl transferase
HO
O
Morphine
OH
C6H9O7
O
C6H9O7
Morphine diglucuronide
Morphine hydrolysis
H3C
H3C
N
N
H
H
-glucuronidase

C6H9O7
O
C6H9O7
Morphine diglucuronide
HO
O
Morphine
OH
TMS derivative of codeine
H3C
H3C
N
N
H
H3C
O
O
Codeine
MW = 299
H
BSTFA
OH
H3C
O
O
TMS-Codeine
MW = 371
O
Si(CH3)3
Mass spectra of TMS-codeine
Heroin metabolism
H3C
N
H
- CH3CO
O
H3C
O
O
O
O
Heroin
H3C
N
CH3
H
H 3C
O
N
HO
- CH3CO
O
Morphine
O
6-Monoacetylmorphine
H
HO
O
OH
CH3
9-Tetrahydrocannabinol (THC)
COOH
CH3
OH
OH
Oxidation
H3C
H3C
O
9-THC
H3C
H3C
O
9-THC-COOH
THC-COOH detection
THC-COOH glucuronide (15%)
Hydrolysis
THC-COOH
BSTFA
TMS-THC-COOH
Thank You!
Questions?