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?