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Forensic
Applications
Therapeutic and Abused Drugs in Urine, Blood, Plasma/Serum for
Acid/Neutral and Basic Drugs for GC or GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
Urine:
To 2mL of urine add internal standard(s) and 1mL of
100mM phosphate buffer (pH 6.0)
Mix/vortex
Sample pH should be 6.0±0.5
Adjust pH accordingly with 100mM monobasic or dibasic
sodium phosphate
Centrifuge as appropriate
Serum, Plasma or Whole Blood:
To 1mL of sample add internal standard(s), 1mL of
phosphate buffer (ph 6.0) and 4mL of DI H
2
O
Mix/vortex and let stand for 5 minutes
Centrifuge for 10 minutes at 2,000rpm and discard pellet
Add 2mL of 100mM phosphate buffer (pH 6.0)
Mix/vortex
Sample pH should be 6.0±0.5
Adjust pH accordingly with 100mM monobasic or
dibasic sodium phosphate
Condition Verify-CX Extraction Column
3mL of CH
3
OH then aspirate
3mL of DI H
2
O then aspirate
1mL of 100mM phosphate buffer (pH 6.0) then aspirate
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
3mL of DI H
2
O
1mL of 100mM acetic acid
Dry column (5 minutes at >10"Hg)
2mL of hexane
Elute Acid and Neutral Drugs (Fraction 1)
3mL of hexane/ethyl acetate (50:50);
Collect eluate at <2mL/minute
Dry Eluate
Evaporate to dryness at <40°C
Reconstitute with 100µL ethyl acetate
Quantitate Acid and Neutral Drugs
Inject 1 to 2µL onto gas chromatograph
Wash Column
3mL of CH
3
OH then aspirate
Dry column (5 minutes at >10"Hg)
Elute Basic Drugs (Fraction 2)
3mL of CH
2
Cl
2
/IPA/NH
4
OH (78:20:2)
Collect eluate at 1 to 2mL/min
NOTE:
Prepare elution solvent fresh daily; add IPA/NH
4
OH mix,
then add CH
2
Cl
2
(pH 11-12)
Dry Eluate
Evaporate to dryness at <40°C using an appropriate
evaporator. Take care not to overheat or over evaporate.
Certain compounds are heat labile, such as the
amphetamines and phencyclidine.
Reconstitute with 100µL of ethyl acetate
Quantitate Basic Drugs
Inject 1 to 2µL onto gas chromatograph
Recommended GC Columns
Part Number
TRACE TR-DoA5 Column, 15m x 0.25mm x 0.25µm
26AF130P
TRACE TR-DoA35 Column, 15m x 0.20mm x 0.33µm
26AC497P
NOTES:
(1)
Fraction 1 (acids and neutrals) and fraction 2 (bases) can be
combined together
(2)
A keeper solvent such as DMF can be used to prevent the
volatilization of amphetamines and phencyclidine; use 30 to 50µL
of high purity DMF in the sample (fraction 2) before evaporation
(3)
A 1% HCl in CH
3
OH solution has been used to prevent
volatization by the formation of the hydrochloric salt of the
drugs. Evaporate fraction 2 to approximately 100µL, then
add 1 drop of the solution. Continue to evaporate to dryness.