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Forensic
Applications
Screening in Whole Blood (Manual Method for Immunoassay)
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 1mL of blood add 4mL of H
2
O (pH 5 to 7)
Mix/vortex
Let sample stand for 5 minutes to lyse red blood cells
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)
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
3mL of acetic acid
Dry column (5 minutes at >10"Hg)
2mL of hexane
Elute Acidic and Neutral Drugs
3mL of hexane/ethyl acetate (50:50)
Collect eluate at <5mL/minute
Remove collection tubes
Wash Column
3mL of CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Basic Drugs
Replace collection tubes from elution of acidic and
neutral drugs
3mL of CH
2
Cl
2
/IPA/NH
4
OH (78:20:2)
Collect eluate at 1 to 2mL/min using minimal vacuum
NOTE:
Elute into the tubes containing the acidic and neutral
drugs. Prepare elution solvent fresh daily; add IPA/NH
4
OH mix,
then add CH
2
Cl
2
(pH 11-12)
Dry Elute – Combine Eluates Acidic and Neutral drugs
and Basic Drugs
Evaporate to a volume 100µL at <40°C
Reconstitute
Add 900µL of normal saline (sample volume is now its
original 1.0mL)
Analyze as Appropriate
Process according to urine drug screening protocols
provided by immunoassay manufacturer
Sertraline and Desmethylsertraline in Serum,
Plasma or Whole Blood for HPLC Analysis
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 4mL of DI H
2
O add 2mL of 100mM phosphate
buffer (pH 6.0)
Add internal standard
Add 1mL of urine, blood or plasma/serum
Mix/vortex
Centrifuge for 10 minutes at 2,000rpm and discard pellet
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)
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
3mL of CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Sertraline and Desmethylsertraline
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
Reconstitute
Reconstitute with 200µL of ethyl acetate/DI H
2
O (1:3)
Mix/vortex vigorously for 30 seconds
Analysis
Inject 100µL onto Isocratic LC system at wavelength 235nm
Mobile phase: 0.25 M potassium phosphate (pH 2.7)
containing 30% CH
3
CN
Flow rate: 2mL/minute
Recommended HPLC Column
Part Number
Hypersil GOLD C8 3µm, 150 x 4.6mm
25203-154630