39
Sertraline and Desemethylsertraline in Blood, Plasma/Serum
for HPLC Analysis
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 4mL DI H
2
O add 2mL of 100mM phosphate buffer
(pH= 6.0), to this add internal standard*
Add 1mL of blood, plasma/serum or urine
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 HyperSep Verify-CX Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 1mL 100mM phosphate buffer (pH= 6.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load at 1mL/minute
Wash Column
1 x 3mL DI H
2
O
1 x 1mL 100mM acetic acid
1 x 3mL CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute
1 x 3mL CH
2
Cl
2
/IPA/NH
4
OH (78:20:2)
Collect eluate at 1mL/minute
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
Quantitate
Reconstitute with 200µL ac
etonitrile:DIH
2
O (1:3)
Mix/vortex vigorously for 30 seconds
Inject 100µL onto LC at wavelength 235nm
Mobile phase: 0.25 M potassium phosphate (pH 2.7)
containing 30% CH
3
CN
Flow rate: 2mL/minute
* Suggested internal standard: Desmethylsertraline
Tacrolimus, Cyclosporin and Rapamycin in Whole Blood
Using 200mg 6mL HyperSep Retain PEP Extraction Column
(Part Number: 60107-212)
Prepare Sample
Add 50mL whole blood and 50mL of 0.1 M ZnSO
4
to a
centrifuge tube
Mix/vortex
Add 500mL methanol and internal standards*
Mix/vortex
Centrifuge
Transfer supernate to a clean tube, add 500mL DI H
2
O
Mix/vortex
Condition HyperSep Retain PEP Extraction Column
1 x 2mL CH
3
OH
1 x 2mL DI H
2
O
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Decant the sample onto the column
Load at 1 to 2mL/minute
Wash Column
1 x 2mL DI H
2
O
Dry column (20 minutes at >10"Hg)
Elute Analytes
Add 750mL of ethyl acetate
Collect eluate at 1 to 2mL/minute
Analysis
Inject onto LC system
* Suggested internal standards: Cyclosporin Cyclosporin-D, Tacrolimus Ascomycin, and
Rapamycin Desmethoxyrapamycin
Recommended HPLC Column
Part Number
Hypersil GOLD 3µm, 150 x 4.6mm
25003-154630
Pharmaceutical/Biotech
Applications