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Tricyclic Antidepressants in Plasma/Serum for HPLC
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 2mL of 100mM phosphate buffer (pH= 6.0) add
internal standard*
Add 1mL of 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 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 or use gravity flow
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 ethyl acetate/DI H
2
O (1:3)
Mix/vortex vigorously for 30 seconds
Inject 100µL onto HPLC
* Suggested internal standards: Trimipramine, Protriptyline
Pharmaceutical/Biotech
Applications