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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:DI

H

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