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Extraction of Total Cortisol from Human Plasma Using Novum SLE & Kinetex Biphenyl

Sample Preparation Details

Part No:8E-S138-5GA
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a 400 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Load:Sample pre-treatment: Dilute 200 uL of human plasma with 200 ul of 50 mM sodium phosphate dibasic heptahydrate, pH unadjusted.
Vortex briefly (3-5 sec).
Sample loading: Load the sample from pretreatment step abive onto the Novum plate and apply a short and gentle pulse of vacuum (~ 5" of Hg for 5-10 sec) or until the sample has completely entered the media.

Wait for 5 minutes.

Final Prep and Analysis: Extraction Procedure
Sample pre-treatment: Dilute 200 uL of human plasma with 200 ul of 50 mM sodium phosphate dibasic heptahydrate, pH unadjusted. Vortex briefly (3-5 sec).
Sample loading: Load the sample from pretreatment step abive onto the Novum plate and apply a short and gentle pulse of vacuum (~ 5" of Hg for 5-10 sec) or until the sample has completely entered the media.
Wait for 5 minutes.
Elution: Dispense 900 uL of dichloromethane onto the SLE media and allow the solvent to elute by gravity (~ 5 min elution time) and collect the eluant.
Repeat with another 900 uL of dichloromethane and collect the eluant.
Apply vacuum at 5" of Hg for 20-30 secs to complete the extraction.
NOTE: Prolonged application of vacuum will result in elution of plasma out of the SLE media and into the final extracted solvent.
Dry down: Evaporate the final extract to complete dryness under slow stream of N2 at 40°C
Reconstitute the dry residue in 200 uL of initial mobile phase fortified with cortisol-D4. Inject: 10 µL on HPLC / Tandem Mass Spec (MS-MS) @ (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)
1 Cortisol 25  79
2 Cortisol-D4 (Internal standard) 50 

Application Detail (App ID: 22787)

Part No:00B-4622-AN
Dimensions:50 x 2.1 mm ID
Elution Type:Gradient
Elution A:10 mm ammonium acetate in water
Elution B:10 mm ammonium acetate in methanol
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 50 50
2 2 5 95
3 3.1 5 95
4 3.11 50 50
5 5 50 50

Flow Rate:0.45 mL/min
Col. Temp: 25 °C
Detection: Tandem Mass Spec (MS-MS) @ (22°C)
Detector Info: AB Sciex API 5000

Analyte Details

Analyte Detail not available

1
Cortisol
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2
Cortisol-D4 (Internal standard)