Automated Extraction of Prednisolone & Cortisone from Plasma Using Novum SLE & Kinetex 2.6u Biphenyl

Sample Preparation Details

Part Number :8E-S138-FGA
Description :Novum SLE, MINI 96-Well Plate, 1/Pk
Load :Load 150.00 µL Sample diluted in 150
Elution 1 :1000 mL Ethyl acetate
Evaporate: :Evaporate to dryness
Reconstitute :150 µL 10mM ammonium acetate in water:methanol(50:50)
Notes :Sample pre-treatment Dilute 150 uL of human plasma (spiked with 25 ng/mL and 125 ng/mL of cortisone and prednisolone respectively) with 150 uL of 50 mM sodium phosphate dibasic heptahydrate, pH unadjusted. Mix briefly (3-5 sec). Sample loading: Load the sample from pre-treatment step above onto the Novum plate and apply a short and gentle pulse of vacuum (~ 10" of Hg for 20 secs) until the sample has completely entered the media. Wait for 5 minutes. Elution: Dispense 1000 uL of ethyl acetate onto the SLE media and allow the solvent to elute by gravity (~ 5 min elution time) and collect the eluant. Apply vacuum at 5" of Hg for 45 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: Reconstitute the dry residue in 150 uL of initial mobile phase fortified with cortisol-D4.

Application Detail (App ID: 22785)

Part No:00B-4622-Y0
Dimensions:50 x 3 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: SCIEX API 5000

Analyte Details

Analyte Detail not available


Cortisol-D4 (Internal standard)