Applications
Extraction of Total Cortisol from Human Plasma Using Novum SLE & Kinetex Biphenyl
22787
Separation Mode: Reversed Phase
Reversed Phase
Biphenyl
HPLC
Anti-Inflammatory Agents
Clinical
Clinical Research
Therapeutic / Clinical

Applications

Extraction of Total Cortisol from Human Plasma Using Novum SLE & Kinetex Biphenyl
Analytes
1
2
Compound Name
Hydrocortisone
Details
LC Conditions (App ID: 22787)
Column:
Brand Name:
Part No:
Elution Type:
Gradient
Mobile Phase:
A: 10 mm ammonium acetate in water
B: 10 mm ammonium acetate in methanol
Gradient Profile:
Step No.Time(min)%A%B
105050
22595
33.1595
43.115050
555050
Flow Rate:
0.45 mL/min
Temperature:
25°C
Detection:
Tandem Mass Spec (MS-MS) @ 22°C
Sample Preparation Details
Description:
Novum SLE, MAX 96-Well Plate, Ea
Part Number:
Pre Treatment Note:
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.
SPE Load:
Load 200.00 µL Sample diluted in 200
SPE Elution 1:
900 mL Methylene Chloride at µL mL/min
SPE Elution 2:
900 mL Methylene Chloride at µL mL/min
SPE Evaporation:
Evaporate to dryness
SPE Reconstitute:
200 µL 10mM ammonium acetate in water:methanol(50:50)

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