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

LC Conditions

Column

Kinetex 2.6 µm Biphenyl 100 Å, LC Column 50 x 2.1 mm, Ea

Brand

Kinetex

Part No

00B-4622-AN

Phase Name

Biphenyl

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

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

Temperature

25°C

Detection

Tandem Mass Spec (MS-MS) @ (22°C)

Detection Info

Sample Preparation Details

Description

Novum SLE, MAX 96-Well Plate, Ea

Part No

8E-S138-5GA

Pretreatment 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.

Load

Load 200.00 µL Sample diluted in 200

Elution 1

900 mL Methylene Chloride at µL mL/min

Elution 2

900 mL Methylene Chloride at µL mL/min

Evaporation

Evaporate to dryness

Reconstitute

200 µL 10mM ammonium acetate in water:methanol(50:50)