Applications

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

Analytes

1Cortisone
2Cortisol-D4 (Internal standard)
3Prednisolone

Compound Name Cortisone
CID 222786
Molecular Formula C21H28O5
Molecular Weight 360
No. Hydrogen Bond Acceptors 5
No. Hydrogen Bond Donors 2

Details

LC Conditions (App ID: 22785)

Column:

Kinetex 2.6 µm Biphenyl 100 Å, LC Column 50 x 3.0 mm, Ea

Brand Name:

Kinetex

Part No:

00B-4622-Y0

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)

Detector Info:

SCIEX API 5000

Sample Preparation Details

Description: Novum SLE, MINI 96-Well Plate, 1/Pk
Part Number: 8E-S138-FGA
Pre Treatment Note: 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.
Sample PreTreatment: 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.
Evaporation: Evaporate to dryness
1 Shake Ethyl acetate

Order Items Used in This Application

00B-4622-Y0

Kinetex 2.6 µm Biphenyl 100 Å, LC Column 50 x 3.0 mm, Ea

Qty:

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

Order Items Used in This Application

00B-4622-Y0

Kinetex 2.6 µm Biphenyl 100 Å, LC Column 50 x 3.0 mm, Ea

Details

LC Conditions

Column:

Kinetex 2.6 µm Biphenyl 100 Å, LC Column 50 x 3.0 mm, Ea

Brand Name:

Kinetex

Part No:

00B-4622-Y0

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)

Detector Info:

SCIEX API 5000

LC Sample Preparation Details

Description:	Novum SLE, MINI 96-Well Plate, 1/Pk
Part Number:	8E-S138-FGA
Phase Name:	SLE
Pre Treatment Note:	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.
SPE Sample PreTreatment:	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.
SPE Evaporation:	Evaporate to dryness

Analytes

1. Cortisone

2. Cortisol-D4 (Internal standard)

3. Prednisolone

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