Applications
Extraction of Timolol from Serum using Strata-X-C Microelution & a Kinetex 2.6u Biphenyl HPLC Column
22984
Separation Mode: Reversed Phase
Reversed Phase
Biphenyl
HPLC
Antihypertensive Agents

Applications

Extraction of Timolol from Serum using Strata-X-C Microelution & a Kinetex 2.6u Biphenyl HPLC Column

Analytes

1
Compound Name
Timolol
CID
33624
Molecular Formula
C13H24N4O3S
Molecular Weight
316
No. Hydrogen Bond Acceptors
7
No. Hydrogen Bond Donors
2

Conditions

LC Conditions (App ID: 22984)
Column:Kinetex 2.6 µm Biphenyl 100 Å, LC Column 50 x 2.1 mm, Ea
Brand Name:Kinetex
Part No:00B-4622-AN
Elution Type:Gradient
Mobile Phase:A: 0.1% formic acid in water
B: 0.1% formic acid in acetonitrile
Gradient Profile:
Step No.Time(min)%A%B
10955
20.5955
33595
43.5595
53.51955
65.5955
Flow Rate:0.5 mL/min
Temperature:25°C
Detection:Tandem Mass Spec (MS-MS) @ 22°C

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.
SPE Load:Load 150.00 µL Sample diluted in 150
SPE Elution 1:1000 mL Ethyl acetate
SPE Evaporation:Evaporate to dryness
SPE Reconstitute:150 µL 10mM ammonium acetate in water:methanol(50:50)

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