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

Sample Preparation Details

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: 22984)

Part No:00B-4622-AN
Dimensions:50 x 2.1 mm ID
Elution Type:Gradient
Elution A:0.1% formic acid in water
Elution B:0.1% formic acid in acetonitrile
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 95 5
2 0.5 95 5
3 3 5 95
4 3.5 5 95
5 3.51 95 5
6 5.5 95 5

Flow Rate:0.5 mL/min
Col. Temp: 25 °C
Detection: Tandem Mass Spec (MS-MS) @ (22°C)
Note:Strata X-C Elution Plate Protocol

200 µL methanol
200uL water
750 µL diluted serum
(375 µL plasma diluted 1:1 with 4% phosphoric acid in water)
200 µL 2% formic acid in water
Wash 2
200 µL methanol
25 µL 5% ammonium hydroxide in acetonitrile:methanol(60:40)
1 µL

Note: Spiked conc. of analyte was 1 ng/mL serum

Analyte Details

Analyte Detail not available