Extraction of Timolol from Serum using Strata-X-C 10 mg Plate & Kinetex 2.6u Biphenyl 50x2.1 Column

Sample Preparation Details

Part No:8E-S029-AGB
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a 10 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Load:Dilute serum 375µL serum with 375µL 4% Phosphoric acid in water
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
Final Prep and Analysis: No dry down step was performed. 1µL of final eluent was injected. Inject: 1 µL on HPLC / Tandem Mass Spec (MS-MS) @ (ambient)


Name Spiked
Log P pKa % Rec %RSC (n=0)
1 Timolol

Application Detail (App ID: 22982)

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 10 mg Plate Protocol

500 µL methanol
500uL water
750 µL diluted serum
(375 µL plasma diluted 1:1 with 4% phosphoric acid in water)
500 µL 2% formic acid in water
Wash 2
500 µL methanol
375 µL(3x125 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