Extraction of Timolol from Serum using Strata-X-C Microelution & a Kinetex 2.6u Biphenyl HPLC Column
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: 0.1% formic acid in water
B: 0.1% formic acid in acetonitrile
Gradient Profile
Step No.
Time(min)
%A
%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
0.50 mL/min
Temperature
25°C
Detection
Tandem Mass Spec (MS-MS) @ (22°C)
Detection Info
Sample Notes
Strata X-C Elution Plate Protocol Condition 200 µL methanol Equilibrate 200uL water Load 750 µL diluted serum (375 µL plasma diluted 1:1 with 4% phosphoric acid in water) Wash1 200 µL 2% formic acid in water Wash 2 200 µL methanol Elute 25 µL 5% ammonium hydroxide in acetonitrile:methanol(60:40) Inject 1 µL Note: Spiked conc. of analyte was 1 ng/mL serum
Sample Preparation Details
Description
Novum SLE, MINI 96-Well Plate, 1/Pk
Part No
8E-S138-FGA
Pretreatment 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.
Load
Load 150.00 µL Sample diluted in 150
Elution 1
1000 mL Ethyl acetate
Evaporation
Evaporate to dryness
Reconstitute
150 µL 10mM ammonium acetate in water:methanol(50:50)
Order Items Used in This Application
- 1. Timolol
-OEChem-05081000432D<br><br> 45 46 0 1 0 0 0 0 0999 V2000<br> 2.3660 -1.8332 0.0000 S 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.8660 2.7056 0.0000 O 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.6261 -0.5731 0.0000 O 0 0 0 0 0 0 0 0 0 0 0 0<br> 6.5282 0.0449 0.0000 O 0 0 0 0 0 0 0 0 0 0 0 0<br> 8.0145 -1.2934 0.0000 N 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.8660 0.7056 0.0000 N 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.0570 -0.8822 0.0000 N 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.3660 -1.8332 0.0000 N 0 0 0 0 0 0 0 0 0 0 0 0<br> 8.7576 -1.9625 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 7.0634 -1.6024 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 6.3203 -0.9332 0.0000 C 0 0 1 0 0 0 0 0 0 0 0 0<br> 3.7320 1.2056 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.0000 1.2056 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 9.5008 -2.6316 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 9.4268 -1.2194 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 8.0885 -2.7056 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.7320 2.2056 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.0000 2.2056 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 5.3692 -1.2423 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.8660 -0.2944 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.6750 -0.8822 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 8.1434 -0.6869 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 6.5749 -1.9841 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 7.3545 -2.1498 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 5.8595 -0.5184 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.9441 0.6230 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.3426 1.3133 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 1.3894 1.3133 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 1.7880 0.6230 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 9.0859 -3.0924 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 9.9615 -3.0465 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 9.9156 -2.1709 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 9.8875 -1.6342 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 9.8416 -0.7586 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 8.9660 -0.8045 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 7.6278 -2.2908 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 7.6737 -3.1664 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 8.5493 -3.1205 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.3426 2.0980 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.9441 2.7882 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 1.7880 2.7882 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 1.3894 2.0980 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.8807 -1.6240 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 5.6603 -1.7897 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 7.1179 0.2365 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 1 7 1 0 0 0 0<br> 1 8 1 0 0 0 0<br> 2 17 1 0 0 0 0<br> 2 18 1 0 0 0 0<br> 3 19 1 0 0 0 0<br> 3 21 1 0 0 0 0<br> 11 4 1 1 0 0 0<br> 4 45 1 0 0 0 0<br> 5 9 1 0 0 0 0<br> 5 10 1 0 0 0 0<br> 5 22 1 0 0 0 0<br> 6 12 1 0 0 0 0<br> 6 13 1 0 0 0 0<br> 6 20 1 0 0 0 0<br> 7 20 2 0 0 0 0<br> 8 21 2 0 0 0 0<br> 9 14 1 0 0 0 0<br> 9 15 1 0 0 0 0<br> 9 16 1 0 0 0 0<br> 10 11 1 0 0 0 0<br> 10 23 1 0 0 0 0<br> 10 24 1 0 0 0 0<br> 11 19 1 0 0 0 0<br> 11 25 1 0 0 0 0<br> 12 17 1 0 0 0 0<br> 12 26 1 0 0 0 0<br> 12 27 1 0 0 0 0<br> 13 18 1 0 0 0 0<br> 13 28 1 0 0 0 0<br> 13 29 1 0 0 0 0<br> 14 30 1 0 0 0 0<br> 14 31 1 0 0 0 0<br> 14 32 1 0 0 0 0<br> 15 33 1 0 0 0 0<br> 15 34 1 0 0 0 0<br> 15 35 1 0 0 0 0<br> 16 36 1 0 0 0 0<br> 16 37 1 0 0 0 0<br> 16 38 1 0 0 0 0<br> 17 39 1 0 0 0 0<br> 17 40 1 0 0 0 0<br> 18 41 1 0 0 0 0<br> 18 42 1 0 0 0 0<br> 19 43 1 0 0 0 0<br> 19 44 1 0 0 0 0<br> 20 21 1 0 0 0 0<br>M END
Compound Name
Timolol
CID
33624
Molecular Formula
C13H24N4O3S
Molecular Weight
316
No. Hydrogen Bond Acceptors
7
No. Hydrogen Bond Donors
2
Similar Applications
Reversed Phase
Beta-blockers using Luna Omega 1.6u PS C18 50x2.1mm
Luna Omega 1.6µm PS C18 100Å, LC Column 50 x 2.1 mm, EA
23727
Reversed Phase
Beta-blockers using Luna Omega Polar C18 50x2.1mm
Luna Omega 1.6µm Polar C18 100Å, LC Column 50 x 2.1 mm, EA
23611