Extraction of Procaine from Serum using Strata-X-C Microelution & a Kinetex 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. Procaine
-OEChem-06211003152D<br><br> 37 37 0 0 0 0 0 0 0999 V2000<br> 3.7320 0.4050 0.0000 O 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.0000 0.4050 0.0000 O 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.5981 2.9050 0.0000 N 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.8660 -4.0950 0.0000 N 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.5981 1.9050 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 5.4641 3.4050 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.7320 3.4050 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.7320 1.4050 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 5.4641 4.4050 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.7320 4.4050 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.8660 -0.0950 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.8660 -1.0950 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.7320 -1.5950 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.0000 -1.5950 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.8660 -3.0950 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.7320 -2.5950 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.0000 -2.5950 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.8101 1.3224 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 5.2087 2.0127 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 5.6762 2.8224 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 6.0747 3.5127 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.1215 3.5127 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.5200 2.8224 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.5200 1.9876 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.1215 1.2973 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 6.0841 4.4050 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 5.4641 5.0250 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.8441 4.4050 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.3520 4.4050 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.7320 5.0250 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.1120 4.4050 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.2690 -1.2850 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 1.4631 -1.2850 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 4.2690 -2.9050 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 1.4631 -2.9050 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 2.3291 -4.4050 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 3.4030 -4.4050 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0<br> 1 8 1 0 0 0 0<br> 1 11 1 0 0 0 0<br> 2 11 2 0 0 0 0<br> 3 5 1 0 0 0 0<br> 3 6 1 0 0 0 0<br> 3 7 1 0 0 0 0<br> 4 15 1 0 0 0 0<br> 4 36 1 0 0 0 0<br> 4 37 1 0 0 0 0<br> 5 8 1 0 0 0 0<br> 5 18 1 0 0 0 0<br> 5 19 1 0 0 0 0<br> 6 9 1 0 0 0 0<br> 6 20 1 0 0 0 0<br> 6 21 1 0 0 0 0<br> 7 10 1 0 0 0 0<br> 7 22 1 0 0 0 0<br> 7 23 1 0 0 0 0<br> 8 24 1 0 0 0 0<br> 8 25 1 0 0 0 0<br> 9 26 1 0 0 0 0<br> 9 27 1 0 0 0 0<br> 9 28 1 0 0 0 0<br> 10 29 1 0 0 0 0<br> 10 30 1 0 0 0 0<br> 10 31 1 0 0 0 0<br> 11 12 1 0 0 0 0<br> 12 13 2 0 0 0 0<br> 12 14 1 0 0 0 0<br> 13 16 1 0 0 0 0<br> 13 32 1 0 0 0 0<br> 14 17 2 0 0 0 0<br> 14 33 1 0 0 0 0<br> 15 16 2 0 0 0 0<br> 15 17 1 0 0 0 0<br> 16 34 1 0 0 0 0<br> 17 35 1 0 0 0 0<br>M END
Compound Name
Procaine
CID
4914
Molecular Formula
C13H20N2O2
Molecular Weight
236
No. Hydrogen Bond Acceptors
4
No. Hydrogen Bond Donors
1
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