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Silliker
Chem, Res. Center Crete, IL – Validation of a LC-MS/MS Method for Vitamin K Analysis
Silliker Laboratories, Chemistry Research Center, Date: 1/30/15
9
Ion Source Gas 1 (GS1): 35.00 psi
Ion Source Gas 2 (GS2): 30.00 psi
Inter Face Heater: on
Entrance Potential: 5.40 V
Collisionally Activated Dissociation Potential (CAD): 4.00 V
Collision Cell Exit Potential (CXP): 4.00
Optimization of HPLC Conditions:
HPLC employing Column YMC CAROTENOID S-3 2.0X150mm, with the
mobile Phase: A: 5mMAmmonium Acetate in Water, and B: 5mMAmmonium Acetate in 75:25
MeOH:IPA with the following gradient provided a very good chromatography (Figure 1).
Gradient: Time
%A %B
Flow Rate (mL/min)
0.10
10 90
0.4
5.00
0 100
0.4
10.00 0 100
0.4
10.10 10 90
0.4
15.00 System Controller
STOP
Figure 1
. A representative chromatograms of VitaminK1, K2-4 and K2-7 in optimized LC and MS
conditions.
Evaluation of the linearity of the analytical response against concentration of the respective
analyte
The linearity of the analytical response, peak areas, (analyte peak area/internal std. peak area in the
case of vit. K1) of the analytes against their respective concentrations was evaluated. All three vitamin
K compounds showed a good linear relationship in their response to the employed concentration in the
XICof +MRM(7pairs): 451.360/187.100DaID: VK1AfromSample3(Solvent Std inj 2) of Extraction140114_01.wiff (...
Max. 4.5e4cps.
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0
83 164 246 327 409 491 572 654 735 817 898 980 1062 1143
Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
4.0e4
4.2e4
4.4e4
Intensity, cps
6.15
6.46
MK7
K1
MK4
VitK-01 w/SLV
FOR ERP USE ONLY
DO NOT DISTRIBUTE