AOAC SPIFAN ERP Meeting Book-March 16, 2016

VitK-01 w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Silliker Chem, Res. Center Crete, IL – Validation of a LC-MS/MS Method for Vitamin K Analysis

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

XICof +MRM(7pairs): 451.360/187.100DaID: VK1AfromSample3(Solvent Std inj 2) of Extraction140114_01.wiff (...

Max. 4.5e4cps.

6.15

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

K1

MK4

6.46

MK7

2000.0 4000.0 6000.0 8000.0

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

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

Silliker Laboratories, Chemistry Research Center, Date: 1/30/15

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