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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
5
j.)
Iso-propanol (IPA)
k.)
Methanol (MeOH)
l.)
Vitamin K1-d7 (5,6,7,8-d4, 2-methyl-d4) (Sigma)
Supplies:
a.)
50ml Centrifuge tubes
b.)
16 x125mm glass culture tubes
Reagents:
a.)
Phosphate Buffer: 54g Potassium phosphate monobasic in 350mL H2O. Adjust pH to 8.0 (+/-0.1) by
slowly adding 40%KOH. Make up to volume to 500mL with H2O.
b.)
6N NaOH: 10N NaOH diluted to 6N with water.
c.)
75:25 MeOH:IPA (Methanol: Isopropanol) solution: Mix methanol 75 ml with 25 mL of iso-propanol
and mix.
d.)
Vitamin K1 Stock Standard Solution: Prepare vitamin K1 standard stock solution in 75:25 MeOH:IPA
solution. 10 mg of vitamin K1cis and all-trans is dissolved in 75:25 MeOH:IPA and make up volume to
10 mL.
e.)
Vitamin K2-4 Stock Standard Solution: Prepare vitamin K2-4 standard stock solution in 75:25
MeOH:IPA solution. 10 mg of vitamin K1 is dissolved in 75:25 MeOH:IPA and make up volume to 10
mL.
f.)
Vitamin K2-7 Stock Standard Solution: Prepare vitamin K2-7 standard stock solution in 75:25
MeOH:IPA solution. 10 mg of vitamin K2-7 is dissolved in 75:25 MeOH:IPA and make up volume to 10
mL
g.)
Vitamin K1 Intermediate Standard Solution (10 ppm): 0.1 mL of vitamin K1stock standard solution
(1000 ppm) is taken into a 10 mL volumetric flask and volume made up to 10 mL with 75:25
MeOH:IPA
h.)
Vitamin K2-4 Intermediate Standard Solution (10 ppm): 0.1 mL of Vitamin K2-4 stock standard
solution (1000 ppm) is taken into a 10 mL volumetric flask and volume made up to 10 mL with 75:25
MeOH:IPA.
i.)
Vitamin K2-7 Intermediate Standard Solution (10 ppm): 0.1 mL of Vitamin K2-7 stock standard
solution (1000 ppm) is taken into a 10 mL volumetric flask and volume made up to 10 mL with 75:25
MeOH:IPA .
j.)
Vitamin K1 Working Standard Solution (1 ppm): 1.0 mL of vitamin K1 intermediate standard solution
(1 ppm) is taken into a 10 mL volumetric flask and volume made up to 10 mL with 75:25 MeOH:IPA
k.)
Vitamin K2-4 Working Standard Solution (1 ppm): 1.0 mL of vitamin K2-4 intermediate standard
solution (1 ppm) is taken into a 10 mL volumetric flask and volume made up to 10 mL with 75:25
MeOH:IPA
l.)
Vitamin K2-7 Working Standard Solution (1 ppm): 1.0 mL of vitamin K2-7 intermediate standard
solution (1 ppm) is taken into a 10 mL volumetric flask and volume made up to 10 mL with 75:25
MeOH:IPA .
m.) Vitamin K1 Internal standard Solution: Dissolve 1 mg of Vitamin K1-d7 (5,6,7,8-d4, 2-methyl-d4) in
100 mL of 75:25 MeOH:IPA to make 10 ppm solution.
VitK-01 w/SLV
FOR ERP USE ONLY
DO NOT DISTRIBUTE