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

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.

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

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