AOAC SPIFAN ERP Meeting Book-March 16, 2016

2011.06 (Fol-22) w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

16x125mm 2.) 50mL centrifuge tubes 3.) PVDF syringe filters 4.) Microcentrifuge tubes; 2mL

Fisher

Cat. No. 14-375-150

Fisher cat. No. 02-681-344 5.) SPE cartridge: Phenomenex Part# 8B-S038UBJ. A mix mode weak anion exchange and reverse phase solid-phase extraction cartridge

Reagents:

1.) Folate Stock Solution Solvent (15.6 mM ammonium acetate buffer with 25% ascorbic acid and 1% mercaptoethanol, pH = 5.5): a.) weigh accurately 1.27g of ammonium acetate and transfer into a 1000mL beaker. b.) In a fume hood, add approximately 700ml of DI water and 4.8 mL of glacial acetic acid slowly. c.) Add 250g of ascorbic acid and 10mL of 2-mercaptoethanol. Stir to completely dissolve using a stir bar. Add concentrated ammonium hydroxide drop wise as necessary to dissolve completely. d.) Check the pH of the solution and adjust to pH 5.5, if necessary, using concentrated ammonium hydroxide or glacial acetic acid. e.) Transfer the solution into a 1L volumetric flask and bring to volume with DI water. 2.) Folate Intermediate Standard Solution Solvent (1.6 mMAmmoniumAcetate buffer, with 1% ascorbic acid, pH 5.5): a.) Weigh accurately 0.12 g ammonium acetate and transfer into a 1000mL beaker. b.) In a fume hood, add approximately 700ml of DI water and 0.5081g (484 uL)of glacial acetic acid slowly. Add 10g of ascorbic acid and stir until completely dissolved. c.) Adjust pH to 5.5 if necessary with concentrated ammonium hydroxide or glacial acetic acid. d.) Transfer the solution to a 1L volumetric flask and bring to volume with DI water. 3.) Solvent for SPE Elution and Folates Standard (Methanol with 10% formic acid and 1% ascorbic acid): a.) Into a 100mL volumetric flask, transfer 10mL of formic acid and 1g of ascorbic acid. Add approximately 50ml methanol. b.) Sonicate in a water bath set at approximately 25 o C until complete dissolution. c.) Bring to volume with methanol. Use solution to elute folates from SPE sorbent after clean up, to make analytical standard of folates and as a blank in UPLC-MS-MS analysis. 4.) Folate Stock Solution (500ug/mL) a.) Accurately weigh out about 5mg of each standard in a separate 10mL low-actinic volumetric flask. b.) Add approximately 7.5mL of the Folates Stock Solution Solvent to each flask and sonicate in a bath set at 40 o C for about 20min or until complete dissolution. Add several drops Ammonium hydroxide to aid in dissolution if necessary. c.) Bring to volume with Folates Stock Solution Solvent and transfer to freezer safe containers. d.) The Folic acid, 13 C-Folic Acid (IS), (6S)-5-Formyl-5,6,7,8-Tetrahydrofolate, 10-methyl- folic acid and 10-formyl-folic acid solutions are stable for up to 180 days stored in the freezer at -70 o C. e.) The (6S,R)-5-Methyl-5,6,7,8-Tetrahydrofolate, and 5,6,7,8-Tetrahydrofolate solutions are

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