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

a.) Pipette appropriate amount of internal standard stock solution 13 C-Folic Acid as well as 13 C5-labeled (6S)-5-methyltetrahydrofolic acid into a 100 mL low-actinic volumetric flask so that final concentration of each of the compounds is 20 ug/mL after making up volume to 50 mL. b.) Add approximately 50mL of Folate Intermediate Standard Solution Solvent and sonicate in water bath set at 40 o C for about 10min to thoroughly mix. c.) Bring to volume with Folate Intermediate Standard Solvent and store approximately -20 o C. Stable for up to 60days. a) Take 20 mL of Solvent for SPE Elution in a 50 mL plastic centrifuge tube. Add 1 ml of ammonium hydroxide to the pipetted solvent for SPE elution to neutralize it. (Neutralized Solvent for SPE Elution). Prepare fresh before use. a.) Pipette 1 mL, ( assuming 20 µg/ml) of internal intermediate standard cocktail solution (above) ( assuming its concentration of 20 µg/ml) into a 10 mL low-actinic volumetric flask. Make up volume to 10 mL with the Neutralized Solvent for SPE Elution. b.) Add approximately 5mL of Neutralized Solvent for SPE Elution and sonicate in water bath set at 40 o C for about 2 min to thoroughly mix. c.) Bring to volume with Neutralized Solvent for SPE Elution and store approximately -20 o C. Stable for up to 60 days a) Take 12 mL of Solvent for SPE Elution in a scintillation vial or a 50 mL plastic centrifuge tube. Add 600 µL of ammonium hydroxide to the pipetted solvent for SPE elution to neutralize it. (Neutralized Solvent for SPE Elution). Prepare fresh before use. d.) Pipette 20 µl, ( assuming 20 µg/ml) of internal intermediate standard cocktail solution (above) ( assuming its concentration of 20 µg/ml) into a 10 mL low-actinic volumetric flask. Make up volume to 10 mL with the Neutralized Solvent for SPE Elution. e.) Add approximately 5mL of Neutralized Solvent for SPE Elution and sonicate in water bath set at 40 o C for about 2 min to thoroughly mix. f.) Bring to volume with Neutralized Solvent for SPE Elution and store approximately -20 o C. Stable for up to 60 days. 9.) Working Internal Std. soln. for calibration standards (40 ng/ml) :

8) Internal Std. solution for calibration standards (2 µg/ml) :

10) Folate Calibration Std. solutions: a) The calibration standard solutions are prepared as detailed in the following table b) The calibration solutions a through G are bprepared as described in the Table. c) The intermediate std. solutions are diluted to 1 mL after adding working internal std. solution.

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