AOAC SPIFAN Nutrients ERP-Final Review (Oct. 2018)

2011.06 (September 2017)-Fol-22 MLT Report

FOR ERP USE ONLY DO NOT DISTRIBUTE

19.) Ascorbic Acid

Fisher

Cat. No. BP351-500

20.) Tris-(2-Carboxyethyl)phosphine, Hydrochloride (TCEP) Fisher

Cat. No. 50-093-0

21.) Charcoal,(Darco G-60 Activated Carbon)

Fisher

Cat. No. D127-500

22.) Formic acid Sigma F0507 G. STANDARD AND REAGENT SOLUTION PREPARATION

Since folate compounds are light sensitive, all samples and standards must be prepared, handled, and stored in the dark or under yellow shielded lighting (see apparatus). If the standards and samples must be transported through or into an area without yellow shielded lighting, they must be wrapped tightly in foil. I - Preparation of Solvent mixture to be used to prepare standard solutions 1. Folate Stock Solvent (15.6mM ammonium acetate buffer with 25% ascorbic acid and 1% mercaptoethanol, pH = 5.5): a. Weigh accurately 0.6 g +/- 0.01g of ammonium acetate and transfer into a 500 mL beaker. b. In a fume hood, add approximately 300 ml of HPLC water and 2.4 mL of glacial acetic acid slowly. c. Add 125 g of ascorbic acid and 5 mL of 2-mercaptoethanol. Stir to completely dissolve using a stirrer. d. Check the pH of the solution and adjust to pH 5.5, if necessary, using concentrated ammonium hydroxide (28-30%). About 30 mL of ammonium hydroxide may be required to adjust pH. e. Transfer the solution into a 500 mL volumetric flask and bring to volume (500 mL) with HPLC water. Mix thoroughly. 2. Folate Intermediate Solvent (1.6 mM Ammonium Acetate buffer, with 1% ascorbic acid, pH 5.5): a. Weigh accurately 0.012 g ammonium acetate and transfer into a 100mL beaker. b. In a fume hood, add approximately 70ml of HPLC water and 48 uL of glacial acetic acid slowly. Add 1g of ascorbic acid and stir until completely dissolved. c. Adjust pH to 5.5 if necessary with 10x water diluted concentrated ammonium hydroxide or 10x water diluted glacial acetic acid. d. Transfer the solution to a 100 mL volumetric flask and bring to volume with HPLC water. Mix thoroughly.

3. Solvent for SPE Elution and Folates Standard (Methanol with 10% formic acid and 1% ascorbic acid):

Collaborative Study Report: Method AOAC 2011.06 (Folate by LC-MS/MS) 27