AOAC OMA# 2011.06 Final Action Review-OMB

2011.06 (September 2017)-Fol-22 MLT Report

FOR ERP USE ONLY DO NOT DISTRIBUTE

and brief (30 seconds) sonication. The solution is stored at -20°C.

c. 13 C 5 -(6S)-5-formyl-5,6,7,8-Tetrahydrofolate Internal Standard Stock soln. (1 mg/mL): 13 C 5 -(6S)-5-formyl-tetrahydrofolate (Merck Cie), the chemical is usually bought in 20 mg amount. This chemical is very expensive and that’s why will be bought by the study coordinators and about 1 mg amount will be provided to each study participants. Each participant will be provided about 1 mg of the chemical in a vial from the study director. The entire 1 mg amount of the received labeled formyl-THF is dissolved in 1 mL of folate stock solvent. Dissolve the chemical completely with the aid of vortex and brief (30 seconds) sonication. The solution is stored at -20°. 7 .) 20 ppm Internal Intermediate Standard Soln. cocktail (20ug/mL of each int. std. folate compound): a.) Take about 5 mL of Folate Intermediate Solvent into a 10 mL low-actinic volumetric flask b.) Transfer accurately 0.2 mL of each of the internal standard stock solutions, i.e., 13 C 5 -Folic Acid, 13 C 5 -labeled (6S)-5-methyl-THF, and 13 C 5 -labeled (6S)- 5-formyl-THF (con. 1000 µg/mL of each) into a 10 mL volumetric flask. c.) Bring to volume (10 mL) with Folate Intermediate Solvent d.) Sonicate in a water bath for about 30 sec. 5x inversion of the flask for a thorough mix of contents in the flask. e.) Calculate actual concentrations of each internal standard, from the reagent’s listed concentrations (i.e. 200 mcg / 10 mL = 20 µg/mL). f.) Store at -20 o C. 8.) 2 ppm Internal Std. solution mixture for calibration standards (2µg/mL of each int. std. folate compound): a) Take 20 mL of Solvent for SPE Elution in a 50 mL centrifuge tube. Add 1 mL of ammonium hydroxide (28-30%) and mix (Neutralized Solvent for SPE Elution). Mix. Prepare fresh before use. b) Pipette accurately 100 µL, of the folate internal intermediate standard solution (concentration = about 20 µg/mL for each folate compound) into a microfuge tube. Add accurately 900 µL of the freshly prepared Neutralized Solvent for SPE Elution. c) Mix thoroughly by a brief vortex for 30 seconds or so. d) Store at -20 o C. 9.) 40 ppb Internal Std. soln. for calibration standards (40 ng/mL each int. std. folate compound):

Collaborative Study Report: Method AOAC 2011.06 (Folate by LC-MS/MS) 31