AOAC OMA# 2011.06 Final Action Review-OMB

2011.06 (September 2017)-Fol-22 MLT Report

FOR ERP USE ONLY DO NOT DISTRIBUTE

a. Take 10mL of concentrated formic acid and 1g of ascorbic acid into a 100mL volumetric flask. Add approximately 50ml methanol. b. Sonicate for about 2 min in a water bath set at approximately 25 o C or until complete dissolution of chemicals. c. Bring to 100 mL volume with methanol. Mix thoroughly. d. This solution is used to elute folates from SPE sorbent after clean up, to make analytical standard of folates and as a blank in LC-MS-MS analysis. II - Preparation of Folate standard solutions 4. Folate Stock Standard Solutions (500ug/mL) a. Accurately weigh and record to 5 decimals ~25mg of each of the standards except pteroyl-tri-glutamate listed in Table 4:A in separate 50mL low-actinic volumetric flasks. Record weights. b. Add approximately 35mL of the Folate Stock Solvent to each flask and sonicate in a water bath for about 1 minute for complete dissolution of the folate chemical. Add least amount of ammonium hydroxide solution (28-30%) to aid in dissolution of folic acid, it may take approximately 30 drops. Let flasks come to room temperature. c. Bring to volume with Folate Stock Solvent. Put stopper on the flasks. Mix the solution thoroughly by 5x inversion of the flask. d. Accurately weigh and record to 5 decimals ~5mg of pteroyl-tri- glutamate in a 10mL low-actinic volumetric flasks. Add approximately 7.5mL of the Folate Stock Solvent and sonicate in a water bath for about 1 minute for complete dissolution. e. Calculate concentration in mcg/mL following the example in Table 4-A below. f. Transfer solutions to suitable clean containers separately and keep in freezer (-20°C). g. Calculate the correct concentration of each of the folate compound’s respective stock solution after adjusting for the moisture content in the obtained folate chemical based on the certificate provided by the supplier. Folic acid may have moisture as much as (7.9%), 5-formyl- THF as much as 14.9%. h. Calculate the correct concentration of each of the folate compound’s respective stock solution after further adjustment for the purity (HPLC) of the obtained folate chemical based on the certificate provided by the supplier. Typically the purity (HPLC based) of the folate compounds obtained from Schircks is usually 98-99%. i. Formyl-THF and CH3-THF chemicals obtained from Schircks (catalogue # 16.221 and 16.235 respectively) and listed in the method for preparation of calibration standards are calcium salt. The correct concentration of formyl-THF and CH3-THF in salt free form is calculated in the enclosed calculation template based on input values of salt forms of these compounds (corrected for moisture and purity). The conversion of Ca-salts of methyl-THF and formyl-THF in the calculation template is done by adjusting for the molecular weight differences in

Collaborative Study Report: Method AOAC 2011.06 (Folate by LC-MS/MS) 28