AOAC OMA# 2011.06 Final Action Review-OMB

2011.06 (September 2017)-Fol-22 MLT Report

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6. METHOD APPLICABILITY: The Folate SMPR applicability statement:

“ Determination of total folate [supplemental folic acid (CAS 59-30-3) and/or 5-methyl- tetrahydrofolate (CAS 68792-52-9), endogenous 5-methyl-tetrahydrofolate polyglutamates, 5- formyl-tetrahydrofolate, and 5-formyl-tetrahydrofolate polyglutamates] in all forms (powders, ready-to-feed liquids, and liquid concentrates) of infant, adult, and pediatric nutritional formula”. The method has been demonstrated to be suitable for the analysis of folate in a wide range of infant formula and nutritional products, as shown with a range of different matrices used in the collaborative study. 7. CONTRIBUTION OF FOLATES OTHER THAN FOLIC ACID : Folic acid accounted for >98% of total folate in five of the MLT samples. 15% of total folate in the NIST SRM 1869a accounted for folates other than folic acid. Soy based infant formula were observed to have about 8-10% contribution from folates other than folic acid. Milk based infant formula and Milk based Child Formula were also observed to have small contribution from non- folic acid folates to an extent of about 6-10%. 8. SAFETY CONSIDERATIONS: There are no safety hazards beyond those typically found in chemistry laboratories. Use appropriate safety equipment in handling acids, bases, and solvents, Refer to MSDS for detailed safety instructions for each of the chemical used. 1. Laboratory 1 prepared all stock standard solutions in 100 mL volume and pointed out that the folate stock standard solvent was barely enough if prepared as mentioned in the protocols. The folate Stock Solvent may be prepared in larger volume. All participants were informed of the method of its preparation in 500 mL volume instead of 250 mL posted initially. 2. Laboratory 2 wanted to know approximate volume of ammonium hydroxide may be required to adjust pH to 5.5 of the Folate Stock solvent. About 30 mL of ammonium hydroxide may be required to adjust pH of 500 mL of the Folate Stock solvent. This was informed to all the participants. 3. Laboratory 5 wanted know how much plasma can be recovered after treatment of 5 mL of plasma with charcoal. It was informed to all participants that usual recovery of the plasma is about 4.3-4.4 mL from 5 mL after the charcoal treatment. 4. Laboratory 1 initially had some issues in gravitation loading and elution of the samples. A suggestion of a push with a syringe plunger or pipette bulb to break air bubble to initiate the flow helped them. This suggestion was communicated to all participants. 5. The HPLC configuration of Laboratory 11 initially could not provide gradient specified in the method and could not reproduce resolution of peaks per Example Chromatogram of 9. COMMENTS FROM COLLABORATORS:

Collaborative Study Report: Method AOAC 2011.06 (Folate by LC-MS/MS) 20