AOAC OMA# 2011.06 Final Action Review-OMB

1884  B handari et al . : J ournal of AOAC I nternational V ol . 101, N o . 6, 2018

used are often calcium salts. Calculate the concentration of both stock standard solutions as salt-free form based on the differences in their respective molecular weights. Stock standard concentration, micrograms per milliliter = [(standard, grams salt free adjusted for purity and moisture contents) × 10 6 ]/(final volume, milliliters). Folate stock standard solutions if stored at –70°C, folic acid, 5-CHO-THF, and Pte-Glu3 are usually stable for 6 months, and 5-CH3-THF is stable for 3 months. The solutions may be stable for less time period if stored at –20°C. The purity of the folic acid standard chemical can be confirmed by making its solution in 0.1 M NaOH (pH 13) at a concentration of 10 μg/mL and reading absorbance at 282 nm in spectrophotometer in a cuvette light path length of 1 cm. Pure folic acid E 1% (in 1 cm light path) at 282 nm = 532.2. ( e )  Preparation of folate intermediate standards (20 μg/mL) .—Add about 5 mL of the intermediate solvent to a 10 mL low-actinic volumetric flask. Add 0.4 mL of each of the stock standard solutions of folic acid, 5-CH3-THF, and 5-CHO-THF to the same flask. Make up the volume to 10 mL with the intermediate solvent and mix the contents. Concen­ tration (micrograms per milliliter) of each folate vitamer in intermediate standard solution = (stock standard solution concentration, micrograms per milliliter × 0.4 mL) / 10 mL. The folate intermediate standard solution can be stored at –20°C for 30 days, and it can be stable for a longer time at –70°C. ( f )  13 C 5 -folic acid internal standard stock solultion (1 mg/mL) .—The chemical is often supplied in a 1 mg amount. The entire 1 mg amount of labeled folic acid is dissolved in 1 mL of the stock solvent. Folic acid is difficult to dissolve. The addition of 10 μL of ammonium hydroxide solution (28–30%) aids in dissolution. Dissolve the chemical in a correct volume of the solvent if its weight is different than 1 mg so that the final concentration is around 1 mg/mL. Sonication and mixing on a vortex mixer for 1 to 2 min may help complete dissolution. ( g )  13 C 5 -(6S)-5-methyl-5,6,7,8-tetrahydrofolate internal standard stock solution (1 mg/mL) .—The labeled methyl-THF may be supplied in a 1 mg amount. The entire 1 mg amount of labeled methyl-THF is dissolved in 1 mL of the stock solvent. Dissolve the chemical in correct volume of the solvent if its weight is different than 1 mg so that the final concentration is around 1 mg/mL. Dissolve the chemical completely with the aid of a vortex mixer and brief (30 s) sonication. ( h )  13 C 5 -(6S)-5-formyl-5,6,7,8-tetrahydrofolate internal standard stock solution (1 mg/mL) .—Weigh 10 mg of labeled formyl-THF in a 10 mL low-actinic volumetric flask. Add about 7 mL of the stock solvent. Dissolve the chemical completely with the aid of a vortex mixer and brief (30 s) sonication. Make up the volume to 10 mL with the stock solvent and mix the solution. Folate internal standard stock solutions if stored at –70°C, folic acid and 5-CHO-THF can be stable for 6 months and 5-CH3-THF for 3 months. The solutions may be stable for less time period if stored at –20°C . The concentration of each of the stock solutions should be calculated based on the known purity of the respective folate chemical. ( i )  Internal standard intermediate solution cocktail ( 20 μg/mL of each folate ).—Add about 5 mL of the intermediate solvent into a 10 mL low-actinic volumetric flask. Transfer exactly 0.2 mL of each of the internal standard stock solutions, i.e., 13 C 5 -FA, 13 C 5 -CH3-THF, and 13 C 5 -CHO-THF, into the same 10 mL volumetric flask. Make up to 10 mL with the intermediate solvent. Thoroughly mix the

solution. Calculate the actual concentrations of each internal standard as the following. The concentration (micrograms per milliliter) of each folate vitamer in the internal standard intermediate solution = (internal standard stock solution concentration in micrograms per milliliter × 0.2 mL) / 10 mL. The folate standard intermediate solution can be stored at –20°C for 30 days, and it can be stable for longer at –70°C. ( j )  Two parts per million internal standard solution for calibration standards (2 μg/mL of each internal standard folate) .—Prepare the solution fresh before use. Store it at 4°C if necessary but not for more than 6 h. Take 20 mL of the solvent for SPE elution into a 50 mL centrifuge tube. Add 1 mL of ammoniumhydroxide (28–30%) andmix (neutralized solvent for SPE elution). Prepare it fresh before use. Pipette exactly 100 μL of the folate internal standard intermediate solution (~20 μg/mL of each folate) into a microcentrifuge tube. Add 900 μL of the freshly prepared neutralized solvent for SPE elution. Mix thoroughly with a vortex mixer for about 30 s. The concentration (micrograms per milliliter) of each folate vitamer in 2 ppm internal standard solution = internal standard intermediate solution concentration in micrograms per milliliter × 0.1 mL. ( k )  Forty parts per billion internal standard solution for calibration standards (40 ng/mLeach folate) .—Prepare the solution fresh before use. Store it at 4°C if necessary but not for more than 6 h. Take 12 mL of the solvent for SPE elution in a scintillation vial or a 50 mL plastic centrifuge tube. Add 600 μL of ammonium hydroxide (28–30%; neutralized solvent for SPE elution) and mix. Prepare it fresh before use. Pipette about 5mLof neutralized solvent for SPE elution into a 10 mL low-actinic volumetric flask. Pipette 20 μL of the internal standard intermediate solution (20 μg/mL each folate) into the same 10 mL volumetric flask. Make up the volume to 10 mL with the neutralized solvent for SPE elution and mix thoroughly. The concentration (micrograms per milliliter) of each folate vitamer in 40 ppb internal standard solution = (concentration of the folate in internal standard intermediate solution in micrograms per milliliter × 0.02 mL) / 10 mL. ( l )  Preparation of calibration standard solutions .— Prepare the solutions fresh before the analysis. Store them at 4°C if necessary but not for more than 6 h. Take 12 mL of the solvent for SPE elution in a scintillation vial or a 50 mL plastic centrifuge tube. Add 600 μL of ammonium hydroxide (neutralized solvent for SPE elution) and mix. Prepare it fresh before use. This solution is also used as an instrument blank in analysis. The calibration standard solutions are prepared as detailed in Table 2011.06A . Set up microcentrifuge tubes labeled calibration standardA through G separately. Standards A and B are prepared using folate intermediate standard solution, whereas calibrants C through G are prepared by dilutions of higher concentration calibrants. Add intermediate standard solution as specified in the Table 2011.06A to the corresponding calibration tubes A and B. Add 20 μL of the 2 ppm internal standard solution to tubes A and B. Add neutralized elution solvent to tubes A and B as specified in Table 2011.06A . Close the tubes and mix thoroughly by vortex mixer for about 20 s. Calibration standards C through G are prepared by dilutions of the specified calibration standards in Table 2011.06A . Pipette the specified calibration standard in the specified volume. Make up the volume to 1 mL with the 40 ppb internal standard solution as specified in Table 2011.06A . Close the tubes and mix thoroughly by vortex mixer for about 20 s. Calculate the exact concentration