AOAC Final Action Methods in 2018

Folate standard intermediate solution can be stored at –20°C for 30 days, and can be stable for longer at –70°C. ( j )  2 ppm Internal standard solution for calibration standards (2 μg/mL of each internal standard folate) .—Prepare solution fresh before use. Store 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 ammonium hydroxide (28–30%) and mix (neutralized solvent for SPE elution). Prepare fresh before use. Pipet exactly 100 μL of 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. Concn (μg/mL) of each folate vitamer in 2 ppm internal standard solution = internal standard intermediate solution concn in μg/mL × 0.1 mL ( k )  40 ppb Internal standard solution for calibration standards (40 ng/mL each folate) .—Prepare solution fresh before use. Store 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 ammonium hydroxide (28–30%; neutralized solvent for SPE elution) and mix. Prepare fresh before use. Pipet about 5 mL of neutralized solvent for SPE elution into a 10 mL low-actinic volumetric flask. Pipet 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. Concn (μg/mL) of each folate vitamer in 40 ppb internal standard solution = (concn of folate in internal standard intermediate solution in μg/mL × 0.02 mL)/10 mL ( l )  Preparation of calibration standard solutions .—Prepare solutions fresh before analysis. Store 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 ammonium hydroxide (neutralized solvent for SPE elution)

and mix. Prepare fresh before use. Solution is also used as an instrument blank in analysis. Calibration standard solutions are prepared as shown in Table  2011.06A . Set up microcentrifuge tubes labeled calibration standard A–G separately. Standards A and B are prepared using folate intermediate standard solution, whereas calibrants C–G are prepared by dilutions of higher concentration calibrants. Add intermediate standard solution as specified in Table 2011.06A to the corresponding calibration tubesAand B.Add 20 μLof 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–G are prepared by dilutions of the specified calibration standards in Table 2011.06A . Pipet 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 of each folate compound, including the internal standard in each calibration standard, by following the dilution of the intermediate standard solution or the respective calibrant in the solution and the used internal standard solution. ( m )  Preparation of substrate solution (Pte-Glu3) to test plasma conjugase activity (20 μg/mL) .—Add about 5mLof the intermediate solvent to a 10 mL low-actinic volumetric flask. Pipet 0.4 mL Pte- Glu3 stock solution (500 mcg/mL). Make up the volume to 10 mL with the intermediate solvent and mix thoroughly. Store at –20°C for 1 month; can be stable at –70°C for longer time periods. Other reagents for folate analysis: ( a )  Protease solution (2 mg/mL) .—Prepare solution fresh before use. Store at 4–8°C if necessary but not for more than 4 h. Dissolve 0.05 g protease in 25 mL water in a 100 mL beaker or Erlenmeyer flask by proper mixing. Each sample requires 1 mL. ( b )  α-Amylase solution (20 mg/mL; 40 units/mL) .—Prepare solution fresh before use. Store at 4–8°C if necessary but not for more than 4 h. Dissolve 0.5 g in about 20 mL water in a 25 mL volumetric flask or a centrifuge tube. Make up the volume to 25 mL. Mix gently for complete mixing. Make sure no foams develop. The amylase solution is transferred to a 50 mL centrifuge tube if not already in

Table 2011.06A. Preparation of folate calibration standard solutions a

Volume of 40 ppb internal standard solution (40 ng/mL), μL

Concn of each folate compound in standard solution, ng/mL

Calibration standard

Volume of intermediate/ calibration standard solution, μL 25 μL intermediate standard solution 10 μL intermediate standard solution 100 μL calibration standard solution A 100 μL calibration standard solution B 100 μL calibration standard solution C 100 μL calibration standard solution D

2 ppm Internal standard solution (2 μg/mL), μL

Volume of neutral elution solvent, μL

Final volume, mL

A

20

0

955

1.0

500

B

20

0

970

1.0

200

C

0

900

0

1.0

50

D

0

900

0

1.0

20

E

0

900

0

1.0

5

F

0

900

0

1.0

2

G

50 μL calibration standard solution D

0

950

0

1.0

1

a  Internal standard concentration in every calibration standard solution (A–G) = 40 ng/mL of each folate.

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