AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

Figure 2011.09A. Example chromatogram for a standard.

binding capacity of cyanocobalamin. Before use, the IAC has to be equilibrated to room temperature. Add 150 μ L cyanocobalamin ST II to 48.85 mL solution B. Inject 5.0 mL of this solution on the IAC. Seal the top column with the related cap. Shake the column for 20 min at 800 rpm on an IKA shaker. Let stand for approximately 15 min. Reattach the column to the suction unit. After the sample solution has passed through the column, wash the column with approximately 10 mL water. Remaining residues of waters are removed by a weak vacuum (approximately 10 min). Elute the vitamin B 12 by injecting 3 mL methanol on to the column. Use an 8 mL brown glass vial as a collection vessel. Wash the column two times with 1 mL methanol. Remaining residues of methanol are removed by a vacuum. Place the vial in the evaporation dry block. Evaporate the methanolic vitamin B 12 solution under nitrogen at approximately 60°C. Reconstitute the sample in 1.0 mL solution A. Decant the solution into a brown glass crimp-top vial with an integrated glass μ L insert and inject 100 μ L to the HPLC. The IAC check is done with three columns per batch. The binding capacity of the IACs is the ratio of analyzed vitamin B 12 amount/known standard amount:

from direct light.) ( a ) Sample preparation .—Mill or homogenize the sample material prior to investigation. The initial weight of the sample depends on the vitamin B 12 content of the sample material. ( b ) Extraction .—Weigh the homogeneous sample into a 100 mL brown glass volumetric flask with a screw cap. Add approximately 50 mL solution B, 50 mg  -amylase, 0.5 g pepsin, and 2 mL KCN solution. Shake the mixture well. Transfer to a water bath for 30 min at 40°C and shake the flask every 10 min. Subsequently transfer the flask to a water bath at 100°C for an additional 30 min and shake every 10 min. After the mixture is cooled down to room temperature in an ice bath, fill up to the mark with solution B. Centrifuge and filter this solution. ( c ) Immunoaffinity cleanup .—Test each new batch of IAC for its specific cyanocobalamin losses prior to use. Follow the procedure outlined in D . Correct the contents of the samples by the factor f that has been determined. Bring the IAC to room temperature prior to use. Twist off the end cap at the bottom of the column and attach the column to the suction unit for the solid-phase extraction. Remove the cap at the top of the column. Once the column liquid has drained, seal the column at the lower end using a stopper supplied for this purpose. Inject 9.0 mL of the sample solution on to the column. Seal the top column with the related cap. Shake the column for 20 min at 800 rpm on an IKA shaker. Let stand for approximately 15 min. Reattach the column to the suction unit. After the sample solution has passed through the column, wash the column with approximately 10 mL water. Remaining residues of waters are removed by a weak vacuum (approximately 10 min). Elute the vitamin B 12 by injecting 3 mL methanol on to the column. Use an 8 mL brown glass vial as a collection vessel. Wash the column two times with 1 mL methanol. Remaining

analyzed vitamin B amount, g k 12 nown standard amount, g

Column recovery, %

100%

column recovery, % 1

Factor f

100

The analytical result of vitamin B 12 (recovery should be between 1.0 and 1.2). F. Sample Preparation and Extraction ( Note : Vitamin B 12 is sensitive to light. Conduct operations under subdued light, or use brown glassware. Keep all solutions away is corrected with the factor f

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