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

AOAC Official Method 2011.09 Vitamin B 12 in Infant Formula and Adult Nutritionals HPLC After Purification on an Immunoaffinity Column First Action 2011 (Applicable for the determination of vitamin B 12 in infant formula and adult nutritionals.) Caution : Solvents employed are common use solvents and reagents. Refer to adequate manuals or material safety data sheets to ensure that the safety guidelines are applied before using chemicals. Potassium cyanide .— Highly toxic when inhaled, swallowed, or when coming into contact with the skin. Develops highly toxic gases when coming into contact with acids. Highly toxic to aquatic organisms and can have longer-term damaging effects in bodies of water. Destroy resides with alkaline NaOCl solution. Wear respiratory protection. Avoid the creation of dust. Avoid inhaling dust. Ensure ventilation is adequate. Trifluoroacetic acid (TFA) .—Causes severe burns and eye damage. Wear protective gloves, clothing, eyewear, and face protection. Use only in effective fume removal device to remove vapors generated. A. Principle Individual vitamers are extracted from the material under investigation using sodium acetate buffer in the presence of α -amylase, pepsin, and potassium cyanide and are transformed into the vitamer cyanocobalamin (R = CN). Extracts are passed through an immunoaffinity column (IAC) that contains specific antibodies that bind selectively to cyanocobalamin (1). The cyanocobalamin is then eluted from the column using methanol. Determination is carried out using HPLC with UV detection. B. Apparatus and Materials ( a ) Analytical balances.— Precision ±0.1 mg. ( b ) Volumetric flask with screw cap. —Brown glass, 100 mL. ( c ) Magnetic stir bar. ( d ) Magnetic stirrer. ( e ) Dispenser.— 50 mL. ( f ) Water baths. ( g ) Funnel. ( h ) Folded filters. ( i ) Pipets. ( j ) Vials. —( 1 ) Brown glass , 6 mL. ( 2 ) 0.1 mL with μ L insert; brown glass. ( k ) Nitrogen evaporation apparatus.— With temperature regulation (evaporation dry block). ( l ) Vial crimper. ( m ) IAC stand or suction chamber.

Table 2011.09B. Conditions for gradient elution Time, min

Flow rate, mL Solution A, % Solution C, %

0.0 3.5

0.25 0.25 0.25 0.25 0.25 0.25 0.25

100 100

0 0

11.0 19.0 20.0 26.0

85 75 90

15 25 10

100 100

0 0

30

( n ) Orbital shaker. —IKA MTS4 (Staufen, Germany) or equivalent. ( o ) pH meter. ( p ) HPLC system. —Composed of pump, injector with sample loop (20–100 μ L), UV-Vis detector, column oven, and analytical software. ( q ) HPLC column. —e.g., ACE 3 AQ (150  3.0 mm; MZ Analytik, Mainz, Germany) or equivalent. ( r ) IACs .—Easi-Extrakt Vitamin B 12 (LGE) from r-Biopharm or equivalent. C. Chemicals and Solvents

( a ) Methanol .—HPLC grade. ( b ) Acetonitrile .—HPLC grade. ( c ) Acetic acid .—100%. ( d ) Ultra-pure water .—Resistance: 0.055 μ s/cm. ( e ) Potassium cyanide, p.a.

( f ) TFA.— ≥ 99%. ( g ) Vitamin B 12

(cyanocobalamin) .—Purity of 99% in accordance with the certificate (Sigma-Aldrich, St. Louis, MO). ( h ) Anhydrous sodium acetate . ( i ) Taka-diastase from Aspergillus oryzae .—Approximately 1.5 units/mg ( α -amylase; Sigma-Aldrich) . ( j ) Pepsin .— ≥ 400 units/mg protein (Sigma-Aldrich). D. Preparation of Reagents and Standard Solutions Note : Make all solutions and eluents for HPLC using ultra-pure water ( ≤ 18 M Ω DI). ( a ) Solution A (0.025% TFA) .—Add 500 μ L TFA to 2000 mL water. ( b ) Solution B (sodium acetate buffer, 50 mM) .—Dissolve 4.1 g sodium acetate in 950 mL water. Adjust solution to pH 4.0 using acetic acid and then make up to 1000 mL. ( c ) Potassium cyanide solution (1%) .—Dissolve 1 g KCN in 100 mL water. ( d ) Cyanocobalamin stock solution I (ST I ).—Weigh out 50 mg cyanocobalamin at a precision of ±0.1 mg into a 50 mL volumetric flask, dissolve in water, and make up to the mark. Decant ST I into brown glass vials. ST I has a shelf life of approximately 18 months at –20°C. ( e ) Cyanocobalamin stock solution II (ST II) .—Add 1.00 mL ST I to 99.0 mL water. ST II is stable for at least 3 months at 2–8°C. ( f ) Working standards .—Prepare working standards in ultra- pure water before each analysis. See Table 2011.09A . E. Recovery Check of IACs Every new batch of IACs has to be checked for its specific

Table 2011.09A. Preparation of working standards

Dilution (in brown glass volumetric flask) 3.0 mL ST II/10 mL 1.0 mL ST II/200 mL 1.0 mL ST II/50 mL 1.0 mL ST II/25 mL

Cyanocobalamin, μ g/100 mL

Working standard

Std 1 Std 2 Std 3 Std 4

1.5

5

20 40

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