Determination of Vitamin B in Infant Formula and …

1 Kirchner et al.: Journal of aoac international Vol. 95, no. 4, 2012 1 Determination of Vitamin B12 in Infant Formula and Adult Nutritionals Using HPLC After Purification on an Immunoaffinity Column: First Action Kirchner, Katharina DegenharDt, and gUenther raffler1 CLF Central Laboratories Friedrichsdorf GmbH, Bahnstrasse 14 30, D-61381 Friedrichsdorf, GermanyMaria nelsonAOAC INTERNATIONAL, 481 N. Frederick Ave, Suite 500, Gaithersburg, MD 20877 Submitted for publication February 8, method was approved by the Expert Review Panel on Infant Formula and Adult Nutritionals as First Action. See Standards News, (2011) Inside Laboratory Management, July/August AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) invites method users to provide feedback on the First Action methods.

2 Feedback from method users will help verify that the methods are fit for purpose and are critical to gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding Corresponding author s e-mail: Formula AND ADULT NUTRITIONALSD uring the Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting, on June 29, 2011, the method Determination of Vitamin B12 in Infant Formula and Adult Nutritionals Using HPLC After Purification on an Immunoaffinity Column was recommended by an Expert Review Panel and adopted as AOAC Official First Action status. The method is applicable for the Determination of Vitamin B12 in milk-based Infant Formula .

3 Vitamin B12 is extracted from the sample in sodium acetate buffer in the presence of potassium cyanide. After purification and concentration with an immunoaffinity column (IAC), Vitamin B12 is determined by LC with UV detection (361 nm). Data supplied by CLF demonstrated linear response over a wide range of concentrations ( 39 g/100 mL). The analytical range is 10 g/100 g, depending on the capacity of the IACs ( g), the input weight, and dilutions. Recovery rates were assessed using National Institute of Standards and Technology SRM 1849, and determined to be , with SD of and CV of Measurement uncertainty (UE) was g/100 g, which was calculated from the validation data. It is an expanded measurement uncertainty and was obtained through multiplication with a coverage factor k.

4 LOQ values were reported as g/100 g. The performance characteristics of the method met the standard method performance requirements set forth by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals; thus, the method was determined to be appropriate for First Action AOAC Board of Directors approved a new pathway to achieve AOAC Official Methods of Analysis First Action status on March 28, 2011. Under this path, methods selected and reviewed using the AOAC volunteer consensus standards development processes can be granted Official First Action status by vetted Expert Review Panels (ERPs). All methods are discussed thoroughly by the ERP, stakeholders, and observers present.

5 Methods are evaluated for completeness of validation and likelihood of meeting standard method performance requirements, including appropriateness for the intended use, clarity of the method description, ruggedness, recovery, analytical range, and LOQ. Methods approved under the new path remain First Action for a period of about 2 years. Information generated through use of the methods will be monitored during this time. The performance of the methods will be reviewed by the ERPs to determine whether Final Action should be recommended to the Official Methods Board (1). Based on the data supplied by CLF, the method Determination of Vitamin B12 in Infant Formula and Adult Nutritionals Using HPLC After Purification on an Immunoaffinity Column was granted First Action status and designated AOAC Official MethodSM The method can be used for the Determination of Vitamin B12 in milk-based Infant Formula .

6 To estimate the concentration of Vitamin B12 in Infant Formula , Vitamin B12 is freed from complexing proteins by heating the samples for a short time in the presence of cyanide. The use of an immunoaffinity extraction step increases selectivity and sensitivity by purifying and concentrating the food extracts. Vitamin B12 can then be determined by LC with UV detection at 361 move from First to Final Action status, it was recommended that the method be combined with AOAC Official First Action Method , Determination of Vitamin B12 in Infant Formula and Adult Nutritionals by Liquid Chromatography/UV Detection with Immunoaffinity Extraction, based on the similarities between the two methods (2).

7 It was also recommended that the use of a correction factor be examined for the immunoaffinity columns (IACs). AOAC Official Method Vitamin B12 in Infant Formula and Adult NutritionalsHPLC After Purification on an Immunoaffinity Column First Action 2011(Applicable for the Determination of Vitamin B12 in Infant Formula and adult nutritionals.)2 Kirchner et al.: Journal of aoac international Vol. 95, no. 4, 2012 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.

8 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 residues 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 PrincipleIndividual vitamers are extracted from the material under investigation using sodium acetate buffer in the presence of -amylase, pepsin, and potassium cyanide and are transformed into the vitamer cyanocobalamin (R = CN).

9 Extracts are passed through an immunoaffinity column (IAC) that contains specific antibodies that bind selectively to cyanocobalamin (3). The cyanocobalamin is then eluted from the column using methanol. Determination is carried out using HPLC with UV Apparatus and Materials(a) Analytical balances. Precision 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) mL with L insert; brown glass.(k) Nitrogen evaporation apparatus. With temperature regulation (evaporation dry block).(l) Vial crimper.(m) IAC stand or suction chamber.

10 (n) Orbital shaker. IKA MTS4 (Staufen, Germany) or equivalent.(o) pH meter.(p) HPLC system. Composed of pump, injector with sample loop (20 100 L), UV-Vis detector, column oven, and analytical software.(q) HPLC column. , ACE 3 AQ (150 mm; MZ Analytik, Mainz, Germany) or equivalent.(r) IACs. Easi-Extrakt Vitamin B12 (LGE) from r-Biopharm or Chemicals and Solvents(a) Methanol. HPLC grade.(b) Acetonitrile. HPLC grade.(c) Acetic acid. 100%.(d) Ultra-pure water. Resistance: s/cm.(e) Potassium cyanide, (f) TFA. 99%.(g) Vitamin B12 (cyanocobalamin). Purity of 99% in accordance with the certificate (Sigma-Aldrich, St. Louis, MO).(h) Anhydrous sodium acetate.(i) Taka-diastase from Aspergillus oryzae.