6. AOACSPIFANMethods-2018Awards

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Butler-Thompson et al.: J ournal of AOAC I nternational V ol. 98, N o. 6, 2015  1655

INFANT FORMULA AND ADULT NUTRITIONALS

Determination of Vitamin B 12 in Infant, Adult, and Pediatric Formulas by HPLC-UV and Column Switching: Collaborative Study, Final Action 2011.10 Linda D. Butler-Thompson, Wesley A. Jacobs, and Karen J. Schimpf 1 Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH, 43219

Collaborators: J. Austad, B. Chen, S. Christiansen, C. Domer, G. Hostetler, D. Labitan, B. Liu, I. Malaviole, B. Marosanović, F. Martin, A. Mittal, J.M. Oberson, A. Ruosch, Y. Shen, G. Wang

Received on April 30, 2015. Accepted by SG July 23, 2015. The method was approved by the AOAC Official Methods Board as Final Action. See “Standards News,” (2014) Inside Laboratory Management , November/December issue. The AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) invites method users to provide feedback on the Final Action methods. 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 author. 1 Corresponding author’s e-mail: Karen.schimpf@abbott.com DOI: 10.5740/jaoacint.15-108 for repeatability were ≤15% RSD at vitamin B 12 concentrations of 0.01 µg/100 g RTF liquid and ≤7% RSD at vitamin B 12 concentrations of 0.2–5.0 µg/100 g RTF liquid. Vitamin B 12 SPIFAN SMPRs for reproducibility were ≤11% RSD in products with vitamin B 12 concentrations ranging from 0.3 to 5.0 µg/100 g RTF liquid. During this collaborative study, the RSD r ranged from AOAC First Action Method 2011.10, Vitamin B 12 in Infant and Pediatric Formulas and Adult Nutritionals, was collaboratively studied. This method uses a pH 4.5 sodium acetate buffer and potassium cyanide at 105°C to extract and convert all biologically active forms of vitamin B 12 present to cyanocobalamin; octylsilyl (C 8 ) or C 18 SPE cartridges to purify and concentrate cyanocobalamin; a combination of size-exclusion and RPLC to isolate cyanocobalamin; and visible absorbance at 550 nm to detect and quantitate cyanocobalamin in infant, pediatric, and adult nutritionals with vitamin B 12 concentrations greater than 0.025 µg/100 g ready-to-feed (RTF) liquid. During this collaborative study, nine to 11 laboratories from eight different countries analyzed blind duplicates of 12 infant, pediatric, and adult nutritional formulas. Per the AOAC Expert Review Panel (ERP) on Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Nutrient Methods the method demonstrated acceptable repeatability and reproducibility and met SPIFAN Standard Method Performance Requirements (SMPRs ® ) for the majority of product matrixes analyzed. Vitamin B 12 SPIFAN SMPRs

2.98 to 9.77%, and the RSD R ranged from 3.54 to 19.5%. During previous single-laboratory validation studies, the method LOQ was estimated to be 0.025 µg/100 g RTF liquid. V itamin B 12 is a generic term used to describe all cobalamins that demonstrate antipernicious anemia activity. These compounds are corrinoids, tetrapyrrole structures where the cobalt ion is chelated by four pyrrole nitrogens. The fifth coordinate covalent bond to cobalt is with a nitrogen of the dimethylbenzimidazole moiety, while the sixth position may be occupied by a cyanide, 5'-deoxyadenosyl, methyl, water, hydroxyl, nitrite, ammonia, or sulfite ligand (1). The AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) has defined vitamin B 12  as any cobalt-containing corrinoid with the biological activity of cyanocobalamin, including cyanocobalamin, aquocobalamin, hydroxycobalamin, methylcobalamin, and adenosylcobalamin. Most nutritional products are fortified with cyanocobalamin, a synthetic form of vitamin B 12 , because of its stability and commercial availability. Methylcobalamin and 5'-deoxyadenosylcobalamin occur naturally and are known to exhibit metabolic activity in humans. Naturally occurring vitamin B 12 originates solely from synthesis by bacteria and other microorganisms growing in soil, water, or sewage and in the rumen and intestinal tract of animals (2). A new chromatographic method capable of determining vitamin B 12 in infant, adult, and pediatric formula powders, ready-to-feed (RTF) liquids, and liquid concentrates was needed after SPIFAN determined that traditional microbiological vitamin B 12 methods were not precise and accurate enough to meet the nutrient specification requirements of infant, pediatric, and adult nutritionals. The SPIFAN vitamin B 12 working group developed Standard Method Performance Requirements (SMPRs ® ) that were then approved by SPIFAN (3). In June 2011, the AOAC Expert Review Panel (ERP) on SPIFAN nutrient methods granted First Action status to multiple vitamin B 12 methods, AOAC 2011.08 (4), 2011.09  (5), AOAC 2011.10 (6), and 2011.16 (7). Single-laboratory validations (SLVs) were completed for AOAC 2011.08 and AOAC 2011.10 (8), and in March 2013, AOAC  2011.10 was selected by the ERP for further evaluation in a multilaboratory collaborative study to determine method reproducibility.