Determination of Vitamin B12 in Infant, Adult, and Pediatric Formulas by HPLC-UV and Column Switching: Collaborative Study, Final Action 2011.10

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 degrees 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 mu g/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 (R)) for the majority of product matrixes analyzed. Vitamin B-12 SPIFAN SMPRs for repeatability were <= 15% RSD at vitamin B-12 concentrations of 0.01 mu g/100 g RTF liquid and <= 7% RSD at vitamin B-12 concentrations of 0.2-5.0 mu g/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 mu g/100 g RTF liquid. During this collaborative study, the RSDr ranged from 2.98 to 9.77%, and the RSDR ranged from 3.54 to 19.5%. During previous single-laboratory validation studies, the method LOQ was estimated to be 0.025 mu g/100 g RTF liquid.