Development and Validation of a Method for Vitamin B₁₂ Measurement in Nutritional Products by HPLC-ICP-MS

A method has been developed and validated for vitamin B₁₂ (cobalamin) measurement in nutritional products (infant formula, toddler and adult nutritive drinks, milk, protein drinks/powders, and breakfast cereals). The optimum procedure was based on collective extraction of the various forms of vitamin B₁₂ as cyanocobalamin (CNCbl) with a sodium acetate buffer (0.25 mol L^-1, pH 4.5) at 95 °C for 30 min in the presence of sodium cyanide, followed by analyte concentration and cleanup on a solid phase extraction (SPE) sorbent. Starch-containing samples were treated with α-amylase (300 µL) and incubated (40 °C, 30 min) prior to heating at 95 °C. The SPE eluate was reconstituted to a smaller volume and subsequently analyzed using high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The analyte was separated isocratically on a C-8 column in under 6 minutes using a mobile phase of 0.1% (v/v) formic acid in methanol and water (30 : 70). The method was single laboratory validated according to U.S. Food and Drug Administration's (FDA) guidelines and compared favorably to AOAC Official Methods 952.20 and 2014.02. The repeatability of the method (< 14% RSD) was demonstrated by interday analysis of samples and reference materials. Recovery values mostly ranged between 80% and 120% for fortified samples and reference materials. Vitamin B₁₂ can be detected and quantified using the current method at levels as low as 0.004 and 0.03 µg CNCbl/100 g (liquid and ready-to-feed/drink samples) and 0.01 and 0.06 µg CNCbl/100 g (solid samples), respectively. The method is simpler and less time-consuming than most official and other published methods with broader application and better analytical performance.