A cyanide-free sample preparation methodology prior to determination of vitamin B12 in infant milk formula using hydrophilic interaction liquid chromatography with fluorescence detection

The analysis of vitamin B₁₂ in infant formulas typically requires the use of cyanide during sample preparation to convert the unstable vitamers (hydroxocobalamin, methylcobalamin and adenosylcobalamin) to cyanocobalamin, the most stable form of vitamin B₁₂. To eliminate the risk to laboratory analysts in handling cyanide, alternative strategies are preferred for the analysis of vitamin B₁₂. This research demonstrates the use of cobalamin-derived α-ribazole (a nucleoside moiety of vitamin B₁₂) to determine total vitamin B₁₂ content. Infant formula samples underwent protein denaturation and sugar removal with subsequent acidic hydrolysis and dephosphorylation employed to release α-ribazole, which was isolated by boronate affinity chromatography then analysed by hydrophilic interaction liquid chromatography with fluorescence detection. The method was validated using bovine- and ovine milk-based infant formula samples. The newly developed method was linear over the range of 0.65-6.48 ng mL^-1 with repeatability of 3.78-5.47% relative standard deviation (RSD_r, n = 10) and an intermediate precision of 3.59-10.0% RSD_iR (n = 10). The limits of detection and quantitation (LOD and LOQ) were 0.4 and 1.2 μg 100 g^-1 of dry weight, respectively. Accuracy was 68.9-76.4% and 68.7-80.0% at 50 and 150% of typical B₁₂ concentrations in infant formula, respectively. The validated method was applied to eleven infant formulas and no statistical difference (p = 0.45, α = 0.05) was found when comparing with the results obtained using the AOAC Official Method 2014.02 high performance liquid chromatography with ultraviolet detection that requires the use of cyanide. These results indicate that the newly validated method is not only reliable but also offers a safer alternative for routine vitamin B₁₂ determination in infant formula while maintaining high accuracy and precision.