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

The analysis of vitamin B 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 B. To eliminate the risk to laboratory analysts in handling cyanide, alternative strategies are preferred for the analysis of vitamin B. This research demonstrates the use of cobalamin-derived α-ribazole (a nucleoside moiety of vitamin B) to determine total vitamin B 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 with repeatability of 3.78-5.47% relative standard deviation (RSD, n = 10) and an intermediate precision of 3.59-10.0% RSD (n = 10). The limits of detection and quantitation (LOD and LOQ) were 0.4 and 1.2 μg 100 g of dry weight, respectively. Accuracy was 68.9-76.4% and 68.7-80.0% at 50 and 150% of typical B 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 B determination in infant formula while maintaining high accuracy and precision.