Development of a Novel Cyanide-free Method for Analysis of Vitamin B12 in Milk-based Infant Formula

Abstract

The analysis of vitamin B12 in infant formulas requires the use of cyanide during the sample preparation process to convert the three unstable vitamers (hydroxocobalamin, methylcobalamin and adenosylcobalamin) to cyanocobalamin, the most stable form of vitamin B12. The undesirable handling of cyanide for the analyst in the laboratory and the associated safety risk indicates a clear necessity for development of a cyanide-free method for vitamin B12 analysis without compromising the analytical quality. This doctoral research demonstrates the possibility of using cobalamin-derived α-ribazole to represent total vitamin B12, since the α-ribazole exists in all vitamin B12 forms removing the necessity for conversion. The absence of a commercial standard of α-ribazole required its in-house isolation from a cyanocobalamin standard. The α-ribazole was released through consecutive acidic hydrolysis and dephosphorylation by alkaline phosphatase. The freed α-ribazole was collected by boronate affinity chromatography and concentrated by lyophilisation. α-Ribazole was identified and characterised by nuclear magnetic resonance spectroscopy and liquid chromatography mass spectrometry to demonstrate its suitability as a standard. This protocol was optimised and adopted in the sample preparation for analysing vitamin B12 in infant formulas. Several extra steps were added to eliminate or limit interferences, including protein denaturation and sugar removal using C18 solid phase extraction. The final analyte was quantified using hydrophilic interaction liquid chromatography with fluorescence detection. The single laboratory validation experiment showed that this cyanide-free method is fit for purpose. Analysis of various milk-based infant formulas and comparison with current procedure demonstrated no bias for vitamin B12 analysis.