Thumbnail Image

Development of a novel cyanide-free method for analysis of vitamin B₁₂ in milk-based infant formula

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.
Type of thesis
The University of Waikato
All items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.