The University of Waikato
Development of a novel cyanide-free method for analysis of vitamin B₁₂ in milk-based infant formula
Abstract
dc:description.abstractThe 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.
Degree
thesis:*- Grantor dc:publisher.institution
- The University of Waikato
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
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- Li, Yanan
- Advisors dc:contributor.advisor
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- Grainger, Megan N.C.
- Manley-Harris, Merilyn
- Gill, Brendon D.
Rights
dc:rights- Statement dc:rights
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- 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.