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oai:researchcommons.waikato.ac.nz:10289/161272026-09-16T21:20:43Zcom_10289_2222col_10289_2223

Development of a novel cyanide-free method for analysis of vitamin B₁₂ in milk-based infant formula Li, Yanan Grainger, Megan N. C. Manley-Harris, Merilyn Gill, Brendon D. 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. The University of Waikato 2023 Thesis en https://hdl.handle.net/10289/16127 https://researchcommons.waikato.ac.nz/bitstreams/5c34f387-2b2d-4233-be80-1937c934e5f8/download 466d2bb133db3c61a046820f50c42ea6 https://researchcommons.waikato.ac.nz/bitstreams/9864a267-98b3-47a0-b82e-641302234abe/download e14202ab27e47ddb00d33097327ba050 https://researchcommons.waikato.ac.nz/bitstreams/611cc2d0-3536-4f30-aa84-66c2484ad26a/download dd03a99ae305680516a5c256117256d1 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. Vitamin B12 deficiency -- Diagnosis Diagnosis, Laboratory -- Evaluation Blood -- Analysis Cyanides Infant formulas -- Analysis Sample preparation (Chemistry)