Grainger, Megan N.C.Manley-Harris, MerilynGill, Brendon D.Li, Yanan2023-11-092023-11-092023https://hdl.handle.net/10289/16127The 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.application/pdfenAll 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 -- DiagnosisDiagnosis, Laboratory -- EvaluationBlood -- AnalysisCyanidesInfant formulas -- AnalysisSample preparation (Chemistry)Thesis2023-11-08