{"id":{"repo_id":"waikato-masters","oai_identifier":"oai:researchcommons.waikato.ac.nz:10289/16127"},"canonical_url":"https://search.dev.ndltd.org/etd/waikato-masters/oai:researchcommons.waikato.ac.nz:10289/16127","repository":{"repo_id":"waikato-masters","name":"University Waikato","base_url":"https://researchcommons.waikato.ac.nz/server/oai/request"},"display":{"title":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Li, Yanan"],"institution":"The University of Waikato","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Grainger, Megan N.C.","Manley-Harris, Merilyn","Gill, Brendon D."],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023","date_published":"2023","updated_at":"2026-07-24T05:57:36Z","subjects":[],"languages":[],"rights":["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."],"rights_urls":["https://researchcommons.waikato.ac.nz/bitstreams/9864a267-98b3-47a0-b82e-641302234abe/download"],"identifier_entries":[]},"links":{"outbound_url":null,"outbound_label":null,"outbound_source":null},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Grainger, Megan N.C.","Manley-Harris, Merilyn","Gill, Brendon D."]},{"key":"dc:creator","label":"Author","values":["Li, Yanan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["The University of Waikato"]},{"key":"dc:relation.isreferencedby","label":"Dc Relation Isreferencedby","values":["https://hdl.handle.net/10289/16127"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["https://researchcommons.waikato.ac.nz/bitstreams/9864a267-98b3-47a0-b82e-641302234abe/download","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."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://researchcommons.waikato.ac.nz/bitstreams/5c34f387-2b2d-4233-be80-1937c934e5f8/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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. 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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. 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