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.
Nucleotides have been routinely supplemented to infant formulas due to the important roles they play in metabolism and to replicate the higher concentrations typically found in human milk. A method utilising anion exchange solid-phase extraction clean-up and liquid chromatography was developed for the rapid, routine determination of supplemented cytidine 5′‑monophosphate, uridine 5′‑monophosphate, inosine 5′‑monophosphate, guanosine 5′‑monophosphate, and adenosine 5′‑monophosphate in bovine milk-based infant formula. Chromatographic analyses were performed using a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique. A single-laboratory validation was performed, with recoveries of 92–101% and repeatability of 1.0–2.3%. An extension study demonstrated the expansion in scope to a wider range of different infant formula products including milk protein and hydrolysate-based products, low and high fat products, soy protein-based and elemental products, adult nutritional and infant formulations, in both ready-to-feed and powder forms.
The development of a method to measure the total potentially available nucleosides (TPAN) in human milk has made an important contribution to further understanding the distribution of nucleosides and nucleotides. This method was applied in a lactation study of bovine milk with colostrum and milk samples collected from two herds over the course of the first month post-partum, pooled within each herd by stage of lactation and the TPAN concentrations were determined. Sample analysis consisted of parallel enzymatic treatments, phenylboronate affinity gel extraction, and liquid chromatography to quantify contributions of nucleosides, monomeric nucleotides, nucleotide adducts, and polymeric nucleotides to the nutritionally available nucleoside pool. Bovine colostrum contained high levels of nucleosides and monomeric nucleotides, which rapidly decreased as lactation progressed into transitional milk. Mature milk was relatively consistent in nucleoside and monomeric nucleotide concentrations from approximately the tenth day post-partum. Differences in concentrations between summer-milk and winter-milk herds were largely attributable to variability in uridine and monomeric nucleotide concentrations.
The TPAN method was subsequently applied to the analysis of mature bovine, caprine, and ovine milk. The contributions to TPAN from polymeric nucleotides, monomeric nucleotides, and nucleotide adducts were then calculated. Ovine milk contained the highest concentration of TPAN (374.1 μmol/dL), with lower concentrations in caprine milk (97.4 μmol/dL) and bovine milk (7.9 μmol/dL). Ovine milk contained the highest concentrations of each of the different nucleoside and nucleotide forms, and bovine milk contained the lowest.
A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography-tandem mass spectrometry was developed. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C18 stationary phase and gradient elution, with mass spectrometric detection, and quantitation by stable isotope labelled internal standard technique. A single laboratory validation study was performed with recoveries of 80.1–112.9% and repeatability relative standard deviations of 1.9–7.2%. The method was validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysate-based infant formula.
Soft-bound thesis submitted: 28 February 2013; Oral examination: 05 September 2013; Hard-bound thesis submitted: 12 September 2013
Natural abundance 15N NMR spectra were obtained during curing of urea-formaldehyde resins. A distinct advantage using 15N NMR spectroscopy is the avoidance of interfering signals from fibre material added ot the resin. The technique using Distortionless Enhancement by Polarisation Transfer (DEPT) confers rapid acquisition times inherent to highly sensitive 1H nuclei and avoids the disadvantages from long nitrogen relaxation times and low natural abundance. The cure of the resin is observed by the loss of primary amine peaks from urea and monomethyl urea.
It is possible to follow the cure of the resin throughout s range of gel times and at a desired pH and temperature. The resin cure is seen in both the resin alone and in the presence of wood fibre. The change in cure rates is observable for water and dichloromethane fibre extracts and the extracted fibre. With the exception of dichlormethane extracted fibre added to the resin, the mechanism appears to be specific acid catalysed.
By itself, or coupled with other methods of analysis, this technique is a powerful method for analysing the cure of urea-formaldehyde and other amino resins.
Supervisors: Grainger, Megan N.C.; Manley-Harris, Merilyn; Gill, Brendon D.
show 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.
Hard-bound thesis submitted: 08 November 2023
Citationshow abstract
Nucleotides have been routinely supplemented to infant formulas due to the important roles they play in metabolism and to replicate the higher concentrations typically found in human milk. A method utilising anion exchange solid-phase extraction clean-up and liquid chromatography was developed for the rapid, routine determination of supplemented cytidine 5′‑monophosphate, uridine 5′‑monophosphate, inosine 5′‑monophosphate, guanosine 5′‑monophosphate, and adenosine 5′‑monophosphate in bovine milk-based infant formula. Chromatographic analyses were performed using a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique. A single-laboratory validation was performed, with recoveries of 92–101% and repeatability of 1.0–2.3%. An extension study demonstrated the expansion in scope to a wider range of different infant formula products including milk protein and hydrolysate-based products, low and high fat products, soy protein-based and elemental products, adult nutritional and infant formulations, in both ready-to-feed and powder forms.
The development of a method to measure the total potentially available nucleosides (TPAN) in human milk has made an important contribution to further understanding the distribution of nucleosides and nucleotides. This method was applied in a lactation study of bovine milk with colostrum and milk samples collected from two herds over the course of the first month post-partum, pooled within each herd by stage of lactation and the TPAN concentrations were determined. Sample analysis consisted of parallel enzymatic treatments, phenylboronate affinity gel extraction, and liquid chromatography to quantify contributions of nucleosides, monomeric nucleotides, nucleotide adducts, and polymeric nucleotides to the nutritionally available nucleoside pool. Bovine colostrum contained high levels of nucleosides and monomeric nucleotides, which rapidly decreased as lactation progressed into transitional milk. Mature milk was relatively consistent in nucleoside and monomeric nucleotide concentrations from approximately the tenth day post-partum. Differences in concentrations between summer-milk and winter-milk herds were largely attributable to variability in uridine and monomeric nucleotide concentrations.
The TPAN method was subsequently applied to the analysis of mature bovine, caprine, and ovine milk. The contributions to TPAN from polymeric nucleotides, monomeric nucleotides, and nucleotide adducts were then calculated. Ovine milk contained the highest concentration of TPAN (374.1 μmol/dL), with lower concentrations in caprine milk (97.4 μmol/dL) and bovine milk (7.9 μmol/dL). Ovine milk contained the highest concentrations of each of the different nucleoside and nucleotide forms, and bovine milk contained the lowest.
A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography-tandem mass spectrometry was developed. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C18 stationary phase and gradient elution, with mass spectrometric detection, and quantitation by stable isotope labelled internal standard technique. A single laboratory validation study was performed with recoveries of 80.1–112.9% and repeatability relative standard deviations of 1.9–7.2%. The method was validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysate-based infant formula.
Soft-bound thesis submitted: 28 February 2013; Oral examination: 05 September 2013; Hard-bound thesis submitted: 12 September 2013
Citationshow abstract
Natural abundance 15N NMR spectra were obtained during curing of urea-formaldehyde resins. A distinct advantage using 15N NMR spectroscopy is the avoidance of interfering signals from fibre material added ot the resin. The technique using Distortionless Enhancement by Polarisation Transfer (DEPT) confers rapid acquisition times inherent to highly sensitive 1H nuclei and avoids the disadvantages from long nitrogen relaxation times and low natural abundance. The cure of the resin is observed by the loss of primary amine peaks from urea and monomethyl urea.
It is possible to follow the cure of the resin throughout s range of gel times and at a desired pH and temperature. The resin cure is seen in both the resin alone and in the presence of wood fibre. The change in cure rates is observable for water and dichloromethane fibre extracts and the extracted fibre. With the exception of dichlormethane extracted fibre added to the resin, the mechanism appears to be specific acid catalysed.
By itself, or coupled with other methods of analysis, this technique is a powerful method for analysing the cure of urea-formaldehyde and other amino resins.
Hard-bound thesis submitted: 26 February 2000
Citation