Analysis of Nucleosides and Nucleotides in Milk and Infant Formula

iii 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 ANALYSIS OF NUCLEOSIDES AND NUCLEOTIDES IN MILK AND INFANT FORMULA ABSTRACT iv 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.iv 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. This work is dedicated to my wife Michelle and to my children, Caitlin, Madelynn, Aidan, and Carys. ―The real voyage of discovery consists not in seeking new landscapes but in having new eyes. ‖ — Marcel Proust, 1923