Development and application of a liquid chromatographic method for analysis of nucleotides and nucleosides in milk and infant formulas

A method for the simultaneous determination in milk of the 5′-mononucleotides adenosine 5′-monophosphate, cytidine 5′-monophosphate, guanosine 5′-monophosphate, inosine 5′-monophosphate and uridine 5′-monophosphate and their corresponding nucleosides is described. Following deproteinisation, the sample extract was analysed by reversed-phase liquid chromatography, whereby chromatographic separation was achieved using a polymer grafted silica C18 column, gradient elution with a simple binary mobile phase and UV detection by photodiode array. Performance parameters included recoveries of 95.5–105.2% and precision evaluated as 3.42–6.38% relative standard deviation. The described technique has been applied to the analysis of bovine and human milk, a range of commercial bovine milk-based infant and follow-on formulas, a seasonal study of skim milk powders and an assessment of alkaline phosphatase influence on nucleotide retention.