The analysis of 5′-mononucleotides in infant formulae by HPLC

A method is described for the determination of four 5′-mononucleotides (cytidine 5′-monophosphate, uridine 5′-monophosphate, adenosine 5′-monophosphate and guanosine 5′-monophosphate) in infant formulae. Nucleosides which may be formed during processing can also be analysed simultaneously. This method is based on deproteinisation of samples and direct analysis by ion-pair HPLC using two Nucleosil 120-C18 columns in series, followed by diode-array detection. This method gives good recoveries of 5′-mononucleotides from spiked infant formula products. However, some chromatographic interferences were observed when analysing hypoallergenic infant formulae containing hydrolysed proteins which made peak quantification difficult. To overcome this problem a strong anion-exchange solid-phase extraction (SPE) column was used. Four SPE columns from different suppliers were evaluated, but the best recoveries of all four 5′-mononucleotides and highest reproducibility of results were obtained with Bakerbond® quaternary amine columns. Nucleosides, which may occur in very low concentrations in hypoallergenic products, are not retained on the SPE columns and so cannot be analysed by this technique.