Development and application of an HPLC/diode array methodology for determination of nucleotides in infant formulae and follow-up milks

This paper describes a procedure performed by high performance liquid chromatography/diode array detection for quantification of 4 nucleotides (cytidine 5-monophosphate, uridine 5-monophosphate, guanosine 5-monophosphate and adenosine 5-monophosphate) in infant formulae and follow-up milks. The sample preparation was simple and involved protein removal and filtration. The chromatographic separation was achieved using reverse-phase column C-18 (S10ODS2). Isocratic elution was used, with 90% buffer A and 10% buffer B. Buffer A consisted of 5 mM tetrabutylammonium hydrogensulphate (TBAHS) and 20 mM potassium dihydrogenphosphate (KH2PO4) and buffer B of 5 mM TBAHS, 100 mM di-potassium hydrogen phosphate (K2HPO4), and 10% (v/v) acetonitrile. The pH of both solutions was adjusted to 5.2. The effluent was monitored using a diode array detector at 260 nm. A linear relationship was found between peak area and concentration of nucleotides, over the concentration range of 2-30 mg/L for cytidine and uridine and of 4-30 mg/L for guanosine and adenosine. Validation of the proposed method was carried out by the standard additions method. The recoveries ranged from 99-103%. The precision of the method was also evaluated and reported a CV% as less than 3%. Twelve samples of commercial brands were successfully monitored applying this methodology.