The determination and distribution of nucleotides in dairy products using HPLC and diode array detection

Abstract

Abstract A method for the determination of adenosine, cytidine, uridine, guanosine and inosine 5′-monophosphates in milk and dairy products was optimized and its performance evaluated. The technique was based on ion-pair reversed-phase HPLC separation of the nucleotides and diode array detection. The chromatographic separation was achieved using a C18 column and a gradient elution with a mixture of two solvents: solvent A, water/glacial acetic acid/tetrabutilammonium hydrogensulphate (TBAHS) and solvent B, methanol/glacial acetic acid/TBAHS. The effluent was monitored using a Diode Array detector set at 260 nm. Validation of the proposed method was carried out by standard additions method, with recoveries of 98.3%. The precision of the method was also evaluated and reported a coefficient of variation (CV) as less than 3.2. Upon development the technique was applied on different dairy products in order to study the distribution of nucleotides therein. The samples included bovine, ovine and caprine milks, the corresponding manually manufactured cheeses, whey cheese and whey. Three commercial cheeses made from bovine, ovine and caprine milks, respectively, and 20 infant formulae were also analysed. In contrast to their absence in cheese upon preparation nucleotides were present in cheese at the end of ripening; this observation led to the extraction of DNA in order to evaluate whether nucleotides were released from degradation of nucleoproteins.