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

A method for the determination of adenosine, cytidine, uridine, guanosine a nd inosine 5'-monophosphates in milk and dairy products was optimized and i ts performance evaluated. The technique was based on ion-pair reversed-phas e HPLC separation of the nucleotides and diode array detection. The chromat ographic separation was achieved using a C18 column and a gradient elution with a mixture of two solvents: solvent A, water/glacial acetic acid/tetrab utilammonium hydrogensulphate (TBAHS) and solvent B, methanol/glacial aceti c acid/TBAHS. The effluent was monitored using a Diode Array detector set a t 260 nm. Validation of the proposed method was carried out by standard add itions method, with recoveries of 98.3%. The precision of the method was al so 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 include d bovine, ovine and caprine milks, the corresponding manually manufactured cheeses, whey cheese and whey. Three commercial cheeses made from bovine, o vine and caprine milks, respectively, and 20 infant formulae were also anal ysed. In contrast to their absence in cheese upon preparation nucleotides w ere present in cheese at the end of ripening; this observation led to the e xtraction of DNA in order to evaluate whether nucleotides were released fro m degradation of nucleoproteins. (C) 2001 Elsevier Science Ltd. All rights reserved.