Development and validation of a HILIC-UV method for the determination of nucleotides in fish samples.

The aim of this work was the development of a simple, novel and accurate method for the determination of adenosine triphosphate (ATP) and its first five catabolites: adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine (Ino) and hypoxanthine (Hx), in fish tissue, based on hydrophilic interaction liquid chromatography (HILIC). For this purpose, a stationary phase for polar and hydrophilic compounds (ZIC-pHILIC) was used. The effect of different chromatographic parameters and the molecular mechanism based on the van't Hoff plot were examined. The t-test and Dixon's Q-test were applied in order to examine statistical differences and outlier values. The recovery of the method ranged between 82.7% and 127% and the %RSD values were lower than 10% for all analytes determined. The method was applied in frozen sea bream samples stored at 0-4 °C. The Ki-, G-, H- and F values were calculated for the estimation of the level of fish freshness.