Hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of hydrophilic ion-pairing reagents for the separation of nucleosides and nucleotide mono-, di- and triphosphates

HILIC XBridge-Amide was found to be the most suitable column for the separation of these analytes, and the optimized mobile phase consisted of an ACN/UHQ water mixture (3 min of isocratic elution using 82:18%, v/v and then a fast gradient from 18% to 22% of water) with 100 mM hexafluoro-2-propanol and 50 mM diethylamine (wwpH 9-wspH 10). In a total analysis time of 8 min, good results were achieved in terms of resolution. Under these optimum conditions, a further comprehensive study of the retention mechanism was carried out.