Short capillary ion‐pair high‐performance liquid chromatography coupled to electrospray (tandem) mass spectrometry for the simultaneous analysis of nucleoside mono‐, di‐ and triphosphates

Abstract

A liquid chromatography/mass spectrometry (LC/MS) method for the analysis of complex mixtures of nucleoside mono‐, di‐ and triphosphates has been developed. A short capillary column (35mm × 0.3mm i.d.) was operated under ion‐pair high‐performance liquid chromatography conditions and hyphenated to (negative) electrospray (tandem) mass spectrometry. As such, the separation of 12 nucleotides was performed by a binary gradient elution using CH₃OH/H₂O and N,N‐dimethylhexylamine (N,N‐DMHA) as ion‐pairing agent. The influence of different N,N‐DMHA concentrations on the chromatographic and mass spectrometric performance was evaluated to achieve optimal LC/MS conditions. In addition it was demonstrated that a controlled admission of ammonium dihydrogen phosphate (NH₄H₂PO₄) improved both chromatographic performance and mass spectrometric detection. Because the system was hyphenated to an orthogonal designed electrospray interface (Z‐spray™), long acquisition times were possible without loss of sensitivity. Copyright © 2002 John Wiley & Sons, Ltd.