LC-MS represents an important technology for the qualitative and quantitative analysis of nucleic acids. For MS, ESI in negative ion mode is used. The chromatographic method of choice is ion-pair (IP) RP chromatography. Chromatographic separations are usually accomplished by gradients of an organic modifier in aqueous solutions of IP reagents. Commonly applied IP reagents are 2.3 mM triethylamine/400 mM 1,1,1,3,3,3-hexafluoro-2-propanol (TEA/HFIP, pH 7.0) and 10-25 mM cyclohexyldimethylammonium acetate (CycHDMAA, pH 8.4). Direct comparison of mass spectrometric performance of the two solvent systems revealed that the TEA/HFIP system offers better detection sensitivity than the CycHDMAA system. This is mainly attributable to the depletion of HFIP during droplet formation and solvent evaporation. Removal of the anionic counterion facilitates oligonucleotide ionization, and the oligonucleotides are desorbed as highly charged ions into the gas phase. TEA/HFIP-based mobile phases are recommended for developing quantitative assays targeting defined oligonucleotides. The CycHDMAA system allows the formation of cyclohexyldimethylammonium adducts. These adducts are cleaved in the gas phase, and this decomposition gives rise to charge state reduction. Ammonium adduct formation is of particular importance in preventing adducting with metal ions. Thus, adducts with metal ions are efficiently suppressed with CycHDMAA. For the TEA/HFIP system, however, such adducting represents a severe problem particularly if large oligonucleotides are analyzed. Thus, CycHDMAA-based mobile phases are recommended for qualitative assays such as LC-MS-based genotyping.