In order to ensure quality, efficacy and safety of therapeutic ON products, impurity profiling is imperative. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the predominant analytical technique used for this purpose in the ON industry. In IP-RPLC, positively charged ion-pair reagents (IPRs) are used to increase retention of the negatively charged ONs on the hydrophobic stationary phase, leading to a length-based separation, as every additional nucleotide increases the apparent hydrophobicity. Impurities that comprise the same number of nucleotides as the FLP are, therefore, typically not or poorly resolved. Separations of sequence variants have been reported, but usually the sequence differs significantly. In this respect, the deaminated product, a sequence variant impurity that only differs on one position and could cause off-target effects, is one of the most challenging impurities to separate and quantify. During deamination, a cytosine or 5-methylcytosine of the ON is converted to uracil or thymine, respectively, corresponding to a loss of ammonia and a gain of water. The molecular mass of this impurity differs less than 1 Da from the FLP, resulting in overlapping isotope patterns in mass spectrometry (MS), hindering their reliable distinction. So far, three approaches have been described to quantify the deamination impurities in ONs. Firstly, weak anion exchange chromatography (WAX) employing a high pH eluent was used to resolve the deaminated products from the FLP. The second, MS-based approach focused on statistical modelling of the subtle changes in the isotope pattern when the impurity co-elutes with the FLP, and recently, supercritical fluid chromatography has been reported to separate DA impurities from the FLP. However, these approaches have limitations, such as MS incompatibility of the WAX method due to the high concentrations of involatile salts used in the eluent, the need of high-resolution and sensitive MS, which is not prevalent in a quality control setting, or it has not been applied to PS ONs. Besides the aforementioned deamination impurity, deamination of adenine and guanine to hypoxantine and xanthine, respectively, can also occur but these processes are much slower.