Introduction Oligonucleotide therapeutics have emerged in recent years as a powerful alternative to small molecule and protein therapeutics. Manufacturing and quality control of oligonucleotide therapeutics requires highly selective and sensitive LC/MS methods for impurity identification and quantification. Here we demonstrate the use of a novel chromatographic system, equipped with a modified surface technology across its entire fluidic path [1], for analysis of modified synthetic oligonucleotides and their impurities. Methods Separations were performed by ion-pairing RP chromatography on a 2.1 x 100 mm UHPLC column with a modified internal surface, packed with 1.7 µm C18 particles. UV chromatograms were acquired on a tunable UV detector at a wavelength of 260 nm. High resolution (> 10,000) ESI-MS spectra were acquired in negative ion mode on a TOF MS instrument using an acquisition time of 0.5 sec. The accuracy of each intact mass measurement was calculated by comparing the experimentally obtained deconvoluted mass against the theoretical average masses of each oligo. Preliminary data The first type of modified oligonucleotide characterized was a 21-mer, bearing 19 modified nucleosides, while the second type was a 100-mer single guide (sg) RNA oligo. Chelating interactions occurring between oligonucleotides and metal surfaces can be mitigated by employing a UHPLC system with a fluidic path containing a hybrid organic/inorganic barrier layer [1] which minimizes the contact of analytes (oligonucleotides) with the metal surfaces present in the fluidic path or the UHPLC column. The data presented here demonstrates that a combination of the bio-inert UHPLC system and column is critical for obtaining accurate results in measuring low-level oligonucleotide impurities (down to 0.2% relative abundance levels) using UV detection at 260 nm. In addition, the same LC-MS platform provides intact mass confirmation of oligonucleotides and their impurities with mass accuracies under 15 ppm. References: 1. Anal Chem, 2021, 93, p5773.