Purity and stability of modified nucleosides in the context of accurate quantification by LC-MS

Reliable quantification of RNA modifications depends on the chemical integrity of synthetic nucleoside standards, yet little is known about their shelf life in aqueous solution. We systematically assessed the short-term stability of 44 canonical and modified nucleosides by LC-UV-MS after storage at -80 °C, -20 °C, 8 °C, 20 °C and 40 °C for up to six months. While canonical nucleosides remained chemically intact, thirteen biologically relevant modifications decomposed in a temperature-dependent manner. 4-thiouridine underwent desulfurisation and dimerisation, 3-methylcytidine deaminated to 3-methyluridine, and N ⁴ -acetylcytidine hydrolysed to cytidine followed by deamination to uridine. mcm ⁵ s ² U displayed desulfurisation, whereas i ⁶ A showed low-level de-prenylation. High-resolution MS established accurate formulas for all degradation products. In order to provide a quantitative basis for nucleoside stability, reaction free energies (ΔG ₂₉₈ ) for four known decomposition reactions, namely deglycosylation, deamination, deacetylation and desulfurisation, were determined in solution phase quantum chemical calculations. The free energies of these reactions correlate well with the experimental data. All plates remain archived and will be re-analysed at 12, 24 and 36 months to establish long-term rate constants. Our data offer practical guidance for the handling of labile standards and present kinetic parameters that will improve inter-laboratory comparability in RNA modification research.