UV-MS Workflow For Impurity Identification And Purity Analysis Of Synthetic Oligonucleotides

Ion-pairing reversed phase methods remain a staple for oligonucleotide purity testing, yet closely related impurities often co-elute with the main peak and go uncounted. Adding high-resolution accurate mass detection to the same separation brings them into view. Working from a commercially approved siRNA, the analysis shows how mass accuracy and isotopic fit identify impurities arising from synthesis, raw materials, and degradation. MS/MS sequencing then localizes modifications. The relative MS response of each co-eluting species is factored into a UV-MS purity value. PMOs call for a different route: chemical hydrolysis stands in where MS/MS sequencing reaches instrument limits.

Examine the data to see how the purity figure for the main strand shifts once co-eluting species are counted.