Use of liquid chromatography and mass spectrometry to characterize oligonucleotides

A method of characterizing a sample, comprising:

• a. providing a sample comprising a population of oligonucleotides of interest of identical sequence and at least one impurity comprising an additional population of oligonucleotides;
• b. subjecting the sample to hydrophilic interaction liquid chromatography (HILIC) and mass spectrometry,
• wherein the HILIC comprises a mobile phase comprising a first buffer comprising 3 to 25 mM ammonium formate in 50% to 80% acetonitrile (ACN), and a second buffer comprising 3 to 25 mM ammonium formate in 20% to 40% ACN,
• thereby generating at least one mass spectrogram corresponding to the population of oligonucleotides of interest;
• and
• c. determining a percentage of total oligonucleotides in the sample corresponding to the population of oligonucleotides of interest.