pH effects on the separation of oligonucleotides by ion-pair reserved phase liquid chromatography mass spectrometry

Significant recent interest has developed in synthetic antisense and small interfering RNA (siRNA) oligonucleotides due to their high therapeutic potential. Characterization and monitoring of low-level impurities produced during oligonucleotide manufacturing is challenging, especially for impurities that co-elute and are isobaric with the parent oligonucleotide. To improve the separation of such impurities, the effects of pH under ion-pair reversed phase (IP-RP) chromatography conditions have been examined here. Effective separation of co-eluting, near-isobaric deamination (DA) impurities from the parent oligonucleotide based on ionization (pKa) differences has been achieved under acidic conditions. The method produces a single chromatographic peak encompassing all individual positional isomers of the DA impurity, which greatly simplifies quantitative analysis. Optimum separation of other co-eluting impurities based on electronegativity/polarity differences (PO) was accomplished under basic conditions. Operation of the mass spectrometer (MS) in the selected ion monitoring (SIM) mode counters the lower sensitivity typically associated with negative ion MS analysis of oligonucleotides under low pH conditions. Analysis of stressed samples (80 °C, 0-7 days) for the extent of deamination over time produced linear relationships with both UV and MS methods of detection. The separation of other near-isobaric impurities from each other, for example, n-d^MeC from n-T, and n-MOE ^MeC from n-MOE T, is demonstrated. The new pH-enabled method is faster and simpler than other methods requiring sample desulfurization or HRMS experiments measuring differences in isotopic peak distributions.