Synthetic oligonucleotides such as antisense and small interfering RNA (siRNA) are an emerging class of therapeutics with a significant potential to treat a wide range of diseases [[1], [2], [3], [4]]. The distinct pharmacological features of the class stem from the ability to modulate gene expression through selective binding to RNA via Watson-Crick base pairing [5,6]. Knowledge of the primary sequence of the target gene facilitates rational identification of candidate oligonucleotide drugs, which is often faster than for small molecule drugs [[1], [2], [3], [4], [5], [6]].

Appropriate analytical methods [7,8] have been developed over the last two to three decades to characterize and monitor low-level impurities potentially formed during oligonucleotide manufacturing [9,10]. Many chromatographic methods are available for the separation of oligonucleotide impurities, with differing level of success depending on the nature of the impurity [[11], [12], [13]]. Ion-pair reversed-phase (IP-RP) and hydrophilic interaction chromatography (HILIC) are among the most widely used HPLC methods, primarily due to their ability to directly interface with mass spectrometry [14,15]. Mass spectrometry can separate co-eluting impurities based on mass differences. However, the separation of co-eluting isobaric and near-isobaric impurities is limited by the resolving power of the mass spectrometer [16,17] and there remains a need for improved chromatographic methods.

A weak anion exchange (WAX) chromatography method combined with chemical derivatization was recently developed to separate the parent phosphorothioate oligonucleotide compound from its major degradation products, i.e., phosphate diester (PO), deamination, and depurination impurities [18]. The PO degradation product is observed following oxidative and thermal stress [14] and deamination and depurination degradation products may be produced under basic, acidic, or thermal conditions [9,19]. The deamination impurity results from the conversion of cytosine or 5-methylcytosine residues to uracil and thymine, respectively. Deamination of adenine is also possible, but its rate is ca. 50 times slower than that of cytosine [16]. The depurination impurity is formed by cleavage of the N-glycosidic bond and the replacement of adenine or guanine by water. From the three major degradation products, the near-isobaric deamination impurity is the most challenging to separate by mass spectrometry. Deamination results in a + 0.98 Da addition to the parent oligonucleotide mass which overlaps with its natural +1 Da isotope (a resolving power of at least 450,000 would be required to separate the two species for a typical 20-mer oligonucleotide of MW ∼ 7 kDa) [16].

In the present work, learnings from the initial WAX method developed to separate DA co-eluting impurities based on their ionization (pKa) differences were exploited to enhance the capabilities of the widely used IP-RP LCMS method [17]. We found that it is possible to increase chromatographic separation between the parent oligonucleotide and impurities by conducting the analysis under different pH regimes. The adverse impact of low pH on negative ion mode MS sensitivity is countered by running the mass spectrometer in the selected ion monitoring (SIM) mode. The approach permits complete separation of PO, deamination and depurinated degradation products from the parent oligonucleotide.