Case study: Verification of SAX method selectivity using 2D-LC-MS for peak purity assessment of oligonucleotide APIs

Accurate analytical characterization is essential throughout oligonucleotide development, supporting impurity profiling, stability assessment, and ultimately the delivery of high-quality APIs. Strong anion exchange (SAX) chromatography is widely used as an orthogonal method to support purity assessment of oligonucleotide APIs because it separates analytes differently from ion-pair reversed-phase (IP-RP) chromatography. However, the use of non-volatile salts in SAX mobile phases prevents direct coupling to mass spectrometry, making it difficult to determine whether a seemingly pure UV peak contains co-eluting impurities.

To address this analytical challenge, Bachem developed a two-dimensional SAX–IP-RP–MS workflow – the first method that brings mass spectrometric detection to SAX-separated material and makes peak purity verification of a SAX method possible. By collecting SAX fractions and re-analyzing them using MS-compatible IP-RP chromatography, the approach enables MS-based assessment of peak purity and provides additional confidence in SAX method performance.

In this case study, we apply the method to a representative duplex siRNA API to evaluate the purity of SAX main peaks that would be classified as pure based solely on UV detection.

Download the case study to discover:

• Why UV detection alone may not be sufficient to confirm purity of SAX main peaks and how co-eluting impurities can remain undetected
• How a 2D SAX–IP-RP–MS workflow enables MS-based evaluation of SAX peak purity
• A worked example on a duplex siRNA, including the PS → PO, methylation, and des-/endo- species revealed under the antisense SAX main peak by 2D SAX–IP-RP–MS
• Why the method is positioned as a development and verification tool, and how it sharpens the routine release strategy without changing it
• A practical view of where 2D SAX–IP-RP–MS fits within Bachem’s broader analytical capabilities for your oligonucleotide program