Development of an assay method for tofersen using IPRP-LC-HRMS with an extracting ion chromatogram processing approach

Abstract

The quality control of oligonucleotides using separation analytical methods is complicated owing to co-elution of structurally similar impurities and active oligonucleotides. Thus, the inadequate separation of impurities from the active pharmaceutical ingredients restricts the applicability of these assay methods. This limitation also hinders achieving compliance with regulatory standards. Accordingly, this research was focused on the development of an assay methodology for tofersen (TSN) to enable accurate quantification despite the presence of co-eluting impurities. An analytical assay method for the determination of TSN was proposed based on ion-pair reverse-phase liquid chromatography coupled with high-resolution mass spectrometry using an extracted ion chromatography processing approach. Nusinersen (NSN) was utilized as the internal standard to mitigate variations in signal intensity of the mass spectrometer. The developed method was validated according to the ICH Q2(R2) and USP regulatory frameworks. The method demonstrated linear response and specificity within the concentration range of 3–10 μg mL⁻¹. The recovery from the formulation matrix, which was determined using the standard addition method with triplicate measurements at three concentration levels, was found to be within the range of 80–120%. Furthermore, the method exhibited precision and robustness for critical liquid chromatography parameters, such as flow rate and injection volume, and mass spectrometer parameters, including drying/desolvation temperature and gas flow.