Development of simple isocratic HPLC methods for siRNA quantitation in lipid-based nanoparticles

[Display omitted] •Simple HPLC methods developed for siRNA determination in lipid nanoparticle formulation.•A practical approach to the traditionally used size exclusion chromatography methodology with greater sensitivity.•The method demonstrated adequate accuracy, precision, sensitivity and robustness. This paper describes the development of simple and user-friendly HPLC methods that can quantitate the amount of small interfering RNA (siRNA) in lipid-based nanoparticle (LNP) formulations. The methods have been used as alternative chromatographic approaches to the size exclusion chromatography in order to perform “fit for purpose” analysis such as determining the amount of released siRNA from LNP formulations as a part of in-vitro release testing. Two HPLC conditions were optimized using reversed phase (a 250 × 4.6 mm Waters XSelect CSH column) and cation exchange columns (a 250 × 4.6 mm Zorbax SCX-300 column) maintained at 30 °C with a mobile phase of 0.1 M ammonium bicarbonate aqueous solution containing 20–30% acetonitrile. All the siRNA variants were excluded from pores in stationary phase and led to a single peak eluted earlier than the solvent front. The impact of chromatographic conditions and column configurations on method specificity, accuracy, and sensitivity have been investigated. Validation data for both methods in terms of precision, linearity, accuracy and sensitivity are also presented.