Simple ion-pairing reversed phase LC-UV method for the relative quantification of capping species in mRNA-based modalities

Messenger RNA (mRNA)-based therapeutics and vaccines rely on proper 5' capping to ensure translational efficiency, stability, and reduced reactogenicity. Current analytical approaches for capping evaluation often rely on mass spectrometry (MS) and fluorinated solvents, which are costly, technically demanding, and not always suitable for quality control (QC) laboratories. Here, we describe the development of a simple, robust, and QC-compatible ion-pairing reversed-phase liquid chromatography method coupled with UV detection (IP-RPLC-UV) for the relative quantification of key 5' capping species in mRNA modalities, including uncapped, Cap 0, Cap 1, and Cap G transcripts, as well as non-templated additions (+G). The method employs targeted cleavage of the 5' end using an oligo hybridization and RNase H digestion strategy, enabling baseline resolution of capping species within 20 min. A systematic screening of various ion-pairing reagents demonstrated that butylammonium acetate offered the optimal balance between retention and resolution, while a design of experiments approach identified critical parameters influencing performance and ensured method robustness. Method sensitivity was confirmed with a lower limit of quantification at 0.01 mg/mL for uncapped species, and repeatability testing showed consistent results across samples. This study introduces the first IP-RPLC-UV method tailored for routine relative quantification of cap structures, providing a practical alternative to MS-based workflows and facilitating the implementation of accessible, reliable analytical control strategies for mRNA drug development.