Transcript-independent quantitative analysis of RNA cap structures by LC-MS/MS

Incorporation of the 5' cap into RNA is a critical quality attribute that requires accurate assessment for therapeutic application. Existing analytical approaches often rely on transcript-specific probes or sequence-dependent workflows, which limit throughput and platform-level applicability. Here, we describe a quantitative, transcript-independent LC-MS/MS assay for RNA cap incorporation designed to support RNA therapeutic process development and quality control, using CleanCap-AG as a representative co-transcriptional capping chemistry. The workflow combines nuclease P1 digestion with external calibration using authentic cap analog standards, enabling quantification of capped (CC-A) and uncapped (ATP) 5' species while correcting for residual unincorporated reagents through paired digested and undigested analyses. The assay supports a 0.01-20 pmol calibration range using commercially available standards and a 10 min LC-MS/MS acquisition, facilitating high analytical throughput. Analytical characterization demonstrated excellent linearity (r ² > 0.995), accuracy (84%-102% recovery), and precision consistent with accepted bioanalytical criteria (RSD <15% at 1 and 10 pmol). Application to multiple RNA transcripts confirmed efficient digestion and accurate cap quantification independent of sequence context. This method provides a rigorously characterized, sequence-agnostic analytical platform for RNA cap analysis compatible with chemical and potentially enzymatic capping strategies, enabling scalable process development, lot comparability assessment, and manufacturing quality control.