Background: The m7G cap structure, which mimics the natural cap of eukaryotic mRNA, is a critical determinant of mRNA vaccine efficacy, safety, and stability. However, its precise quantification remains challenging due to complex impurity profiles and the high physicochemical similarity between the target cap and related impurities. Although liquid chromatography mass spectrometry (LC-MS) is widely employed for this purpose, current methodologies still face significant limitations, including labor-intensive sample preparation, low analytical throughput, poor reproducibility in quantifying low-level impurities, and a lack of universally applicable strategies across diverse mRNA vaccine platforms. Methods: We systematically optimized sample preparation and LC-MS detection workflows. RNase H-mediated cleavage was compared with DNAzymes, guide DNA probes were rationally designed, and thermostable RNase H was introduced for one-step denaturation and cleavage. To establish an accurate, efficient, and universal sample preparation workflow. Chromatographic conditions were optimized using an ion-pairing reagent system to suppress ESI-MS metal adducts. Eliminating sample purification improves recovery, reduces manual handling errors, and boosts assay efficiency. Results: Through optimally designed guide DNA probes, RNase H cleavage specificity reached ≥98% with high cleavage efficiency, offering higher efficiency than DNAzyme. Furthermore, the incorporation of thermostable RNase H enabled a single-step workflow combining high-temperature denaturation and site-specific cleavage, substantially streamlining sample preparation. On the chromatographic side, optimization of the ion-pairing reagent system effectively suppressed metal adduct formation in electrospray ionization mass spectrometry (ESI-MS). This advancement enabled direct injection of the 5' cap fragments without purification, achieving high-recovery quantification while demonstrating broad compatibility across mainstream LC-MS platforms. The optimized assay reduces the total analytical workflow from 4~6 h to under 1.5 h. Conclusions: Combining high accuracy, robustness, and broad platform compatibility, this method offers a universal, high-throughput analytical solution for mRNA vaccine quality control and continuous process development.
