Rapid LC–MS Quantification of mRNA Vaccine Capping Efficiency via High-Specificity RNase H Cleavage and Metal Adduct Suppressed Chromatography

作者：Ren Yang, Xiaohong Wu, Xi Zhang, Shengqing Fu, Kaiping Gu, Zhe Lv, X Q Li, Qunying Mao · 发表于：Vaccines · 年份：2026 · DOI：10.3390/vaccines14070581 · 研究领域：RNA and protein synthesis mechanisms、Molecular Biology Techniques and Applications、RNA Research and Splicing

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 enab...