{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T05:16:54Z","timestamp":1783055814135,"version":"3.54.6"},"reference-count":43,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T00:00:00Z","timestamp":1782777600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100017242","name":"Youth Development Research Foundation of NIFDC","doi-asserted-by":"publisher","award":["2024B3"],"award-info":[{"award-number":["2024B3"]}],"id":[{"id":"10.13039\/100017242","id-type":"DOI","asserted-by":"publisher"}]},{"name":"State Key Laboratory of Drug Regulatory Science Project","award":["2026SKLDRS03090"],"award-info":[{"award-number":["2026SKLDRS03090"]}]},{"DOI":"10.13039\/501100012166","name":"National Key R&D Program of China","doi-asserted-by":"crossref","award":["2021YFC2302404"],"award-info":[{"award-number":["2021YFC2302404"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100012166","name":"National Key R&D Program of China","doi-asserted-by":"crossref","award":["2023YFC2606004"],"award-info":[{"award-number":["2023YFC2606004"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>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 \u226598% 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\u2032 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.<\/jats:p>","DOI":"10.3390\/vaccines14070581","type":"journal-article","created":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T00:50:37Z","timestamp":1782867037000},"page":"581","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid LC\u2013MS Quantification of mRNA Vaccine Capping Efficiency via High-Specificity RNase H Cleavage and Metal Adduct Suppressed Chromatography"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8134-4010","authenticated-orcid":false,"given":"Ren","family":"Yang","sequence":"first","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing 102629, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-6471-1285","authenticated-orcid":false,"given":"Xiaohong","family":"Wu","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing 102629, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaowei","family":"Zhang","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing 102629, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shengqing","family":"Fu","sequence":"additional","affiliation":[{"name":"Tianjin Pharmaceutical and Cosmetic Evaluation and Inspection Center, Tianjin 300308, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaiping","family":"Gu","sequence":"additional","affiliation":[{"name":"Department of Analytical Science, Suzhou Abogen Biosciences Co., Ltd., Suzhou 215123, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhe","family":"Lv","sequence":"additional","affiliation":[{"name":"Sinovac Life Sciences Co., Ltd., Beijing 102601, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoli","family":"Li","sequence":"additional","affiliation":[{"name":"Shandong WeigaoLitong Biological Products Co., Ltd., Weihai 276300, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qunying","family":"Mao","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing 102629, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"736","DOI":"10.1038\/s41586-024-08304-0","article-title":"The Mechanism of mRNA Cap Recognition","volume":"637","author":"Gentry","year":"2025","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1002\/wrna.19","article-title":"Enzymology of RNA Cap Synthesis","volume":"1","author":"Ghosh","year":"2010","journal-title":"WIREs RNA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"596","DOI":"10.1073\/pnas.1515152113","article-title":"Structural Basis for m7G Recognition and 2\u2032-O-Methyl Discrimination in Capped RNAs by the Innate Immune Receptor RIG-I","volume":"113","author":"Devarkar","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"452","DOI":"10.1038\/nature09489","article-title":"2\u2032-O Methylation of the Viral mRNA Cap Evades Host Restriction by IFIT Family Members","volume":"468","author":"Daffis","year":"2010","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"7511","DOI":"10.1093\/nar\/gkw551","article-title":"mRNA Capping: Biological Functions and Applications","volume":"44","author":"Ramanathan","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1038\/nrd.2017.243","article-title":"mRNA Vaccines\u2014A New Era in Vaccinology","volume":"17","author":"Pardi","year":"2018","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1038\/s41586-021-03791-x","article-title":"Systems Vaccinology of the BNT162b2 mRNA Vaccine in Humans","volume":"596","author":"Arunachalam","year":"2021","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2377","DOI":"10.1038\/s41467-022-30059-3","article-title":"Comparative Effectiveness over Time of the mRNA-1273 (Moderna) Vaccine and the BNT162b2 (Pfizer-BioNTech) Vaccine","volume":"13","author":"Islam","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"5021","DOI":"10.1007\/s00216-016-9605-x","article-title":"Label-Free Analysis of mRNA Capping Efficiency Using RNase H Probes and LC-MS","volume":"408","author":"Beverly","year":"2016","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1544","DOI":"10.1056\/NEJMoa2024671","article-title":"Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates","volume":"383","author":"Corbett","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e34","DOI":"10.1093\/nar\/gkad019","article-title":"Versatile Strategy Using Vaccinia Virus-Capping Enzyme to Synthesize Functional 5\u2032 Cap-Modified mRNAs","volume":"51","author":"Ohno","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1114","DOI":"10.1016\/j.cell.2017.02.017","article-title":"Modified mRNA Vaccines Protect against Zika Virus Infection","volume":"168","author":"Richner","year":"2017","journal-title":"Cell"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1088","DOI":"10.1038\/s41586-025-10004-2","article-title":"Individualized mRNA Vaccines Evoke Durable T Cell Immunity in Adjuvant TNBC","volume":"651","author":"Sahin","year":"2026","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2603","DOI":"10.1056\/NEJMoa2034577","article-title":"Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine","volume":"383","author":"Polack","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1016\/j.omtn.2023.07.006","article-title":"Mesenchymal Stem Cell Engineering by ARCA Analog-Capped mRNA","volume":"33","author":"Andrzejewska","year":"2023","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/S0076-6879(07)31011-2","article-title":"Synthesis of Anti-Reverse Cap Analogs (ARCAs) and Their Applications in mRNA Translation and Stability","volume":"Volume 431","author":"Stepinski","year":"2007","journal-title":"Methods in Enzymology"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e39","DOI":"10.1002\/cpz1.39","article-title":"Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap\u00ae Analog by In Vitro Transcription","volume":"1","author":"Henderson","year":"2021","journal-title":"Curr. Protoc."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e202200005","DOI":"10.1002\/tcr.202200005","article-title":"Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines","volume":"22","author":"Shanmugasundaram","year":"2022","journal-title":"Chem. Rec."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1144","DOI":"10.1261\/rna.079173.122","article-title":"RNase H-Based Analysis of Synthetic mRNA 5\u2032 Cap Incorporation","volume":"28","author":"Chan","year":"2022","journal-title":"RNA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"151860","DOI":"10.1016\/j.bbrc.2025.151860","article-title":"Capping in mRNA Analyzed Using Pistol Ribozyme and LC-MS","volume":"766","author":"Cui","year":"2025","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Vlatkovic, I., Ludwig, J., Boros, G., Szab\u00f3, G.T., Reichert, J., Buff, M., Baiersd\u00f6rfer, M., Reinholz, J., Mahiny, A.J., and \u015eahin, U. (2022). Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA. Pharmaceutics, 14.","DOI":"10.3390\/pharmaceutics14020328"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1007\/s11033-026-11455-0","article-title":"Process and Analytical Strategies for the Safe Production of mRNA Vaccines and Therapeutics","volume":"53","author":"Nowak","year":"2026","journal-title":"Mol. Biol. Rep."},{"key":"ref_23","unstructured":"(2026, June 11). Analytical Procedures for Quality of mRNA Vaccines and Therapeutics. Available online: https:\/\/www.uspnf.com\/notices\/analytical-procedures-mrna-vaccines-20240802."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"124922","DOI":"10.1016\/j.jchromb.2026.124922","article-title":"Simple Ion-Pairing Reversed Phase LC-UV Method for the Relative Quantification of Capping Species in mRNA-Based Modalities","volume":"1271","author":"Maurer","year":"2026","journal-title":"J. Chromatogr. B"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e142","DOI":"10.1093\/nar\/gkr695","article-title":"Generating the Optimal mRNA for Therapy: HPLC Purification Eliminates Immune Activation and Improves Translation of Nucleoside-Modified, Protein-Encoding mRNA","volume":"39","author":"Muramatsu","year":"2011","journal-title":"Nucleic Acids Res."},{"key":"ref_26","first-page":"3","article-title":"Electrospray Ionisation Mass Spectrometry: Principles and Clinical Applications","volume":"24","author":"Ho","year":"2003","journal-title":"Clin. Biochem. Rev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1667","DOI":"10.1002\/rcm.7596","article-title":"Reduction of Metal Adducts in Oligonucleotide Mass Spectra in Ion-pair Reversed-phase Chromatography\/Mass Spectrometry Analysis","volume":"30","author":"Birdsall","year":"2016","journal-title":"Rapid Commun. Mass Spectrom."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1038\/nature16471","article-title":"Crystal Structure of a DNA Catalyst","volume":"529","author":"Steuerwald","year":"2016","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"4262","DOI":"10.1073\/pnas.94.9.4262","article-title":"A General Purpose RNA-Cleaving DNA Enzyme","volume":"94","author":"Santoro","year":"1997","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2006","DOI":"10.1038\/s41467-017-02203-x","article-title":"Crystal Structure of an RNA-Cleaving DNAzyme","volume":"8","author":"Liu","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2883","DOI":"10.1093\/nar\/gkg378","article-title":"Optimisation of the 10-23 DNAzyme-Substrate Pairing Interactions Enhanced RNA Cleavage Activity at Purine-Cytosine Target Sites","volume":"31","author":"Cairns","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Pang, J., Guo, Q., and Lu, Z. (2022). The Catalytic Mechanism, Metal Dependence, Substrate Specificity, and Biodiversity of Ribonuclease H. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.1034811"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1005","DOI":"10.1016\/j.cell.2005.04.024","article-title":"Crystal Structures of RNase H Bound to an RNA\/DNA Hybrid: Substrate Specificity and Metal-Dependent Catalysis","volume":"121","author":"Nowotny","year":"2005","journal-title":"Cell"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"465574","DOI":"10.1016\/j.chroma.2024.465574","article-title":"Evaluation of Ion Pairing Reversed-Phase Liquid Chromatography for the Separation of Large RNA Molecules","volume":"1740","author":"Maurer","year":"2025","journal-title":"J. Chromatogr. A"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/0378-4347(93)80024-X","article-title":"Reversed-Phase and Ion-Pair Separations of Nucleotides, Nucleosides and Nucleobases: Analysis of Biological Samples in Health and Disease","volume":"618","author":"Werner","year":"1993","journal-title":"J. Chromatogr. B Biomed. Sci. App."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"464475","DOI":"10.1016\/j.chroma.2023.464475","article-title":"Ion-Pair Reversed-Phase and Hydrophilic Interaction Chromatography Methods for Analysis of Phosphorothioate Oligonucleotides","volume":"1712","author":"Gilar","year":"2023","journal-title":"J. Chromatogr. A"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1007\/s13361-016-1500-3","article-title":"The Role of Fluorinated Alcohols as Mobile Phase Modifiers for LC-MS Analysis of Oligonucleotides","volume":"28","author":"Basiri","year":"2017","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"465074","DOI":"10.1016\/j.chroma.2024.465074","article-title":"Impact of Ion-Pairing Systems Choice on Diastereomeric Selectivity of Phosphorothioated Oligonucleotides in Reversed-Phase Liquid Chromatography","volume":"1730","author":"Gilar","year":"2024","journal-title":"J. Chromatogr. A"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e125","DOI":"10.1093\/nar\/gkz731","article-title":"A General LC-MS-Based RNA Sequencing Method for Direct Analysis of Multiple-Base Modifications in RNA Mixtures","volume":"47","author":"Zhang","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1258","DOI":"10.1038\/s41557-022-01037-5","article-title":"Nanopore Microscope Identifies RNA Isoforms with Structural Colours","volume":"14","author":"Keyser","year":"2022","journal-title":"Nat. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3629","DOI":"10.1093\/nar\/24.18.3629","article-title":"Synthesis of a Fluorescent 7-Methylguanosine Analog and a Fluorescence Spectroscopic Study of Its Reaction with Wheatgerm Cap Binding Proteins","volume":"24","author":"Ren","year":"1996","journal-title":"Nucleic Acids Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"127339","DOI":"10.1016\/j.vaccine.2025.127339","article-title":"Assay for Rapid Quantification of Capped and Tailed Intact mRNA","volume":"61","author":"Gao","year":"2025","journal-title":"Vaccine"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1261\/rna.2436411","article-title":"A Quantitative Assay for Measuring mRNA Decapping by Splinted Ligation Reverse Transcription Polymerase Chain Reaction: qSL-RT-PCR","volume":"17","author":"Blewett","year":"2011","journal-title":"RNA"}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/7\/581\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T04:22:38Z","timestamp":1783052558000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/7\/581"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,30]]},"references-count":43,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["vaccines14070581"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14070581","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,30]]}}}