Determination of antigen components in inactivated SARS-CoV-2 vaccine using ultra-high-performance liquid...

Accurate and reliable quantification of antigen components in vaccines is critical in vaccine quality control and evaluation of immunogenic consistency. However, conventional immunoassays often suffer from limited specificity, trace-level antigen concentrations, and indirect quantification. In this study, we demonstrated an ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the determination of effective antigen components in inactivated SARS-CoV-2 vaccines. Specifically, the vaccine samples were denatured and digested with trypsin to generate tryptic peptides. Then, the signature peptides derived from the nucleocapsid protein and their stable isotope-labeled internal standards were selectively captured and separated using anti-peptide antibody-conjugated magnetic beads. The signature peptides were characterized by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, which confirmed their amino acid sequence and multi-charged ionization states. Quantitative analysis was then performed using UHPLC-MS/MS in positive electrospray ionization mode with multiple reaction monitoring. Chromatographic separation of the signature peptides was achieved on an ACQUITY Premier Peptide BEH C₁₈ column using 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile as the mobile phases. The method was validated and exhibited excellent linearity for the signature peptides over the concentration range of 1-60 μg/L, with correlation coefficients higher than 0.999. The recovery ranged from 75.1 % to 86.3 %, with intra-day precision (RSD) of 0.8-1.0 % and inter-day precision of 1.3-3.6 %. Finally, the method was successfully applied to determine the effective antigen components in inactivated SARS-CoV-2 vaccine samples. The concentrations of the signature peptide ADETQALPQR ranged from 4.95 to 12.95 µg/L across the three vaccine batches analyzed, corresponding to 4.38-11.47 nmol/L of nucleocapsid protein. The results indicated that the method exhibited great promise for the determination of active antigenic proteins in inactivated SARS-CoV-2 vaccine samples and provided an alternative analytical platform for vaccine quality control.