20.500.12556/RUL-180956 Development and validation of SEC-UV/HRMS procedure for simultaneous determination of BSA and its association products Monitoring peptide and protein self-association is essential for understanding biological function, formulation stability, and aggregation mechanisms. While size-exclusion chromatography (SEC) is routinely used to quantify protein-size variants under native conditions, its hyphenation to high-resolution mass spectrometry (HRMS) for simultaneous structural characterization remains limited. Here, we report the development and validation of a robust SEC-UV/HRMS method optimized for native-like analysis of bovine serum albumin (BSA) monomers and higher-order oligomers using standard-flow electrospray ionization. Systematic evaluation of source parameters, mobile-phase composition, and chromatographic conditions enabled retention of native BSA structure, minimized in-source unfolding, and enhanced MS sensitivity, allowing detection of oligomers up to the heptamer. A short, narrow-bore 200 Å UHPLC SEC separation column was used. Low-flow separations (~0.05 mL/min) enabled efficient ionization and 10 min run times. An accelerated 60 °C stress-testing protocol demonstrated that SEC-MS can semi-quantitatively monitor oligomerization dynamics, complementing UV-based quantification and revealing transient species not resolved by UV alone. The method showed acceptable linearity, precision, and sample stability, and comparison with SEC-RALS/LALS confirmed molecular-weight trends across aggregation states. Overall, the developed SEC-UV/HRMS workflow provides a rapid, sensitive, and widely accessible approach for UV-based quantification of monomer- and HRMS-based characterizing protein aggregation in research and quality control in pharmaceutical laboratories. size exclusion chromatography proteins high-resolution mass spectrometry velikostno izključitvena kromatografija proteini visokoločljivostna masna spektrometrija true false true Angleški jezik Slovenski jezik Članek v reviji 2026-03-20 12:11:44 2026-03-20 12:11:46 2026-03-23 03:51:55 0000-00-00 00:00:00 2026 0 0 20 str. iss. 6, art. 1001 Vol. 31 Mar. 2026 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 543.544:543.51 1420-3049 10.3390/molecules31061001 272258051 RAZ_Hodnik_Blaz_2026.pdf RAZ_Hodnik_Blaz_2026.pdf 1 3754D55AAADDEA9B22003547ED708246 996d5d17a36b8fcd085c31e85e0f2c8c8e5ee3505c89a319a98d5660c70fcd68 c953f3b2-244d-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=230983 https://www.mdpi.com/1420-3049/31/6/1001 1 7eecc1ad-244d-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=230981 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0