Development and validation of SEC-UV/HRMS procedure for simultaneous determination of BSA and its association products

Hodnik, Blaž (author), Čamič, Žiga (author), Pompe, Matevž (author)

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.

• Language: English
• Keywords: size exclusion chromatography, proteins, high-resolution mass spectrometry
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: 20 str.
• Numbering: Vol. 31, iss. 6, art. 1001
• PID: 20.500.12556/RUL-180956
• UDC: 543.544:543.51
• ISSN on article: 1420-3049
• DOI: 10.3390/molecules31061001
• COBISS.SI-ID: 272258051
• Publication date in RUL: 20.03.2026

Record is a part of a journal

• Title: Molecules
• Shortened title: Molecules
• Publisher: MDPI
• ISSN: 1420-3049
• COBISS.SI-ID: 18462981

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: velikostno izključitvena kromatografija, proteini, visokoločljivostna masna spektrometrija

Projects

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: P1-0153-2020
• Name: Raziskave in razvoj analiznih metod in postopkov