# Development and Validation of SEC-UV/HRMS Procedure for Simultaneous Determination of BSA and Its Association Products

## TL;DR

This paper introduces a new SEC-UV/HRMS method to quickly and accurately analyze BSA and its aggregates, useful for pharmaceutical research and quality control.

## Contribution

A validated SEC-UV/HRMS method for native-like analysis of BSA and its oligomers using standard-flow electrospray ionization.

## Key findings

- The method detects BSA oligomers up to the heptamer with minimal in-source unfolding.
- Low-flow separations enabled efficient ionization and 10-minute run times.
- SEC-MS semi-quantitatively monitors oligomerization dynamics, revealing transient species.

## Abstract

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.