ABSTRACT Human serum albumin (HSA) is the most abundant protein in human plasma, used as an excipient and carrier in pharmaceutical formulations to enhance drug solubility and pharmacokinetic profile. Size exclusion chromatography (SEC) is commonly employed to evaluate protein aggregation; however, methods that allow simultaneous determination of HSA content and oligomeric composition within a single analytical run remain limited. In the present study, a stability‐indicating SEC method was developed and validated for the concurrent quantification of HSA and assessment of its oligomeric distribution. Chromatographic separation was performed using an SEC column with a mobile phase composed of 0.1 M Na 2 HPO 4 (pH 6.8) and acetonitrile (900:100, v/v). The analysis was performed at a column temperature of 30°C, using UV detection at 220 nm and a flow rate of 0.8 mL min −1 , with a total run time of 30 min. The method demonstrated excellent linearity over a wide concentration range of 1.25–3750 µg mL −1 . Accuracy and precision met the acceptance criteria specified by ICH guidelines. Stability‐indicating capability was confirmed through forced degradation studies supported by mass balance analysis, enabling reliable detection and quantification of oligomeric and degradation products. The method was successfully applied to both excipient‐grade HSA and albumin‐bound nanoparticle formulations, confirming its suitability for diverse matrices. By combining assay determination and oligomeric profiling in a single chromatographic run, it reduces time and solvent use, making it suitable for quality control, formulation development, and stability studies in pharmaceutical and nanomedicine applications.
Patel et al. (Wed,) studied this question.