Analytical validation of total mucin concentration assay using SEC MALLS dRI for diagnosing and monitoring...

Mucins play a pivotal role in the pathophysiology of mucoobstructive lung diseases. Accurate quantification of total mucin concentrations in clinical sputum samples is critical for developing objective biomarkers for diagnosis, prognosis, and therapeutic monitoring. By using sputum samples and mucin standards, the analytical performance of the measurements of total mucin concentration by Size Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering and Differential Refractometer [SEC-(MALLS)-dRI] method was assessed using universal validation metrics, including precision, accuracy, recovery, parallelism, specificity, linearity, and sample stability. Possible sample contamination sources, such as saliva, blood, and DNA, were also evaluated. The method demonstrated excellent precision across low, medium, and high concentrations (CV% ≤ 2.6%) and high recovery (116%). It exhibited strong linearity over a broad dynamic range (~30-15,000 µg/mL) and stability for up to 12 months at - 20 °C in naïve samples and 4 °C in 4 M GuHCl. Measurement interference was negligible, up to 20% saliva, 2% blood, and 2% DNA. This study validates the SEC-(MALLS)-dRI method as a robust, reliable approach for quantifying total mucin concentrations in clinical sputum samples. The demonstrated analytical validity establishes its use as a biomarker platform for clinical and research applications, aiding in the diagnosis and management of hypersecretory/mucoobstructive lung diseases.