Refining the CTAB method for hyaluronic acid analysis: A cost-effective alternative to carbazole and LC-MS assays

Reliable determination of hyaluronic acid (HA) in culture broth is critical for monitoring biotechnological production processes. Among the commonly used methods carbazole method is accessible but lacks specifity, while LC-MS offers high sensitivity but is costly and time-intensive. This study focuses on optimizing a benchtop turbidimetric assay using cetyltrimethylammonium bromide (CTAB) for accurate and efficient HA quantification. Key reaction parameters, including concentrations of CTAB, NaOH, and NaCl, measurement conditions such as wavelength and time and the suitable method of capsular HA release were systematically optimized using standard HA solutions and culture broth samples. Dynamic light scattering (DLS) analysis provided insights into the kinetics and mechanisms of HA-CTAB complex formation. The optimized CTAB protocol showed superior precision, accuracy, and resistance to interference compared to the carbazole assay, and exhibited strong concordance with LC-MS results. This refined CTAB method provides a cost-effective, practical, and robust solution for routine HA analysis in complex matrices.