Objective  To establish a highly specific and accurate method for determining collagen content in recombinant collagen dressings, addressing challenges from complex matrices and severe excipient interference.

Methods A method combining acid hydrolysis, pre-column derivatization with phenylisothiocyanate, and reversed-phase high-performance liquid chromatography was developed. Glycine and proline were detected at 254 nm, and collagen content was calculated based on the stoichiometric relationship between amino acids and protein. Validation included specificity, linearity, precision, accuracy, limit of detection (LOD), limit of quantitation (LOQ), and robustness.

Results Both glycine and proline exhibited excellent linearity in the range of 0.10–1.00 μmol/mL (R² > 0.999). Repeatability relative standard deviations (RSDs) were 0.8% for both amino acids, while intermediate precision RSDs were 2.1% and 1.2%, respectively. Spike recovery ranged from 92.4% to 100.6%. The LOQ and LOD are 0.22 mg/g and 0.03 mg/g respectively. Target amino acids were detected in all four commercially available recombinant collagen dressings and the accuracy was all above 80%, the negative control sample did not show any detection.

Conclusion This method demonstrates high specificity, sensitivity, accuracy, and strong anti-interference capability, providing a reliable technical platform for quality control of recombinant collagen in complex medical device matrices.