Determination of Collagen Content in Recombinant Human Collagen Dressing by High Performance Liquid Chromatography

Objective To establish a high performance liquid chromatography method for the determination of recombinant type Ⅲ human collagen in recombinant human collagen dressing for quality control of the product.

Methods  HPLC was utilized, and its parameters were as follows: an Agilent Eclipse XDB-C18 column (4.6×150 mm, 5 μm) was used with 0.1% TFA solution and 0.1% TFA acetonitrile solution as mobile phase at 30 ℃, the flow rate was 1.0 mL/min, and the detection wavelength was 214 nm.

Results This method demonstrates high specificity. For dressings with a recombinant type Ⅲ human collagen content greater than 0.1 mg/mL, after hydrolysis treatment, a good linear relationship between concentration and peak area was observed within the range of 0.1 to 1.2 mg/mL (R²=0.9994). The average spiked recovery ranged from 90.91% to 94.14%, with a detection limit (LOD) of 0.05 mg/mL and a quantification limit (LOQ) of 0.1 mg/mL. For dressings with a recombinant type Ⅲ human collagen content of 0.1 mg/mL or less, after enzymatic digestion treatment, a good linear relationship between concentration and peak area was obtained within the range of 0.05 to 1.0 mg/mL (R²=0.9995). This validates the method’s suitability for guiding the detection of low-concentration dressing proteins.

Conclusion The method is rapid, accurate and reproducible, and can be used to determine the content of type Ⅲ collagen in dressings.