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      高效液相色谱法测定重组人源胶原蛋白敷料中胶原蛋白含量

      Determination of collagen content in recombinant human collagen dressing by high performance liquid chromatography

        摘要
      • 摘要:
        目的 建立一种高效液相色谱法用于定量检测重组人源化胶原蛋白敷料中重组Ⅲ型人源化胶原蛋白的含量，对该胶原蛋白以及相关产品进行质量控制。
        方法 采用Agilent Eclipse XDB-C18色谱柱（4.6×150 nm, 5 μm）；以0.1%三氟乙酸水溶液和0.1%三氟乙酸乙腈溶液为流动相；柱温30 ℃；流速1.0 mL/min；检测波长214 nm。
        结果 该法专属性强，对重组Ⅲ型人源化胶原蛋白含量大于0.1 mg/mL以上的敷料，水解法处理后，在0.1~1.2 mg/mL的范围内其浓度与峰面积具有良好的线性关系（R2=0.9994），加标平均回收率在90.91%~92.92%，检出限和定量限分别为0.05 mg/mL、0.1 mg/mL。对重组Ⅲ型人源化胶原蛋白含量≤0.1 mg/mL以下的敷料，酶解法处理后，在0.05 mg/mL~1.0 mg/mL的范围内其浓度与峰面积具有良好的线性关系（R2=0.9 995），可以用来指导低浓度敷料蛋白的检测。
        结论 该方法快速、准确、重复性好，可以用来检测敷料中胶原蛋白的含量。

         

        Abstract:
        Objective To establish an HPLC method for the determination of type Ⅲ collagen in dressings for quality control of the product.
        Methods A Agilent Eclipse XDB-C18 column(4.6×150 nm, 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−1, the detection wavelength was 214 nm.
        Results The method was sensitive, after hydrolysis,for dressings with recombinant type Ⅲ humanized collagen content greater than 0.1mg/mL, The linear range was 0.1−1.2 mg/mL （R2=0.9 994）for type Ⅲ collagen, the average recovery was 90.91%~92.92%. The LOD and LOQ was 0.05 mg/mL and 0.1 mg/mL. After enzymatic hydrolysis, The linear range was 0.05~1.0 mg/mL （R2=0.9995）for type Ⅲ collagen, Can be used to guide 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.

         

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