北京生物医学工程

体积排阻高效液相色谱法测定酸溶性I型胶原蛋白含量的研究

Determination of acid soluble type I collagen withsize-exclusionhigh- performance liquid chromatography

作者：吴晓丽林红赛黄永富刘曦岳卫华

单位：北京市医疗器械检验所(北京 101111)

关键词：酸溶性I型胶原蛋白；胶原蛋白含量；体积排阻高效液相色谱法；磷酸钠缓冲液；优化

分类号：R318

出版年·卷·期（页码）：2020·39·2（123-130）

摘要：

目的建立酸溶性I型胶原蛋白含量测定的体积排阻高效液相色谱法（size-exclusion high- performance liquid chromatography, SE-HPLC）并进行方法确认，为快速、准确测定酸溶性I型胶原蛋白含量提供技术参考。方法通过优化流动相组成、流速及柱温等因素来改善峰形，提高柱效，确定最佳检测条件，采用含DAD检测器的Agilent 1260高效液相色谱仪进行检测，检测波长为210 nm，外标法定量并进行方法学研究。结果选择300 mmol/L磷酸钠-150 mmol/L氯化钠（pH 6.0）为流动相，设定流速为0.4 mL/min，柱温为20℃时，酸溶性I型胶原蛋白在体积排阻色谱柱Agilent Bio SEC-5（300A，4.6 mm×300 mm，5μm）(1A=0.1 nm)上的分离能获得最佳柱效。方法学研究结果表明，酸溶性I型胶原蛋白在1.5～120.0 μg/mL范围内，与峰面积呈良好的线性关系（r=0.999 9），定量限为0.6 μg/mL。用建立的方法连续进样6次，检测供试品溶液的I型胶原蛋白含量，相对标准偏差(relative standard deviation, RSD)为1.1%；加标回收率为92.8%～101.3%。结论该方法简便、快速，准确度、精密度和稳定性良好，可用于制备医疗器械产品的酸溶性I型胶原蛋白含量的测定。

Objective Development of size-exclusion high-performance liquid chromatography (SE-HPLC) for acid soluble type I collagen was established and validated. The establishment of this test method provided technical reference for rapid and accurate determination of acid soluble type I collagen content. Methods By optimizing the composition of mobile phase, flow rate and column temperature, the peak shape and column efficiency were improved, and the determination of optimum conditions for chromatographic detection were confirmed. Agilent 1260 high performance liquid chromatography equipped with a DAD detector was used for detection. The detection wavelength was 210 nm. The external standard method was used for quantitative analysis, and then the methodological study was carried out. Results The optimum column efficiency of acid soluble type I collagen was obtained in the condition of size exclusion column Agilent Bio SEC-5 (300 ?, 4.6 mm×300 mm, 5 μm)(1 ?=0.1 nm), 300 mmol/L sodium phosphate-150 mmol/L sodium chloride (pH 6.0) as the mobile phase with a flow rate of 0.4 mL/min and the column temperature of 20℃.The acid soluble type I collagen content showed good linear relationship to the peak area with a concentration range of 1.5-120.0 μg/mL (r=0.999 9). The limit of quantitation was 0.6 μg/mL. The relative standard deviation (RSD) of the results of sample solution content by the established method tested for 6 times was 1.1%. The recovery rate was 92.8% to 101.3%. Conclusions SE-HPLC for determination of acid soluble type I collagen was developed，which was simple, rapid, accurate, precise and stable, and might be suitable for the determination of the content of acid soluble type I collagen in medical products.

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