北京生物醫(yī)學(xué)工程

體積排阻高效液相色譜法測定酸溶性I型膠原蛋白含量的研究

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

作者：吳曉麗林紅賽黃永富劉曦岳衛(wèi)華

單位：北京市醫(yī)療器械檢驗所(北京 101111)

關(guān)鍵詞：酸溶性I型膠原蛋白；膠原蛋白含量；體積排阻高效液相色譜法；磷酸鈉緩沖液；優(yōu)化

分類號：R318

出版年·卷·期（頁碼）：2020·39·2（123-130）

摘要：

目的建立酸溶性I型膠原蛋白含量測定的體積排阻高效液相色譜法（size-exclusion high- performance liquid chromatography, SE-HPLC）并進行方法確認(rèn)，為快速、準(zhǔn)確測定酸溶性I型膠原蛋白含量提供技術(shù)參考。方法通過優(yōu)化流動相組成、流速及柱溫等因素來改善峰形，提高柱效，確定最佳檢測條件，采用含DAD檢測器的Agilent 1260高效液相色譜儀進行檢測，檢測波長為210 nm，外標(biāo)法定量并進行方法學(xué)研究。結(jié)果選擇300 mmol/L磷酸鈉-150 mmol/L氯化鈉（pH 6.0）為流動相，設(shè)定流速為0.4 mL/min，柱溫為20℃時，酸溶性I型膠原蛋白在體積排阻色譜柱Agilent Bio SEC-5（300A，4.6 mm×300 mm，5μm）(1A=0.1 nm)上的分離能獲得最佳柱效。方法學(xué)研究結(jié)果表明，酸溶性I型膠原蛋白在1.5～120.0 μg/mL范圍內(nèi)，與峰面積呈良好的線性關(guān)系（r=0.999 9），定量限為0.6 μg/mL。用建立的方法連續(xù)進樣6次，檢測供試品溶液的I型膠原蛋白含量，相對標(biāo)準(zhǔn)偏差(relative standard deviation, RSD)為1.1%；加標(biāo)回收率為92.8%～101.3%。結(jié)論該方法簡便、快速，準(zhǔn)確度、精密度和穩(wěn)定性良好，可用于制備醫(yī)療器械產(chǎn)品的酸溶性I型膠原蛋白含量的測定。

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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