《整形手术用交联透明质酸钠凝胶》标准中蛋白质含量的检测方法研究

Study on the Detection Method of Protein Content in the Standard of Cross-linked Sodium Hyaluronate Gel for Plastic Surgery

目的:建立整形手术用交联透明质酸钠凝胶产品中蛋白质含量的标准检测方法,为行业标准YY/T 0962-2014《整形手术用交联透明质酸钠凝胶》的修订提供实验依据。方法:采用酸解法处理样品,考察了酸的种类、酸解时间等影响因素,用0.5mol/L硫酸溶液且在(95±5)℃恒温干燥箱45min中将样品完全溶解,利用考马斯亮蓝G-250与蛋白质结合显色的原理,以牛血清白蛋白为对照品,采用分光光度法在波长595nm处测定样品中蛋白质的含量,且委托第三方三所实验室对方法进行验证,对数据结果进行双因子方差分析。结果:该方法专属性较高,蛋白浓度在2~10μg/ml,回归方程y=0.0125x-0.0033(r=0.9997),校正曲线的线性关系良好,样品测定重复性RSD均值为3.19%,回收率结果98.70%,检出限为0.96μg/ml,四所实验室对同批次样品进行测定,采用双因子方差分析,得到P值为0.253>0.05,认为不同实验室采用同一方法对同批次样品的蛋白测定结果不存在显著性差异。结论:该方法是一种简单、快速、准确测定整形手术用交联透明质酸钠凝胶产品中蛋白含量的检测方法。

Objective To establish a standard detection method for protein content in cross-linked sodium hyaluronate gel products for plastic surgery,and to provide experimental basis for the revision of industry standard YY/T 0962-2014 Crosslinked sodium hyaluronate gel for plastic surgery.Methods The sample was treated by acid hydrolysis method,and the influencing factors such as the type of acid and acid hydrolysis time were investigated.The sample was completely dissolved in a 0.5mol/L sulfuric acid solution and(95±5)℃constant temperature oven for 45min.The principle of blue G-250 combined with protein for color development,used bovine serum albumin as a control,used spectrophotometry to determine the protein content in the sample at a wavelength of 595nm,and entrusted three laboratories to the method perform verification and perform two-way ANOVA on the data results.Results The method has high specificity,protein concentration in the range of 2-10μg/ml,regression equation y=0.0125x-0.0033(r=0.9997),the linear relationship of the calibration curve is good,and the average RSD of the sample measurement repeatability was 3.19%,the recovery rate was 98.70%,and the detection limit was 0.96μg/ml.The four laboratories measured the same batch of samples and used a two-factor analysis of variance to obtain P=0.253>0.05.It was considered that there was no significant difference in the protein determination results of the same batch of samples by the same method in different laboratories.Conclusion This method is a simple,rapid and accurate method for determining the protein content of cross-linked sodium hyaluronate gel products for plastic surgery.