摘要
医用交联透明质酸钠凝胶难溶于水,不能直接用福林酚法和考马斯亮蓝法等现行标准中的方法测定其中蛋白质含量,鉴于此,用硫酸溶液将医用交联透明质酸钠凝胶解交联后,比较了两种方法的检测效果。结果显示,交联透明质酸钠的降解产物(D-葡萄糖醛酸、N-乙酰基-D-葡萄糖酰胺),交联剂1,4-丁二醇二环氧甘油醚的水解产物和添加剂盐酸利多卡因等会严重影响福林酚法的测定结果,因此选择了考马斯亮蓝法。取医用交联透明质酸钠凝胶约0.5 g,按样品和解交联剂质量体积比2∶1(g∶mL)加入0.5 mol·L^(-1)硫酸溶液0.25 mL,密封后在沸水浴中加热10 min。加入与上述硫酸溶液等体积的1 mol·L^(-1)氢氧化钠溶液,混匀后加入0.10 g·L^(-1)考马斯亮蓝G-250溶液5.0 mL,放置10 min。以空白牛血清白蛋白标准溶液为参比,用紫外-可见分光光度计测量上述体系在595 nm处的吸光度,用牛血清白蛋白标准溶液系列制作标准曲线。结果显示,牛血清白蛋白的质量浓度在2.04~10.22 mg·L^(-1)内与其对应的吸光度呈线性关系,检出限(3.3s/k)为1.353 mg·L^(-1)。以阴性医用交联透明质酸钠凝胶为基质进行3个浓度水平的加标回收试验,回收率为99.3%~100%,测定值的相对标准偏差(n=6)为1.3%~3.5%。
The medical cross-linked sodium hyaluronate gel was insoluble in water and could not be directly measured by current standard methods such as Lowry method and Bradford method.The determined results of medical cross-linked sodium hyaluronate gel obtained by the 2 methods of Lowry and Bradford after de-crosslinked by sulfuric acid solution were compared.It was shown that the degradation products of cross-linked sodium hyaluronate(D-glucuronic acid and N-acetyl-D-glucosamide),the hydrolysate of cross-linking agent 1,4-butanediol diglycidyl ether and additive of lidocaine hydrochloride could seriously affect the determined result of Lowry method,based on this fact,the method of Bradford was selected for determination.The medical cross-linked sodium hyaluronate gel of 0.5 g was taken,and 0.5 mol·L^(-1) sulfuric acid solution(de-crosslinking agent)of 0.25 mL was added according to the ratio of sample mass to de-crosslinking agent volume at 2∶1(g∶mL).After sealing,the sample was heated in the boiling water bath for 10 min.Then 1 mol·L^(-1) sodium hydroxide solution equal in volume to the above-mentioned sulfuric acid solution was added.After mixing well,5.0 mL of 0.10 g·L^(-1) Coomassie brilliant blue G-250 solution was added,and the mixed solution was placed for 10 min.The absorbance of the above system at 595 nm was measured by UV-Vis spectrophotometer with blank standard solution of bovine serum albumin as reference,and the standard curve was made with bovine serum albumin standard solution series.As found by results,linear relationship between values of absorbance and mass concentration of bovine serum albumin was kept in the range of 2.04-10.22 mg·L^(-1),with detection limit(3.3s/k)of 1.353 mg·L^(-1).Test for the spiked recovery was made at three concentration levels with medical cross-linked sodium hyaluronate gel as matrix,giving recoveries in the range of 99.3%-100%,and RSDs(n=6)of the determined values were in the range of 1.3%-3.5%.
作者
郭利娟
刘爱娟
孙兴霞
刘叶
陈方
沈永
GUO Lijuan;LIU Aijuan;SUN Xingxia;LIU Ye;CHEN Fang;SHEN Yong(Shandong Institute of Medical Device and Pharmaceutical Packaging Inspection,Jinan 250101,China;NMPA Key Laboratory for Safety Evaluation of Biomaterials and Medical Devices,Jinan 250101,China;Shandong Key Laboratory of Biological Evaluation for Medical Devices,Jinan 250101,China)
出处
《理化检验（化学分册）》
CAS
CSCD
北大核心
2022年第2期148-151,共4页
Physical Testing and Chemical Analysis(Part B:Chemical Analysis)
基金
国家重点研发计划项目(2016YFC1103205)。
关键词
解交联
考马斯亮蓝法
蛋白质
医用交联透明质酸钠凝胶
de-cosslinking
the method of Bradford
protein
medical cross-linked sodium hyaluronate gel
