柱前衍生-高效液相色谱法测定重组Ⅲ型人源胶原蛋白修复凝胶中胶原蛋白  

Determination of collagen content in recombinant typeⅢhuman collagen repair gel by precolumn derivatization-high-performance liquid chromatography

作　　者：张晓芸 孙星宇 杨政 徐礼友 谷琴 王晓晨 丁逸梅[1,2] ZHANG Xiaoyun;SUN Xingyu;YANG Zheng;Xu Liyou;GU Qin;WANG Xiaochen;DING Yimei(Jiangsu Provincial Institute of Material Medica,Nanjing Tech University,Nanjing 210009,China;School of Pharmaceutical Sciences,Jiangsu Nanjing Tech University,Nanjing 211816,China;Jiangsu high-wit biotechnology,Nanjing 210018,China;Shangdong Center for Food and Drug Evaluation&Inspection,Jinan 250022,China)

机构地区：[1]南京工业大学江苏省药物研究所,南京210009 [2]南京工业大学药学院,南京211816 [3]江苏海智生物医药有限公司,南京210018 [4]山东食品药品审评查验中心,济南250022

出　　处：《化学分析计量》2023年第12期84-89,共6页Chemical Analysis And Meterage

摘　　要：建立柱前衍生化高效液相色谱测定重组Ⅲ型人源胶原蛋白修复凝胶中胶原蛋白含量。将样品以6 mol/L盐酸溶液在110℃下加热水解24 h,采用12 mol/L氢氧化钠溶液中和,以2,4-二硝基氟苯(DNBF)为衍生化试剂,在弱碱性环境下,于60℃反应1 h,采用高效液相色谱法检测,色谱柱为Kromasil 100-5型C18柱(250 mm×4.6 mm,5μm),柱温为30℃,流动相A相为0.05 mol/L乙酸铵溶液,B相为乙腈,梯度洗脱,流量为1.0 mL/min,进样体积为10μL,检测波长为360 nm。以脯氨酸为对照品,利用色谱峰面积外标法计算样品中脯氨酸的含量,再进行相对分子质量折算得到重组Ⅲ型人源胶原蛋白的含量。脯氨酸质量浓度在10~40μg/mL范围内线性相关系数为0.9996,低、中、高三个浓度水平平均回收率分别为96.00%、96.05%、94.98%,相对标准偏差为2.29%(n=9)。脯氨酸对照品和样品的衍生化溶液于室温放置24 h均保持稳定。该方法具有测定结果准确、专属性强等优点,可应用于重组Ⅲ型人源胶原蛋白修复凝胶中胶原蛋白含量检测。A high performance liquid chromatography(HPLC)with pre-column derivatization method was established for the quantitative determination of collagen content in recombinant typeⅢhuman collagen repair gel.the sample was hydrolyzed with 6 mol/L hydrochloric acid solution at 110℃for 24 h,and neutralized with 12 mol/L sodium hydroxide solution,then reacted with DNBF reagent at 60℃for 1 h and detected by HPLC.The chromatographic column was Kromasil 100-5 C18 Column(250 mm×4.6 mm,5μm),the column temperature was 30℃,mobile phase A was 0.05 mol/L ammonium acetate solution and mobile phase B was acetonitrile for gradient elution.Flow rate was 1.0 mL/min,injection volume was 10μL,and detection wavelength was 360 nm.Using proline as the reference substance,the content of proline in sample was calculated using external standard method of chromatographic peak area,and the molecular weight was converted to obtain the content of collagen.The chromatographic peak area of proline was linear with the mass concentration in the range of 10-40μg/mL with the correlation coefficient of 0.9996.The average recovery at low,medium,and high concentration levels were 96.00%,96.05%and 94.98%,respectively,and relative standard deviation was 2.29%(n=9).The derivatization solution of proline reference substance and test substance remained were stable within 24 h of room temperature exposure.This method has many advantages,such as scientific and reasonable,accurate determination results,and strong specificity.It can be used to detect the content of collagen in the recombinant typeⅢhuman collagen repair gel.

关 键 词：衍生化 重组Ⅲ型人源胶原蛋白 修复凝胶 脯氨酸 

分 类 号：O657.7[理学—分析化学]