生物学杂志

生物学杂志 ›› 2026, Vol. 43 ›› Issue (3): 95-.doi: 10.3969/j.issn.2095-1736.2026.03.095

• 技术方法 • 上一篇    下一篇

基于液质联用的皮内注射重组胶原蛋白检测方法研究

高建萍1， 凡孝菊2， 储　筠2， 翟源心2， 王欣慧2， 闫昊前1， 乐　浩2，汤　鼎2， 钱　松2， 张贵锋1
  

  1. 1. 中国科学院过程工程研究所 生物药制备与递送全国重点实验室， 北京 100190；
    2. 江苏创健医疗科技股份有限公司， 常州 213200
  • 出版日期:2026-06-18 发布日期:2026-06-16
  • 通讯作者: 张贵锋，博士，研究员，研究方向为生物材料，E-mail:gfzhang@ipe.ac.cn
  • 作者简介:高建萍，博士，工程师，研究方向为生物材料，E-mail:jpgao@ipe.ac.cn
  • 基金资助:
    北京市自然科学基金-昌平创新联合基金资助项目（L234071， L234021）； 常州市科技计划-领军型创新人才引进培育项目A类（CQ20230004）； 常州市科技计划-领军型创新人才引进培育项目B类（CQ20230005）； 常州市科技计划-科技支撑计划项目（CE20235002）

#br# Study on detection method of recombinant collagen by internal injection based on HPLC-MS

GAO Jianping1, FAN Xiaoju2, CHU Yun2, ZHAI Yuanxin2, WANG Xinhui2, YAN Haoqian1,LE Hao2, TANG Ding2, QIAN Song2, ZHANG Guifeng1   

  1. 1. State Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering,
    Chinese Academy of Sciences, Beijing 100190, China; 2. Jiangsu Trautec Medical Technology Co., Ltd.,
    Changzhou 213200, China
  • Online:2026-06-18 Published:2026-06-16

摘要： 本文以重组Ⅲ型胶原蛋白为研究对象，通过液质联用与酶解相结合的方法识别重组Ⅲ型胶原蛋白的特征多肽，并以特征多肽作为定量标准品建立重组Ⅲ型胶原的定量检测方法，将该方法应用于大鼠皮内注射的重组Ⅲ型胶原残留量的定量检测。结果表明，重组Ⅲ型胶原蛋白的特征多肽为GESGPAGPAGAPGPAGSK，该多肽在0.1~200 μg/mL质量浓度范围内线性关系良好，相关系数均大于0.99，重复性和精密度的相对标准偏差分别为2.65%和4.89%，加标回收率均在96%~118%，特征多肽的定量限和检出限分别为0.5 ng/mL和0.05 ng/mL。大鼠皮内注射5 h后皮肤中残留的重组Ⅲ型胶原蛋白含量在0.79~3.94 μg/mg。该方法精密度高、重现性好、灵敏度高，不受动物体内本身胶原的影响，可实现在大鼠体内注射后残留的重组Ⅲ型胶原的定量检测。

关键词: 液质联用, 重组胶原蛋白, 皮内注射, 特征多肽

Abstract: In this study, recombinant type Ⅲ collagen was used as the subject of this study. The marker peptide was digested by trypsin and identified with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This peptide was then used as a quantitative reference standard to establish the quantification method. Subsequently, the method was applied to quantify the residual amounts of recombinant type Ⅲ collagen (rhCOL-Ⅲ) following intradermal injection in rats. The results indicated that the marker peptide of rhCOL-Ⅲ was GESGPAGPAGAPGPAGSK. The peptide showed good linearity in the mass concentration range of 0.1－200 μg/mL, with a correlation coefficient over 0.99. The relative standard deviation (RSD) of reproducibility and precision were 2.65% and 4.89%, respectively. The spiked recoveries were all within the range of 96%－118%. The limit of quantification (LOQ) and the limit of detection (LOD) for the marker peptide were 0.5 ng/mL and 0.05 ng/mL, respectively. After 5 hours of intradermal injection, the concentration of rhCOL-Ⅲ remaining in the skin ranged from 0.79 μg/mg to 3.94 μg/mg. The method had high precision, good reproducibility and high sensitivity. Furthermore, it is unaffected by the animal’s endogenous collagen. This method can be used for the quantitative detection of rhCOL-Ⅲ remaining afterin vivoinjection in rats.

Key words: HPLC-MS/MS, recombinant collagen, internal injection, marker peptide

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