YY/T 1805.3-2022Tissue engineering medical device products—Collagen protein - Part 3: Quantification of collagen based on marker peptide detection—Liquid chromatography-mass spectrometry (English PDF)

Foreword

This document is in accordance with the provisions of GB/T 1.1-2020 "Guidelines for Standardization Work Part 1: Structure and Drafting Rules of Standardization Documents" drafted.

This document is part 3 of YY/T 1805 "Collagen for Tissue Engineering Medical Device Products". YY/T 1805 has been released The following parts.

--- Part 2: Type I collagen molecular weight detection by sodium dodecyl sulfate polyacrylamide gel electrophoresis;

--- Part 3: Liquid chromatography-mass spectrometry for the detection of collagen content based on the determination of characteristic peptides.

Introduction

Collagen-based materials for tissue engineering medical device products include tissue-extracted collagen and genetically recombined collagen.

Collagen is widely used in the research and development and product preparation of various medical device products. Collagen-based materials have different properties, such as.

Molecular weight, purity, species, type differences, triple helix structure, cell adhesion properties, etc. These properties are directly related to the quality and use of collagen performance. YY/T 1805 "Collagen for Tissue Engineering Medical Device Products" aims to establish the detection and characterization of some special properties of collagen The method consists of four parts.

--- Part 1: Terms and Definitions. The purpose is to provide various types of collagen including tissue-extracted collagen and genetically recombinant collagen. Terms and Definitions.

--- Part 2: Type I collagen molecular weight detection by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purpose is to bring Provides a method that can be used for the determination of collagen molecular weight.

--- Part 3: Liquid chromatography-mass spectrometry for the detection of collagen content based on the determination of characteristic peptides. The purpose is to give a specific It is a liquid chromatography-mass spectrometry detection method that indirectly reflects the content of collagen by quantitative determination of characteristic peptides. The method can also be used for species Identification of source and type.

--- Part 4: Characterization of collagen helical structure. The purpose is to establish collagen structure analysis with different parameters and indicators And triple-helix structure characterization methods were used to evaluate the presence, relative content and integrity of the triple-helix structure.

The hydroxyproline method is commonly used in the detection of collagen content, and the content of collagen can be reflected by the detection of hydroxyproline content.

In this document, based on the synthesis of specific collagen-characterized peptide reference substances, the use of liquid chromatography-mass spectrometry for collagen characterization The determination of peptide content indirectly reflects the content of collagen.

1 Scope

This document specifies a method for the determination of the characteristic polypeptide content of different types of collagen by liquid chromatography-mass spectrometry.

This document is applicable to the determination of the content of characteristic polypeptides of different types of collagen in tissue-extracted and purified collagen and its collagen products. Determination.

Note. The samples of recombinant collagen and acellular matrix after collagen extraction can refer to this method for the determination of the corresponding collagen characteristic polypeptide content.

2 Normative references

The contents of the following documents constitute essential provisions of this document through normative references in the text. Among them, dated citations documents, only the version corresponding to that date applies to this document; for undated references, the latest edition (including all amendments) applies to this document.

GB/T 6682 Analysis Laboratory Water Specifications and Test Methods

YY/T 1453 Method for Characterization of Type I Collagen in Tissue Engineering Medical Device Products Pharmacopoeia of the People's Republic of China (2020 Edition)

3 Terms and Definitions

Terms and definitions defined in YY/T 1453 apply to this document.

4 Overview

The collagen samples were dissolved, heated and denatured, and then digested with trypsin to release the characteristic polypeptides, which were then determined by liquid chromatography-mass spectrometry.

Qualitative and confirmed by the retention time of different types of collagen characteristic peptides and the mass-to-charge ratio of secondary product ion fragments, using external standard method or internal standard method. standard method for quantification.

Using the synthesized different types of collagen characteristic peptides as the standard, the quantitative detection by external standard method was realized;

The isotope-labeled characteristic polypeptides and non-isotopically-labeled characteristic polypeptides in the column are mixed in a certain proportion to realize quantitative detection by the internal standard method. within the isotope The standard method helps to improve the interference of matrix effects on the accuracy of the assay.

This document takes the isotopic internal standard method as an example to give the standards for the detection of the characteristic polypeptide content of bovine type I, bovine type II and porcine type I collagen respectively.

Operating procedures and requirements. Among them, the content of bovine type I collagen characteristic polypeptides is detected according to Appendix A, and the content of bovine type II collagen characteristic polypeptides is tested.

The measurement is carried out according to Appendix B, and the content of characteristic polypeptides of porcine type I collagen is measured according to Appendix C. For different animal sources or different types Collagen can also be screened for suitable characteristic peptides for quantitative detection after verification.