ISO 6631:2025

International
Standard
ISO 6631
First edition
Tissue-engineered medical
2025-11
products — Quantification of bovine
type I collagen marker peptide with
liquid chromatography — Tandem
mass spectrometry
Produits médicaux issus de l'ingénierie tissulaire —
Quantification du marqueur peptidique du collagène de type I
bovin par chromatographie en phase liquide — Spectrométrie de
masse en tandem
Reference number
© ISO 2025
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2  Normative references . 1
3  Terms and definitions . 1
4  Principles and significances . 2
4.1 Principles .2
4.2 Significances .2
5 Reagent and apparatus . 2
5.1 Reagents and preparation .2
5.2 Apparatuses .4
6 Experimental procedures . 4
6.1 Sample preparation .4
6.1.1 Test Samples .4
6.1.2 Recovery control sample (RCS) .4
6.2 Denaturation treatment .4
6.2.1 Solid sample .4
6.2.2 Liquid sample .5
6.2.3 RCS .5
6.3 Trypsin digestion .5
6.4 LC-MS/MS detection .5
6.4.1 LC-MS/MS operating conditions .5
6.4.2 Qualitative and quantitative detection .7
7 Data collection and calculation . 7
7.1 Standard curve .7
7.2 Result calculation .7
8  Acceptable criteria of experimental result . 8
9 Test report . 8
Bibliography .10

iii
Foreword
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This document was prepared by Technical Committee ISO/TC 150, Implants for surgery, Subcommittee SC 7,
Tissue-engineered medical products.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
Introduction
Collagen is a primary biopolymer in the extracellular matrix of mammals, and provides a scaffold for cell
attachment and migration, as well as specific mechanical properties. It is prevalent in tendons, skin, blood
vessel, cornea and coordinates with minerals to construct bones, teeth and cartilage. The collagen molecule
is formed by three chains building a triple helix. According to their amino sequences and structures, collagen
can be categorized into different types. So far, at least 28 different types of collagen have been identified in
vertebrates. The different collagen types are characterized by considerable complexity and diversity in their
structure, their splice variants, and the presence of additional non-helical domains, their assembly and their
function. The most abundant and widespread family of collagens with about 90 % of the total collagen is
[4,5]
represented by the fibril-forming collagens, among which type I is the most common fibrillar collagen.
The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and
weak antigenicity, made collagen one of the most useful biomaterials. It was widely used as raw materials
for constructing tissue-engineered medical products (TEMPs) for tissue regeneration, reconstruction
or replacement, such as skin graft, bone substitutes, and artificial blood vessels and valves, etc. Collagen
based wound dressing include hydrogel, haemostatic sponges/powder, membranes and film. The main
applications of collagen as drug delivery systems are collagen shields in ophthalmology, sponges for burns/
wounds, mini-pellets and tablets for protein delivery, gel formulation in combination with liposomes
for sustained drug delivery, as controlling material for transdermal delivery, and nanoparticles for gene
delivery and basic matrices for cell culture systems. To improve collagen physical and chemical properties,
[6]
chemical modification, crosslinking, or hybridizing with synthetic polymer are also used. Collagen-based
biomaterials are expected to become a widely used matrix substance for various biomedical applications in
the future. Therefore, quality control of collagens is of great importance from safety, regulatory, and process
control perspectives. The specific peptide content of collagen products, is important parameter that related
to collagen purity, identification (whether is bovine specific or/and type I collagen-specific) and is useful for
quality control of collagen.
Among all the collagen types, type I collagen is the most common used in the fields of TEMPs and regenerative
medicine. Type I collagen is normally a heterotrimeric molecule, composed of two α1 (I) chains and one α2
(I) chain. Most collagens with native states are insoluble in water. Collagen extracted from different animal
sources shows similarity in structure and properties. Thus, it is difficult to qualify a particular type or
animal source (species) of collagen. To date, several ways for quantifying collagen and/or collagen types
or even particular collagen chains have been reported, such as the quantification of hydroxyproline, which
accounts for approximately 10 % of the collagen molecule, or a method based on a spectrophotometric
assay of collagen stained by a strong anionic dye in picric acid solution. However, since hydroxyproline is a
common amino acid in all types of collagen, the hydroxyproline quantification method shows low specificity.
Other methods include the radioactive labelling of proline and enzyme immunoassays using antibodies
specific to each collagen type, which are very expensive and require many complicated steps, or not fit for
some formulations of collagen-containing biomaterials. Any type of collagen has its particular amino acid
sequences in their chains, which are different from other types from the same animal. Same type collagen
from different species of animals also shows different sequences in their chains. Thus, the sequential
differences can be used for characterization of its type or animal source. Enzymatic digestion of collagen
coupled with various separation methods such as liquid chromatography (LC), will be able to achieve some
marker reference peptides, which is particular for a specific-type and/or specific-animal sources collagen.
Marker peptides can be used for qualitative and quantitative analysis of the original collagen with mass
[7]
spectrometry.
Qualitative identification and quantification of type I collagen using mass spectrometry has been applied in
many fields, including quantification of collagen in some tissues, identification of animal source of gelatins,
[8-10]
or metabolic kinetic analysis in vivo of collagen-containing medicine. The method for determination of
donkey collagen
...