Textiles - Qualitative and quantitative proteomic analysis of some animal hair fibres - Part 3: Peptide detection using LC-MS without protein reduction (ISO 20418-3:2020)

This document specifies a qualitative and quantitative procedure to determine the composition of
animal hair fibre blends (made of wool, cashmere, yak, alpaca, camel or angora) by LC-MS without
protein reduction.
NOTE 1 The composition of non-animal hair fibres can be measured by ISO 1833 (all parts). Both results are
combined to determine the total fibre composition.
The method is based on a preliminary identification, by light microscopy, of all fibres in the blend on the
basis of their morphology, according to ISO/TR 11827[4]. It is not applicable if fibres of the same animal
species (such as blends of cashmere and mohair) are present.
NOTE 2 In this case, the quantitative analysis is performed using microscopical analysis [for example,
ISO 17751 (all parts)].

Textilien - Qualitative und quantitative Proteomanalyse einiger Tierhaarfasern - Teil 3: Peptiddetektion mit LC-MS ohne Proteinreduktion (ISO 20418-3:2020)

Dieses Dokument legt ein qualitatives und quantitatives Verfahren zur Bestimmung der Zusammensetzung von Tierhaarfasermischungen (aus Wolle, Kaschmir, Yak, Alpaka, Kamel oder Angora) mittels LC MS ohne Proteinreduktion fest.
ANMERKUNG 1 Die Zusammensetzung von Haarfasern nichttierischen Ursprungs kann mithilfe von ISO 1833 (alle Teile) bestimmt werden. Beide Ergebnisse werden kombiniert, um die Gesamtfaserzusammensetzung zu bestimmen.
Das Verfahren beruht auf einer Vorabidentifizierung, mittels Lichtmikroskopie, aller Fasern in der Mischung auf der Grundlage ihrer Morphologie nach ISO/TR 11827 [4]. Es ist nicht anzuwenden, wenn Fasern von der gleichen Tierart vorliegen (wie zum Beispiel Mischungen aus Kaschmir und Mohair).
ANMERKUNG 2 In diesem Fall wird die quantitative Analyse mittels mikroskopischer Analyse durchgeführt [zum Beispiel ISO 17751 (alle Teile)].

Textiles - Analyse protéomique qualitative et quantitative de certaines fibres animales - Partie 3: Détection des peptides par LC-MS sans réduction protéique (ISO 20418-3:2020)

Le présent document spécifie une méthode qualitative et quantitative pour déterminer la composition des mélanges de fibres animales ? poils - (composés de laine, de cachemire, de yack, d'alpaga, de chameau ou d'angora) par LC-MS sans réduction protéique.
NOTE 1 La composition des fibres autres que les poils animaux peut être mesurée conformément à l'ISO 1833 (toutes les parties). Les résultats de ces deux méthodes sont ensuite combinés pour déterminer la composition globale des fibres.
La méthode repose sur une identification préliminaire, par microscopie optique, de toutes les fibres présentes dans le mélange sur la base de leur morphologie, conformément à l'ISO/TR 11827[4]. Elle n'est pas applicable si des fibres de la même espèce animale (telles que des mélanges de cachemire et de mohair) sont présentes.
NOTE 2 Dans ce cas, l'analyse quantitative est réalisée par microscopie [tel que décrit dans l'ISO 17751 (toutes les parties), par exemple].

Tekstilije - Kvalitativna in kvantitativna proteomska analiza nekaterih živalskih vlaken - 3. del: Odkrivanje peptida z uporabo LC-MS brez zmanjšanja proteina (ISO 20418-3:2020)

General Information

Status
Published
Public Enquiry End Date
29-Sep-2019
Publication Date
10-Aug-2020
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
15-Jul-2020
Due Date
19-Sep-2020
Completion Date
11-Aug-2020

Buy Standard

Standard
SIST EN ISO 20418-3:2020
English language
32 pages
sale 10% off
Preview
sale 10% off
Preview

e-Library read for
1 day
Draft
oSIST prEN ISO 20418-3:2019
English language
30 pages
sale 10% off
Preview
sale 10% off
Preview

e-Library read for
1 day

Standards Content (sample)

SLOVENSKI STANDARD
SIST EN ISO 20418-3:2020
01-september-2020

Tekstilije - Kvalitativna in kvantitativna proteomska analiza nekaterih živalskih

vlaken - 3. del: Odkrivanje peptida z uporabo LC-MS brez zmanjšanja proteina (ISO

20418-3:2020)

Textiles - Qualitative and quantitative proteomic analysis of some animal hair fibres -

Part 3: Peptide detection using LC-MS without protein reduction (ISO 20418-3:2020)

Textilien - Qualitative und quantitative Proteomanalyse einiger Tierhaarfasern - Teil 3:

Peptiddetektion mit LC-MS ohne Proteinreduktion (ISO 20418-3:2020)

Textiles - Analyse protéomique qualitative et quantitative de certaines fibres animales -

Partie 3: Détection des peptides par LC-MS sans réduction protéique (ISO 20418-
3:2020)
Ta slovenski standard je istoveten z: EN ISO 20418-3:2020
ICS:
59.060.01 Tekstilna vlakna na splošno Textile fibres in general
SIST EN ISO 20418-3:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN ISO 20418-3:2020
---------------------- Page: 2 ----------------------
SIST EN ISO 20418-3:2020
EN ISO 20418-3
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2020
EUROPÄISCHE NORM
ICS 59.060.01
English Version
Textiles - Qualitative and quantitative proteomic analysis
of some animal hair fibres - Part 3: Peptide detection using
LC-MS without protein reduction (ISO 20418-3:2020)

Textiles - Analyse protéomique qualitative et Textilien - Qualitative und quantitative

quantitative de certaines fibres animales - Partie 3: Proteomanalyse einiger Tierhaarfasern - Teil 3:

Détection des peptides par LC-MS sans réduction Peptiddetektion mit LC-MS ohne Proteinreduktion (ISO

protéique (ISO 20418-3:2020) 20418-3:2020)
This European Standard was approved by CEN on 7 June 2020.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20418-3:2020 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN ISO 20418-3:2020
EN ISO 20418-3:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

---------------------- Page: 4 ----------------------
SIST EN ISO 20418-3:2020
EN ISO 20418-3:2020 (E)
European foreword

This document (EN ISO 20418-3:2020) has been prepared by Technical Committee ISO/TC 38

"Textiles" in collaboration with Technical Committee CEN/TC 248 “Textiles and textile products” the

secretariat of which is held by BSI.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by January 2021, and conflicting national standards shall

be withdrawn at the latest by January 2021.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 20418-3:2020 has been approved by CEN as EN ISO 20418-3:2020 without any

modification.
---------------------- Page: 5 ----------------------
SIST EN ISO 20418-3:2020
---------------------- Page: 6 ----------------------
SIST EN ISO 20418-3:2020
INTERNATIONAL ISO
STANDARD 20418-3
First edition
2020-06
Textiles — Qualitative and
quantitative proteomic analysis of
some animal hair fibres —
Part 3:
Peptide detection using LC-MS without
protein reduction
Textiles — Analyse protéomique qualitative et quantitative de
certaines fibres animales —
Partie 3: Détection des peptides par LC-MS sans réduction protéique
Reference number
ISO 20418-3:2020(E)
ISO 2020
---------------------- Page: 7 ----------------------
SIST EN ISO 20418-3:2020
ISO 20418-3:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 20418-3:2020
ISO 20418-3:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Symbols and abbreviated terms ........................................................................................................................................................... 2

5 Principle ........................................................................................................................................................................................................................ 2

6 Reagents ........................................................................................................................................................................................................................ 3

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Test method ............................................................................................................................................................................................................... 4

8.1 Sampling ....................................................................................................................................................................................................... 4

8.2 Preliminary identification ............................................................................................................................................................. 4

8.3 Wash for degreasing ........................................................................................................................................................................... 4

8.4 Powderization of fibres ................................................................................................................................................................... 4

8.5 Trypsin digestion .................................................................................................................................................................................. 4

8.6 Marker peptides ..................................................................................................................................................................................... 5

8.7 LC-MS analysis ......................................................................................................................................................................................... 5

8.8 Evaluation of the validity of observed data .................................................................................................................... 5

8.9 Calculation of correction factor ................................................................................................................................................ 5

8.9.1 Correction factor .............................................................................................................................................................. 5

8.9.2 Correction factor for Cas1 against She1 and Yak1 (kc)................................................................... 6

8.9.3 Correction factor for Yak2 against She2, She3 and Cas2 (ky) ................................................... 6

8.9.4 Correction factor for alpaca and camel (ka) ............................................................................................. 6

8.9.5 Correction factor for angora (kr) ....................................................................................................................... 6

8.10 Calculation of blending ratio ....................................................................................................................................................... 6

8.10.1 Calculation of blending ratio of cashmere, sheep wool and yak ............................................. 6

8.10.2 Calculation by She1, Cas1, Yak1 and kc ........................................................................................................ 7

8.10.3 Calculation by She2, She3, Cas2, Yak2 and ky ......................................................................................... 7

8.10.4 Calculation by She1, She2, She3, Cas2, Yak1 ............................................................................................ 7

8.10.5 Calculation of blending ratio of camel, alpaca and angora .......................................................... 8

9 Test report ................................................................................................................................................................................................................... 9

Annex A (informative) Marker peptides of cashmere, sheep wool and yak fibres ............................................10

Annex B (informative) Example of LC-MS analysis conditions ...............................................................................................13

Annex C (informative) Analysis of camel, alpaca and angora fibres .................................................................................16

Annex D (informative) Example of marker peptide mass chromatograms ...............................................................19

Annex E (informative) Results of interlaboratory study ...............................................................................................................23

Bibliography .............................................................................................................................................................................................................................24

© ISO 2020 – All rights reserved iii
---------------------- Page: 9 ----------------------
SIST EN ISO 20418-3:2020
ISO 20418-3:2020(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 38, Textiles, in collaboration with the

European Committee for Standardization (CEN) Technical Committee CEN/TC 248, Textiles and textile

products, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna

Agreement).
A list of all parts in the ISO 20418 series can be found on the ISO website.

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 © ISO 2020 – All rights reserved
---------------------- Page: 10 ----------------------
SIST EN ISO 20418-3:2020
ISO 20418-3:2020(E)
Introduction

Cashmere is a long slender fibre obtained from cashmere goats and is expensive because of its high

quality and rarity. Mislabelling or adulteration of cashmere products blended with other cheaper

animal fibres such as sheep wool and yak have been repeatedly reported worldwide.

Current official methods to identify specific animal fibres are based on microscopic observations.

However, the microscopy-based identification is becoming increasingly difficult due to a wider use

of chemical or physical treatments in the manufacturing process. Given these issues, several other

methods have also been studied either to distinguish fibre structures by the use of near-infrared

spectroscopy or terahertz spectroscopy, or to distinguish DNA sequences by the use of polymerase

chain reaction. Nevertheless, each method has shown some complications when applied. Therefore, it is

required to develop novel identification methods.

Animal fibres consist mainly of proteins called keratins and some associated proteins. Therefore, the

most promising methods to identify fibres are based on the analysis of proteins contained in textiles.

Commonly, proteins are analysed by being subjected to digestion by trypsin, resulting in smaller

molecules, i.e. peptides, which will be later characterized through mass spectrometry. Accordingly,

identification methods using either matrix-assisted laser desorption/ionization time-of-flight mass

spectrometer or liquid chromatography/mass spectrometer (LC-MS) have been studied. When

comparing these options, the latter type of instrument is less expensive and more readily available in

testing laboratories as a versatile analytical instrument than the former. Moreover, LC-MS has a high

quantitative capability, and is therefore preferable to calculate the blending ratio of animal fibres.

Keratins are highly insoluble due to the disulphide bonds they tend to form, both at an intramolecular

as well as at an intermolecular level. Thus, keratins are generally extracted in the presence of reducing

agents. However, this reducing step is considered as time-consuming and arduous. In this document,

an alternative method in which cysteine-free peptides are selected for identification markers is used,

thereby eliminating the need of the reducing step and enabling rapid preparation of LC-MS samples.

Both ISO 20418-1 and this document describe procedures using LC-MS, but they differ regarding the

method utilized to extract the peptides. In ISO 20418-1, proteins are first extracted from fibres with

a thiourea/urea/dithiothreitol (DTT) solution, and then digested by trypsin to obtain peptides. In the

process described here, peptides are directly extracted by trypsin digestion of mechanically powdered

fibres. The method has been shown to be useful even for highly processed samples and is applicable to

various types of animal hairs such as goat (cashmere or mohair), wool and yak.
© ISO 2020 – All rights reserved v
---------------------- Page: 11 ----------------------
SIST EN ISO 20418-3:2020
---------------------- Page: 12 ----------------------
SIST EN ISO 20418-3:2020
INTERNATIONAL STANDARD ISO 20418-3:2020(E)
Textiles — Qualitative and quantitative proteomic analysis
of some animal hair fibres —
Part 3:
Peptide detection using LC-MS without protein reduction
1 Scope

This document specifies a qualitative and quantitative procedure to determine the composition of

animal hair fibre blends (made of wool, cashmere, yak, alpaca, camel or angora) by LC-MS without

protein reduction.

NOTE 1 The composition of non-animal hair fibres can be measured by ISO 1833 (all parts). Both results are

combined to determine the total fibre composition.

The method is based on a preliminary identification, by light microscopy, of all fibres in the blend on the

[4]

basis of their morphology, according to ISO/TR 11827 . It is not applicable if fibres of the same animal

species (such as blends of cashmere and mohair) are present.

NOTE 2 In this case, the quantitative analysis is performed using microscopical analysis [for example,

ISO 17751 (all parts)].
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 1833-1, Textiles — Quantitative chemical analysis — Part 1: General principles of testing

ISO 3696, Water for analytical laboratory use — Specification and test methods

ISO 17751 (all parts), Textiles — Quantitative analysis of cashmere, wool, other specialty animal fibers and

their blends
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
animal hair fibre

type of keratin fibre for textile use, such as wool, cashmere, yak, alpaca, camel or angora

3.2
Bovidae

biological family of cloven-hoofed, ruminant mammals including cashmere goat, sheep and yak

© ISO 2020 – All rights reserved 1
---------------------- Page: 13 ----------------------
SIST EN ISO 20418-3:2020
ISO 20418-3:2020(E)
3.3
Camelidae
biological family of even-toed ungulate mammals including camel and alpaca
3.4
protein
polymer of amino acids that play many critical roles in the body
3.5
peptide
small protein (3.4) consisting of approximately less than 50 amino acids
3.6
marker peptide

portion of a protein (3.4) used for its identification, recovery and purification

3.7
mass chromatogram
chromatogram for a specific mass-to-charge ratio
3.8
total ion chromatogram
TIC

chromatogram with each data point created by summing up intensities of all mass spectral peaks

belonging to the same scan
3.9
selected ion monitoring
SIM

mass spectrometry scanning mode in which only a limited m/z range is transmitted/detected by the

instrument
4 Symbols and abbreviated terms
A peak area
W blending ratio
Br Bovidae rate
Cr Camelidae rate
ka correction factor for alpaca and camel
kc correction factor for cashmere
kr correction factor for angora
ky correction factor for yak

m/z mass to charge ratio, where m is the mass, expressed in atomic mass unit, and z is the charge

number of ions
5 Principle

The mechanically powdered fibres are directly subjected to trypsin digestion without prior reduction.

The analysis of the digested peptides is performed with LC-MS. The percent composition is calculated

from the peak areas of the species-specific marker peptides.
2 © ISO 2020 – All rights reserved
---------------------- Page: 14 ----------------------
SIST EN ISO 20418-3:2020
ISO 20418-3:2020(E)
6 Reagents
The following analytical grade reagents shall be used.
6.1 Acetone, with purity greater than or equal to 99,5 %.
6.2 Water, grade 3 quality specified in ISO 3696.
6.3 Ammonium hydrogen carbonate (NH HCO ) solution (25 mmol/l)
4 3
— 197,5 mg of NH HCO , with purity greater than or equal to 96,0 %.
4 3
— Make up 100 ml by adding water (6.2).
6.4 Trypsin, sequencing-grade porcine trypsin modified by reductive methylation.
6.5 Acetonitrile, with purity greater than or equal to 99,8 %.
6.6 Formic acid, with purity greater than or equal to 98 %.
6.7 Trypsin solution
— Trypsin (6.4) 20 μg.
— 0,1 % formic acid (6.6) 200 μl.
7 Apparatus
The usual laboratory apparatus and, in particular, the following.

7.1 Heating mantle, capable of operating at a temperature range of 50 °C to 150 °C.

7.2 Mill, beads mill, cryogenic grinder or an equivalent, capable of crushing materials into an extremely

fine powder.
7.3 Membrane filter, for aqueous solutions, with a pore size of 0,45 µm.
7.4 Heat block, capable of heating microtubes at 37 °C.
7.5 Tube mixer, capable of vortex microtubes and LC vials for about 30 min.
7.6 Centrifugal evaporator, capable to deliver 5 000 g.

7.7 LC-MS, liquid chromatography–mass spectrometer, capable of detecting m/z (u) range from

200 m/z (u) to 1 500 m/z (u).
NOTE (u) is for unified atomic mass unit, SI unit.
7.8 LC vial, shall be glass or polymethylpentane.
7.9 LC column, octadecyl (C-18)-silica reversed phase column.
7.10 Balance, with a resolution of at least 0,001 g.
© ISO 2020 – All rights reserved 3
---------------------- Page: 15 ----------------------
SIST EN ISO 20418-3:2020
ISO 20418-3:2020(E)
7.11 Recovery flask (eggplant flask or round-bottom flask).
8 Test method
8.1 Sampling

The general requirement is that the test specimen shall be representative for the lot of material from

which it is taken. The method to obtain a fibre test specimen differs depending on the sample form. The

terms relating to sampling for the various types of samples shall be in accordance with ISO 1833-1.

8.2 Preliminary identification

The preliminary qualitative analysis of the animal hair fibre shall be carried out based on their

morphology, which is determined using light microscopy, according to ISO 17751 (all parts), after

removal of non-animal fibre.
8.3 Wash for degreasing

8.3.1 Reflux 1 g of the fibres in a recovery flask (7.11) on a heating mantle (7.1) with 200 ml of acetone

(6.1) for 30 min. This washing step may be omitted in the case of clean samples. Quantity of the fibres

can be changed.

8.3.2 Take the degreased fibres out of the recovery flask and dry them in the air. Alternatively, the

[5]
sample preparation method of ISO 20418-1 can be used.
8.4 Powderization of fibres

Crush the dried fibre sample (8.3.2) using a mill (7.2) to get a fine powder with an average length of

100 µm or less by checking under the microscope and mix thoroughly for securing representative

sampling of the fibres.
8.5 Trypsin digestion

8.5.1 Weigh about 10 mg of the crushed sample and place it into a microtube. If more than 10 mg of

the sample is used, increase the volumes of the NH HCO solution in 8.5.2 and Trypsin solution in 8.5.3

4 3
proportionally.
8.5.2 Add 300 µl of the NH HCO solution (6.3) and vortex for 10 min to 30 min.
4 3

8.5.3 Add 10 µl of the Trypsin solution (6.7) to the sample and incubate at 37 °C for 20 h to 24 h.

8.5.4 Centrifuge the tryptic solution for 3 min using the centrifugal evaporator (7.6). Filter the

supernatant through a membrane filter (7.3) to remove residual fibres.

NOTE Centrifugal filter, syringe filter or other means of filtration can be used.

8.5.5 Transfer the solution to an LC vial, then dry it using the centrifugal evaporator (7.6). If the LC vial

does not fit in the dryer, the solution can be dried in other types of container such as a microtube. The

sample is transferred to an LC vial after dissolution. Alternatively, a freeze dryer or nitrogen flux can be

used as the drying method, instead of the centrifugal evaporator.

8.5.6 Add 40 µl of water containing 0,1 % formic acid and 5 % acetonitrile and vortex for 30 min, for

subsequent LC-MS measurements. Sonication shall not be a substitute for vortex when LC-MS sample is

dissolved.
4 © ISO 2020 – All rights reserved
---------------------- Page: 16 ----------------------
SIST EN ISO 20418-3:2020
ISO 20418-3:2020(E)
8.6 Marker peptides

8.6.1 Select the peptides which are used as markers for differential identification of fibres, as specified

in Annex A and Annex C. The result of the preliminary identification by microscopy (8.2) and Table 1 can

be used as references for this selection.
Table 1 — Correlation of marker peptides and identifiable animal taxa
Species Family Class
Fibre
Name Marker Name Marker Name Marker
She1,
Sheep wool Ovis aries (She2,
She3)
Cashmere/ Cas1, Bovidae Fbv1
Capra hircus
mohair (Cas2)
Yak1,
Yak Bos grunniens
(Yak2)
Mammalia Cmm1
Alp1,
Alpaca Vicugna pacos
(Alp2)
Camelidae Fcm1
Cam1,
Camel Camelus ferus
(Cam2)
Oryctolgus Ang1,
Angora rabbit Leporidae —
cuniculus (Ang2)

Marker peptides with suffix 1 are preferable when conducting quantitative analysis.

8.6.2 Optimize LC-MS parameters and confirm retention times of target peaks by using either

synthesized peptides (with amino acid sequences shown in Annex A and Annex C) or peptides extracted

from pure animal hair fibre samples.
8.7 LC-MS analysis

8.7.1 Inject 5 µl of the sample onto an LC column (7.9). Use water containing 0,1 % formic acid and

acetonitrile containing 0,1 % formic acid to form a gradient with increasing concentration of acetonitrile

for chromatography. The initial concentration of acetonitrile is 5 %. An example of LC parameters is

indicated in Annex B.

8.7.2 Operate mass spectrometer in SIM mode. The selected markers (preferably those with suffix

1), which are described in Annex A and Annex C, shall be monitored. An example of MS parameters is

indicated in Annex B.

8.7.3 Integrate the peak area of each marker peptide. The peaks of additional marker peptides (those

with suffix 2 and 3), which are also described in Annex A and Annex C, can be used when it is difficult to

use the target peak.
8.8 Evaluation of the validity of observed data
See Annex C.
8.9 Calculation of correction factor
8.9.1 Correction factor

Peak areas of marker peptides are not expected to be proportional to the amounts of corresponding

animal fibres in the following combinations of marker peptides, for reasons such as the difference in

© ISO 2020 – All rights reserved 5
---------------------- Page: 17 ----------------------
SIST EN ISO 20418-3:2020
ISO 20418-3:2020(E)

the protein species from which each marker peptide is derived (see 8.9.2). In these cases, a correction

factor for a peptide against other peptides should be experimentally obtained for the quantification of

...

SLOVENSKI STANDARD
oSIST prEN ISO 20418-3:2019
01-september-2019

Tekstilije - Kvalitativna in kvantitativna proteomska analiza nekaterih živalskih

vlaken - 3. del: Odkrivanje peptida z uporabo LC-MS brez zmanjšanja proteina
(ISO/DIS 20418-3:2019)

Textiles - Qualitative and quantitative proteomic analysis of some animal hair fibers -

Part 3: Peptide detection using LC-MS without protein reduction (ISO/DIS 20418-3:2019)

Textilien - Qualitative und quantitative Proteomanalyse einiger Tierhaarfasern - Teil 3:

Peptiddetektion mit LC-ESI-MS ohne Proteinreduktion (ISO/DIS 20418-3:2019)

Textiles - Analyse protéomique qualitative et quantitative de certaines fibres animales -

Partie 3: Détection des peptides par LC-MS sans réduction protéique (ISO/DIS 20418-

3:2019)
Ta slovenski standard je istoveten z: prEN ISO 20418-3
ICS:
59.060.01 Tekstilna vlakna na splošno Textile fibres in general
oSIST prEN ISO 20418-3:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 20418-3:2019
---------------------- Page: 2 ----------------------
oSIST prEN ISO 20418-3:2019
DRAFT INTERNATIONAL STANDARD
ISO/DIS 20418-3
ISO/TC 38 Secretariat: SAC
Voting begins on: Voting terminates on:
2019-06-18 2019-09-10
Textiles — Qualitative and quantitative proteomic analysis
of some animal hair fibres —
Part 3:
Peptide detection using LC-MS without protein reduction

Textiles — Analyse protéomique qualitative et quantitative de certaines fibres animales —

Partie 3: Détection peptidique par LC-MS sans réduction de protéines
ICS: 59.060.01
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 20418-3:2019(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2019
---------------------- Page: 3 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Symbols and abbreviated terms ........................................................................................................................................................... 2

5 Principle ........................................................................................................................................................................................................................ 2

6 Reagents ........................................................................................................................................................................................................................ 3

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Test method ............................................................................................................................................................................................................... 4

8.1 Sampling ....................................................................................................................................................................................................... 4

8.2 Preliminary identification ............................................................................................................................................................. 4

8.3 Wash for degreasing ........................................................................................................................................................................... 4

8.4 Powderization of fibres ................................................................................................................................................................... 4

8.5 Trypsin digestion .................................................................................................................................................................................. 4

8.6 Marker peptides ..................................................................................................................................................................................... 5

8.7 LC-MS analysis ......................................................................................................................................................................................... 5

8.8 Evaluation of the validity of observed data .................................................................................................................... 5

8.9 Calculation of correction factor ................................................................................................................................................ 6

8.9.1 Correction factor .............................................................................................................................................................. 6

8.9.2 Correction factor for Cas1 against She1 and Yak1 (kc)................................................................... 6

8.9.3 Correction factor for Yak2 against She2, She3 and Cas2 (ky) ................................................... 6

8.9.4 Correction factor for alpaca and camel (ka) ............................................................................................. 6

8.9.5 Correction factor for angora rabbit (kr) ...................................................................................................... 6

8.10 Calculation of blending ratio ....................................................................................................................................................... 7

8.10.1 Calculation of blending ratio of cashmere, sheep wool and yak ............................................. 7

8.10.2 Calculation by She1, Cas1, Yak1 and kc ........................................................................................................ 7

8.10.3 Calculation by She2, She3, Cas2, Yak2 and ky ......................................................................................... 7

8.10.4 Calculation by She1, She2, She3, Cas2, Yak1 ............................................................................................ 7

8.10.5 Calculation of blending ratio of camel , alpaca and angora rabbit ........................................ 8

9 Test report ................................................................................................................................................................................................................... 9

Annex A (informative) Marker peptides of cashmere, sheep wool and yak fibres ............................................10

Annex B (informative) An example of LC-MS analysis conditions .......................................................................................13

Annex C (informative) Analysis of camel, alpaca and angora rabbit fibres ...............................................................16

Annex D (informative) Example of marker peptide mass chromatograms ...............................................................19

Annex E (informative) Results of International Round Trial ...................................................................................................24

Bibliography .............................................................................................................................................................................................................................25

© ISO 2019 – All rights reserved iii
---------------------- Page: 5 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,

as well as information about ISO's adherence to the World Trade Organization (WTO) principles in the

Technical Barriers to Trade (TBT) see the following URL: www .iso .org/iso/foreword .html.

The committee responsible for this document is ISO/TC 38, Textiles.
A list of all parts in the ISO 20418- series can be found on the ISO website.
iv © ISO 2019 – All rights reserved
---------------------- Page: 6 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)
Introduction

Cashmere is a long slender fibre obtained from cashmere goat and is expensive because of the high

quality and the rarity. Mislabelling or adulteration of cashmere products blended with other cheaper

animal fibres such as sheep wool and yak has been repeatedly reported worldwide.

The current official methods to identify specific animal fibres are based on microscopic observation.

In recent years, however, the microscopy-based identification is becoming more and more difficult as

chemical or physical treatment in the manufacturing process, which often complicates microscopic

identification of animal fibres, has become widely used.

Therefore, the development of novel identification methods has been desired. Though several methods

such as near infrared spectroscopy and terahertz spectroscopy to distinguish the difference in fibre

structures and the use of polymerase chain reaction to distinguish the difference in DNA sequences

have been studied, each method has some difficulty in its practical application.

The animal fibres consist mainly of proteins called keratins and some associated proteins. Therefore, to

analyse proteins contained in textiles is generally regarded as one of the most promising identification

methods. The general method to analyse proteins includes the digestion of proteins by trypsin to

convert to smaller molecules, i.e., peptides, followed by the detection of resulting peptides in mass

spectrometers. Accordingly, identification method using either matrix-assisted laser desorption/

ionization time-of-flight mass spectrometer or liquid chromatography/electrospray ionization mass

spectrometer (LC-MS) has been studied. The latter type of instrument is less expensive and more

readily available in testing laboratories as a versatile analytical instrument. Moreover, LC-MS has high

quantitative capability and is therefore preferable to calculate the blending ratio of animal fibres.

Keratins have many intermolecular and intramolecular disulfide bonds, which make the proteins

hardly soluble. Therefore, keratins are generally extracted in the presence of reducing agents. This

reducing step, however, needs much time and effort. In this part of the standard, an alternative method

in which cysteine-free peptides are selected for identification markers, thereby eliminating the need

for reducing step and enabling rapid preparation of LC-MS samples, is presented.

Both ISO 20418-1 and this document use LC-MS, but are different in the extracting method of peptides. In

ISO 20418-1, proteins are first extracted from fibres by a thiourea/ urea/ dithiothreitol (DTT) solution,

and then digested by trypsin to obtain peptides. In this document, peptides are directly extracted by

trypsin digestion of mechanically powdered fibres. The method has been shown to be useful even for

highly processed samples and is applicable to various types of animal hairs such as goat (cashmere or

mohair), wool and yak.
© ISO 2019 – All rights reserved v
---------------------- Page: 7 ----------------------
oSIST prEN ISO 20418-3:2019
---------------------- Page: 8 ----------------------
oSIST prEN ISO 20418-3:2019
DRAFT INTERNATIONAL STANDARD ISO/DIS 20418-3:2019(E)
Textiles — Qualitative and quantitative proteomic analysis
of some animal hair fibres —
Part 3:
Peptide detection using LC-MS without protein reduction
1 Scope

This document specifies a qualitative and quantitative procedure to determine the composition of

animal hair fibre blends by LC-MS without protein reduction.

The composition of non-animal hair fibres can be measured by ISO 1833- series; then both results are

combined to determine the whole composition of fibres.

The method is based on a preliminary identification of all fibres in the blend on the basis of their

morphology, by light microscopy. In case of fibres of the same animal species are present (e.g. blends

of cashmere and mohair), the method is not applicable and the quantitative analysis can be performed

using microscopical analysis (e.g. ISO 17751- series).
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 1833-1, Textiles — Quantitative chemical analysis — Part 1: General principles of testing

ISO 3696, Water for analytical laboratory use — Specification and test methods

ISO 17751-1, Textiles — Quantitative analysis of cashmere, wool, other specialty animal fibers and their

blends — Part 1: Light microscopy method

ISO 17751-2, Textiles — Quantitative analysis of cashmere, wool, other specialty animal fibers and their

blends — Part 2: Scanning electron microscopy method
3 Terms and definitions

For the purposes of this document, the following terms and definitions apply. ISO and IEC maintain

terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http: //www .electropedia .org/
— ISO Online browsing platform: available at https: //www .iso .org/obp
3.1
animal hair fibres

cashmere, sheep wool, yak, and some other animal hair fibres such as camel, alpaca, and angora rabbit

3.2
Bovidae

biological family of cloven-hoofed, ruminant mammals including cashmere goat, sheep and yak

© ISO 2019 – All rights reserved 1
---------------------- Page: 9 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)
3.3
Camelidae
biological family of even-toed ungulates mammals including camel and alpaca
3.4
proteins
polymers of amino acids that play many critical roles in the body
3.5
peptides
small proteins consisting of approximately less than 50 amino acids
3.6
marker peptides
specific peptides for the identification of animal hair fibres
3.7
LC-MS

high performance liquid chromatography–mass spectrometer equipped with an electrospray ionization

ion source
3.8
mass chromatogram
chromatogram for a specific mass-to-charge ratio (m/z)
3.9
total ion chromatogram (TIC)

chromatogram with each data point created by summing up intensities of all mass spectral peaks

belonging to the same scan
3.10
selected ion monitoring (SIM)

mass spectrometry scanning mode in which only a limited m/z range is transmitted/detected by the

instrument
4 Symbols and abbreviated terms
A peak area
Br Bovidae rate
Cr Camelidae rate
ka correction factor for alpaca and camel
kc correction factor for cashmere
kr correction factor for angora rabbit
ky correction factor for yak
5 Principle

The mechanically powdered fibres are directly subjected to trypsin digestion without prior reduction.

The analysis of the digested peptides is performed with LC-MS. The percent composition is calculated

from the peak areas of the species-specific marker peptides.
2 © ISO 2019 – All rights reserved
---------------------- Page: 10 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)
6 Reagents
Following analytical grade reagents should be used.
6.1 Acetone, 99,5 % (GC)
6.2 Water, grade 3 quality specified in ISO 3696
6.3 Ammonium hydrogen carbonate (NH HCO ) solution (25 mmol/l)
4 3
— NH HCO , 96,0 % (T) 197,5 mg
4 3
— Make up 100 ml by adding water (6.2)
6.4 Trypsin, sequencing-grade porcine trypsin modified by reductive methylation
6.5 Acetonitrile, 99,9 % (GC)
6.6 Formic acid, 98 % (T)
6.7 Trypsin solution
— Trypsin (6.4) 20 μg
— 0,1 % formic acid (6.6) 200 μl
7 Apparatus
The usual laboratory apparatus and, in particular, the following.

7.1 Heating mantle, capable of operating at a temperature range of 50 °C to 150 °C

7.2 Mill, beads mill, cryogenic grinder or an equivalent, capable of crushing materials into an extremely

fine powder
7.3 Membrane filter, for aqueous solutions, with a pore size of 0,45 µm
7.4 Heat block, capable of heating micro tubes at 37 °C
7.5 Tube mixer, capable of vortex micro tubes and LC vials for about 30 min
7.6 Centrifugal evaporator
7.7 LC-MS, capable of detecting m/z range from 200 to 1 500
7.8 LC vial, shall be manufactured from glass or polymethylpentane
7.9 LC column, octadecyl (C-18)-silica reversed phase column
7.10 Balance, with 1 mg readability or better
7.11 Recovery flask (eggplant flask or round bottom flask)
© ISO 2019 – All rights reserved 3
---------------------- Page: 11 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)
8 Test method
8.1 Sampling

The general requirement is that the test specimen shall be representative for the lot of material from

which it is taken. The method of obtaining a fibre test specimen differs depending upon the sample

form. The terms relating to sampling for the various types of samples are namely given in ISO 1833-1.

8.2 Preliminary identification

The preliminary qualitative analysis of the fibre composition is carried out on the basis of their

morphology by light microscopy, according to ISO 17751- series.
8.3 Wash for degreasing

8.3.1 Reflux 1 g of the fibres in a recovery flask (7.11) on a heating mantle with 200 ml acetone

for 30 min.
NOTE 1 Quantity of the fibres can be changed.
NOTE 2 This washing step may be omitted in the case of clean samples.
8.3.2 Take the degreased fibres out of a recovery flask and dry them in air.
NOTE Sample preparation method of ISO 20418-1 can be used alternatively.
8.4 Powderization of fibres

Crush about 1 g of the dried fibre sample using a mill (7.2) to a fine powder with an average length of

100 µm or less and mix thoroughly for securing representative sampling of the fibres.

8.5 Trypsin digestion

8.5.1 Weigh 10 mg of the crushed samples and place the test specimen into a micro tube. If more than

10 mg of the sample is used, increase the volumes of NH HCO solution in 8.5.2 and Trypsin solution in

4 3
8.5.3 accordingly.
8.5.2 Add 300 µl NH HCO solution (6.3) and vortex for 10 min to 30 min.
4 3

8.5.3 Add 10 µl Trypsin solution (6.7) to the sample and incubate at 37 °C for 20 h to 24 h.

8.5.4 Centrifuge the tryptic solution at 5 000 G for 3 min. Filter the supernatant through a membrane

filter (7.3) to remove residual fibres.

NOTE Centrifugal filter, syringe filter or other means of filtration can be used.

8.5.5 Transfer the quantity of solution corresponding to 2 mg of fibre to a LC vial. Dry the sample in a

centrifugal evaporator.

NOTE 1 When LC vial does not fit in a dryer, the solution can be dried in other types of container such as micro

tubeand transfered sample to a LC vial after dissolution.

NOTE 2 Alternatively, freeze dryer or nitrogen flux can be used as the drying method, instead of centrifugal

evaporator.
4 © ISO 2019 – All rights reserved
---------------------- Page: 12 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)

8.5.6 Add 40 µl of water containing 0,1 % formic acid and 5 % acetonitrile and vortex for 30 min, for

subsequent LC-MS measurements. Sonication shall not be a substitute for vortex when LC-MS sample is

dissolved.
8.6 Marker peptides

8.6.1 Select the peptides which are used as markers for differential identification of fibres from

Annex A and Annex C. The result of preliminary identification by microscopy (8.2) and Table 1 can be

referred to for the selection.
Table 1 — Correlation of marker peptides and identifiable animal taxa.
species family class
fibre
name marker name marker name marker
She1,
sheep wool Ovis aries (She2,
She3)
cashmere/ Cas1, Bovidae Fbv1
Capra hircus
mohair (Cas2)
Yak1,
yak Bos grunniens
(Yak2)
Mammalia Cmm1
Alp1,
alpaca Vicugna pacos
(Alp2)
Camelidae Fcm1
Cam1,
camel Camelus ferus
(Cam2)
Oryctolgus Ang1,
angora rabbit Leporidae —
cuniculus (Ang2)

Marker peptides with suffix 1 are preferable when conducting quantitative analysis.

8.6.2 Optimize LC-MS parameters and confirm retention times of target peaks by using either

synthesized peptides (with amino acid sequences shown in Annex A and Annex C) or peptides extracted

from pure animal hair fibre samples.
8.7 LC-MS analysis

8.7.1 Inject 5 µl sample to a LC column (7.9). Use water containing 0,1 % formic acid and acetonitrile

containing 0,1 % formic acid to form a gradient with increasing concentration of acetonitrile for

chromatography. Initial concentration of acetonitrile is 5 %. An example of LC parameters is indicated in

Annex B.

8.7.2 Operate mass spectrometer in SIM mode. The selected markers (preferably those with suffix

1), which are described in Annex A and Annex C, shall be monitored. An example of MS parameters is

indicated in Annex B.

8.7.3 Integrate peak area of each marker peptides. The peak of additional marker peptides (those with

suffix 2 and 3), which are also described in Annex A and Annex C, can be used when it is difficult to take

the target peak.
8.8 Evaluation of the validity of observed data
See Annex C.
© ISO 2019 – All rights reserved 5
---------------------- Page: 13 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)
8.9 Calculation of correction factor
8.9.1 Correction factor

Peak areas of marker peptides are not expected to be proportional to the amounts of corresponding

animal fibres in the following combinations of marker peptides, for reasons such as the difference in

the protein species from which each marker peptide is derived (8.9.2). In these cases, a correction

factor for a peptide against other peptides should be experimentally obtained for the quantification of

their blending ratio. Moreover, the correction factor should be updated when analysis conditions are

modified.
8.9.2 Correction factor for Cas1 against She1 and Yak1 (kc)

8.9.2.1 Analyse blend samples with 50 % cashmere and 50 % sheep wool at least three times.

8.9.2.2 Calculate the value of A /A from the peak area of She1 (A ) and the peak area of Cas1

she1 cas1 she1
(A ) for each analysis.
cas1

8.9.2.3 Correction factor for cashmere (kc) shall be calculated as the average of each A /A .

she1 cas1
8.9.3 Correction factor for Yak2 against She2, She3 and Cas2 (ky)

8.9.3.1 Analyse blend samples with 50 % cashmere and 50 % yak at least three times.

8.9.3.2 Calculate the value of A /A from the peak area of Cas2 (A ) and the peak area of Yak2

cas2 yak2 cas2
(A ) for each analysis.
yak2

8.9.3.3 Correction factor for yak (ky) shall be calculated as the average of each A /A .

cas2 yak2
8.9.4 Correction factor for alpaca and camel (ka)

8.9.4.1 Analyse blend samples with 50 % alpaca and 50 % sheep wool at least three times.

8.9.4.2 Calculate the value of A /A from the peak area of Fbv1 (A ) and the peak area of F

fbv1 fcm1 fbv1 cm1
(A ) for each analysis.
fcm1

8.9.4.3 Correction factor for alpaca and camel (ka) shall be calculated as the average of each A /A .

fbv1 fcm1
EXAMPLE See Annex C.
8.9.5 Correction factor for angora rabbit (kr)

8.9.5.1 Analyse blend samples with 50 % angora rabbit and 50 % sheep wool at least three times.

8.9.5.2 Calculate the value of A /A from the peak area of Fbv1 (A ) and the peak area of Ang1

fbv1 ang1 fbv1
(A ) for each analysis.
ang1

8.9.5.3 Correction factor for angora rabbit (kr) shall be calculated as the average of each A /A .

fbv1 ang1
EXAMPLE See Annex C.
6 © ISO 2019 – All rights reserved
---------------------- Page: 14 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)
8.10 Calculation of blending ratio
8.10.1 Calculation of blending ratio of cashmere, sheep wool and yak
Calculate the blending ratios from one of the following three subclauses.
8.10.2 Calculation by She1, Cas1, Yak1 and kc
Calculate the blending ratios from the following formula.
sheep wool (%) = A / (A + kc × A + A ) × 100
she1 she1 cas1 yak1
cashmere (%) = kc × A / (A + kc × A + A ) × 100
cas1 she1 cas1 yak1
yak (%) = A / (A + kc × A + A ) × 100
yak1 she1 cas1 yak1
where
A is peak area of She1
she1
A is peak area of Cas1
cas1
A is peak area of Yak1
yak1
8.10.3 Calculation by She2, She3, Cas2, Yak2 and ky
Calculate the blending ratios from the following formula.
sheep wool (%) = (A + A ) / (A + A + A + ky × A ) × 100
she2 she3 she2 she3 cas2 yak2
cashmere (%) = A / (A + A + A + ky × A ) × 100
cas2 she2 she3 cas2 yak2
yak (%) = ky × A / (A + A + A + ky × A ) × 100
yak2 she2 she3 cas2 yak2
where
A is peak area of She2
she2
A is peak area of She3
she3
A is peak area of Cas2
cas2
A is peak area of Yak2
yak2
8.10.4 Calculation by She1, She2, She3, Cas2, Yak1
Calculate the blending ratios from the following formula when A is not 0.
she1
A = A + A
she2+3 she2 she3
sheep wool (%) = 1 / (1 + A / A + A / A ) × 100
cas2 she2+3 yak1 she1
cashmere (%) = (A / A ) / (1 + A / A + A / A ) × 100
cas2 she2+3 cas2 she2+3 yak1 she1
© ISO 2019 – All rights reserved 7
---------------------- Page: 15 ----------------------
oSIST prEN ISO 20418-3:2019
ISO/DIS 20418-3:2019(E)
yak (%) = (A / A ) / (1 + A / A + A / A ) × 100
yak1 she1 cas2 she2+3 yak1 she1
where
A is peak area of She1
she1
A is peak area of She2
she2
A is peak area of She3
she3
A is peak area of Cas2
cas2
A is peak area of Yak1
yak1
8.10.5 Calculation of blending ratio of camel , alpaca and angora rabbit

Calculate blending ratio from the following formula. Marker peptides of camel, alpaca and angora rabbit

with suffix 1 can be replaced by those with suffix 2. Use A in combination with A and A in

alp1 cam1 alp2
combination
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.