Foodstuffs - Detection of food allergens by liquid chromatography - mass spectrometry (LC-MS) methods - General considerations

This document establishes an overall framework covering qualitative and quantitative methods for the determination of food allergens and allergenic ingredients using mass spectrometry-based methods for the determination of specific peptides/proteins. This document provides general guidelines and performance criteria applicable to this methodology. Guidelines, minimum requirements and performance criteria laid down in this document are intended to ensure that comparable and reproducible results are obtained by different analysts, instrumentation and laboratories.

Lebensmittel - Nachweis von Lebensmittelallergenen mit flüssigkeitschromatographisch-massenspektrometrischen (LC-MS) Verfahren - Allgemeine Betrachtungen

Dieses Dokument erstellt einen Gesamtrahmen für die qualitativen und quantitativen Verfahren zur Bestimmung von Lebensmittelallergenen und allergenen Inhaltsstoffen unter Anwendung von auf der Massenspektrometrie basierenden Verfahren für die Bestimmung spezifischer Peptide/Proteine. Dieses Dokument bietet allgemeine Leitlinien und Leistungskriterien, die auf diese Verfahren anwendbar sind. Die in diesem Dokument angeführten Leitlinien, Mindestanforderungen und Leistungskriterien sind dafür vorgesehen, sicherzustellen, dass von verschiedenen Analytikern, Geräten und Laboratorien vergleichbare und reproduzierbare Ergebnisse erhalten werden.

Produits alimentaires - Détection des allergènes alimentaires par des méthodes de chromatographie en phase liquide couplée à la spectrométrie de masse (CL-SM) - Considérations générales

Le présent document établit un cadre général couvrant les méthodes qualitatives et quantitatives pour la détermination des allergènes alimentaires et ingrédients allergisants par des méthodes de spectrométrie de masse pour la détermination de peptides/protéines spécifiques. Le présent document fournit les lignes directrices générales et les critères de performance applicables à cette méthode. Les lignes directrices, les exigences minimales et les critères de performance exposés dans le présent document ont pour but de garantir l'obtention de résultats comparables et reproductibles, par différents analystes, instruments et laboratoires.

Živila - Odkrivanje prisotnosti alergenov v živilih s tekočinsko kromatografijo masno spektrometrijo (LC-MS) - Splošne ugotovitve

Ta dokument podaja splošen okvir kvalitativnih in kvantitativnih metod za določanje alergenov in alergenih sestavin z uporabo metod na osnovi masne spektrometrije za določitev specifičnih peptidov/proteinov. Ta dokument podaja splošne smernice in merila učinkovitosti za to metodologijo. Smernice, minimalne zahteve in merila učinkovitosti iz tega dokumenta so namenjeni zagotavljanju, da se pridobijo primerljivi ter ponovljivi rezultati s strani različnih analitikov, z različnimi instrumenti in v različnih laboratorijih.

General Information

Status
Published
Public Enquiry End Date
19-Apr-2021
Publication Date
23-Aug-2022
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
05-Aug-2022
Due Date
10-Oct-2022
Completion Date
24-Aug-2022

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SLOVENSKI STANDARD
SIST EN 17644:2022
01-september-2022
Živila - Odkrivanje prisotnosti alergenov v živilih s tekočinsko kromatografijo
masno spektrometrijo (LC-MS) - Splošne ugotovitve

Foodstuffs - Detection of food allergens by liquid chromatography - mass spectrometry

(LC-MS) methods - General considerations

Lebensmittel - Nachweis von Lebensmittelallergenen mit flüssigkeitschromatographisch-

massenspektrometrischen (LC-MS) Verfahren - Allgemeine Betrachtungen

Produits alimentaires - Détection des allergènes alimentaires par des méthodes de

chromatographie en phase liquide couplée à la spectrométrie de masse (CL-SM) -
Considérations générales
Ta slovenski standard je istoveten z: EN 17644:2022
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN 17644:2022 en,fr,de

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

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SIST EN 17644:2022
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SIST EN 17644:2022
EN 17644
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2022
EUROPÄISCHE NORM
ICS 67.050
English Version
Foodstuffs - Detection of food allergens by liquid
chromatography - mass spectrometry (LC-MS) methods -
General considerations

Produits alimentaires - Détection des allergènes Lebensmittel - Nachweis von Lebensmittelallergenen

alimentaires par des méthodes de chromatographie en mit flüssigkeitschromatographisch-

phase liquide couplée à la spectrométrie de masse (CL- massenspektrometrischen (LC-MS) Verfahren -

SM) - Considérations générales Allgemeine Betrachtungen
This European Standard was approved by CEN on 22 May 2022.

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

© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17644:2022 E

worldwide for CEN national Members.
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SIST EN 17644:2022
EN 17644:2022 (E)
Contents Page

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

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 General laboratory requirements ............................................................................................................. 8

4.1 Principle ............................................................................................................................................................. 8

4.2 Apparatus and equipment ........................................................................................................................... 8

4.3 Material and reagents .................................................................................................................................... 9

5 Method development ..................................................................................................................................... 9

5.1 General ................................................................................................................................................................ 9

5.2 Sample preparation ..................................................................................................................................... 10

5.2.1 Grinding ........................................................................................................................................................... 10

5.2.2 Extraction/purification .............................................................................................................................. 10

5.2.3 Enzymatic digestion .................................................................................................................................... 10

5.3 Detection ......................................................................................................................................................... 11

5.3.1 General ............................................................................................................................................................. 11

5.3.2 Selection of the target proteins/peptides ........................................................................................... 11

5.3.3 Selection of measured MRM transitions .............................................................................................. 12

5.3.4 Internal standard (IS) ................................................................................................................................. 13

6 Method validation procedure .................................................................................................................. 13

6.1 General ............................................................................................................................................................. 13

6.2 Method validation parameters ................................................................................................................ 14

6.2.1 Measurand ...................................................................................................................................................... 14

6.2.2 Limit of detection and limit of quantification .................................................................................... 14

6.3 Selectivity ........................................................................................................................................................ 15

6.4 Calibration curves ........................................................................................................................................ 15

6.5 Trueness .......................................................................................................................................................... 15

6.6 Precision .......................................................................................................................................................... 15

6.7 Measurement uncertainty ........................................................................................................................ 16

6.8 Robustness...................................................................................................................................................... 16

6.9 Fit-for-purpose/applicability .................................................................................................................. 16

7 Routine analysis of allergenic food ingredients................................................................................ 16

7.1 General ............................................................................................................................................................. 16

7.2 Validation of an analytical run ................................................................................................................ 17

7.3 Test report ...................................................................................................................................................... 18

Bibliography ................................................................................................................................................................. 19

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SIST EN 17644:2022
EN 17644:2022 (E)
European foreword

This document (EN 17644:2022) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - Horizontal methods”, the secretariat of which is held by DIN.

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 2023, and conflicting national standards shall

be withdrawn at the latest by January 2023.

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.

Any feedback and questions on this document should be directed to the users’ national standards body.

A complete listing of these bodies can be found on the CEN website.

According to the CEN-CENELEC Internal Regulations, the national standards organisations 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.
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SIST EN 17644:2022
EN 17644:2022 (E)
Introduction

Specific peptides or groups of peptides deriving from specific proteins can serve as markers for the

presence of food or food ingredients provoking allergic reactions. This document describes the procedure

for the development of qualitative and/or quantitative mass spectrometry-based methods for the

determination of protein-derived peptides as markers for potentially allergenic food ingredients or

constituents by analysing the protein/s extracted from a sample. Appropriate procedures for the

extraction of the targeted proteins are an essential part of each method. This document describes general

considerations for the application of liquid chromatography mass spectrometry-based methods in

qualitative or quantitative targeted analysis of specific peptides (derived from specific proteins) that are

representative for a food allergen. The document includes recommendations for method validation and

for the conversion of the analytical results to units of mg protein/kg food.
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SIST EN 17644:2022
EN 17644:2022 (E)
1 Scope

This document establishes an overall framework covering qualitative and quantitative methods for the

determination of food allergens and allergenic ingredients using mass spectrometry-based methods for

the determination of specific peptides/proteins. This document provides general guidelines and

performance criteria applicable to this methodology. Guidelines, minimum requirements and

performance criteria laid down in this document are intended to ensure that comparable and

reproducible results are obtained by different analysts, instrumentation and laboratories.

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.

EN 15842, Foodstuffs - Detection of food allergens - General considerations and validation of methods

3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 15842 and the following apply.

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

— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
high-performance liquid chromatography–mass spectrometry
HPLC-MS

analytical chemistry technique that combines the separation capabilities of high-performance liquid

chromatography with the detection capabilities of mass spectrometry (MS)
Note 1 to entry: The abbreviation LC-MS is also used.
3.2
tandem mass spectrometry
MS/MS
sequential combination of two mass analyses

Note 1 to entry: Different mass spectrometer instrument types exist, combining different principles of mass

detection, e.g. quadrupole, time-of-flight, ion trap, Fourier-Transform mass spectrometer.

3.3
multi-stage mass spectrometry
sequential combination of more than two mass analyses
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SIST EN 17644:2022
EN 17644:2022 (E)
3.4
targeted mass spectrometry

mass spectrometry application analysing only specific ions (m/z) at specific retention times (RT)

Note 1 to entry: The targets are specified in an inclusion list.
Note 2 to entry: The opposite is untargeted MS measuring any ion present.
Note 3 to entry: In general, targeted MS increases method sensitivity.
3.5
peptide
amide that consists of two or more amino acids

Note 1 to entry: Peptides are formed by amide bonding of the amino group of one amino acid (AA) with the

carboxyl group of another AA.

Note 2 to entry: Peptides are usually obtained by enzymatic hydrolysis of proteins during sample preparation for

mass spectrometry-based methods.
3.6
marker peptide

peptide that is specific/unique for a definite protein and used as analyte in mass spectrometry-based

methods

Note 1 to entry: Portion of a protein used for its identification, recovery, quantification and purification.

[SOURCE: ISO 20418-1:2018, 3.6, modified – The definition has been moved to a Note to entry and a new

definition has been added.]
3.7
analyte
substance or chemical constituent that is subjected to measurement
[SOURCE: CEN/TS 15968:2010, 3.1]
3.8
measurand
quantity intended to be measured

Note 1 to entry: The specification of a measurand requires knowledge of the kind of quantity, description of the

state of the phenomenon, body, or substance carrying the quantity, including any relevant component, and the

chemical entities involved.

Note 2 to entry: In the second edition of the VIM and in IEC 60050-300:2001, the measurand is defined as the

“particular quantity subject to measurement”.

Note 3 to entry: The measurement, including the measuring system and the conditions under which the

measurement is carried out, might change the phenomenon, body, or substance such that the quantity being

measured may differ from the measurand as defined. In this case, adequate correction is necessary.

[SOURCE: ISO/IEC Guide 99:2007, 2.3, modified – removed Examples and Note 4 to entry.]

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SIST EN 17644:2022
EN 17644:2022 (E)
3.9
incurred samples

material produced by adding a specific amount of allergenic ingredient to a relevant matrix before it is

processed by food manufacturing techniques
3.10
mass-to-charge ratio
m/z

dimensionless quantity formed by dividing the ratio of the mass (m) of an ion to the unified atomic mass

unit by its charge number (z)

[SOURCE: IUPAC Recommendations 2013: Definitions of terms relating to mass spectrometry, Pure Appl.

Chem., Vol. 85, No. 7, pp. 1515–1609]
3.11
retention time

time taken for an analyte to pass through a chromatography column from injection to detection

3.12
single reaction monitoring
SRM

determination of a targeted analyte by measuring the precursor-ion to product-ion transition in MS/MS

fragmentation

Note 1 to entry: The mass filters in the mass spectrometer are adjusted to the ion masses of, respectively, the

precursor ion and the product ion (“mass windows”), increasing the selectivity and sensitivity of the measurement.

Note 2 to entry: SRM is monitoring only a single fixed mass window.
Note 3 to entry: SRM is also called selected reaction monitoring.
Note 4 to entry: Parent ion is another expression for precursor ion.
Note 5 to entry: Fragment ion is another expression for product ion.
3.13
multiple reaction monitoring
MRM
simultaneous measurement of multiple SRMs

Note 1 to entry: MRM scans rapidly over multiple (narrow) mass windows and thus captures traces of multiple

product/fragment ion masses in parallel.

Note 2 to entry: MRM can also be called parallel reaction monitoring (PRM) in a high resolution mass

spectrometer.
3.14
basic local alignment search tool
BLAST

bioinformatic algorithm for the comparison of primary sequence information such as amino acid

sequences of peptides or/and proteins or nucleotide sequences of DNA and/or RNA molecules

Note 1 to entry: BLAST is maintained by the National Center for Biotechnology Information (NCBI), U.S. National

Library of Medicine, Bethesda MD, USA.
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SIST EN 17644:2022
EN 17644:2022 (E)
3.15
internal standard
ISTD

substance, which is similar in the chemical behaviour (chemical structure, polarity) and analytical

response to a certain target analyte

Note 1 to entry: Stable-isotope labelled ISTDs are preferred in mass spectrometry-based methods, which are

then sometimes called absolute quantitation (AQUA)-methods.
[SOURCE: EN 62697-1:2012, 3.12, modified – Note 1 to entry has been changed.]
3.16
standard addition
procedure in which a known amount of an analyte is added to a test sample

Note 1 to entry: To perform standard addition procedure, the test sample is divided in two (or more) test

portions. One test portion is analysed as such, whereas known amounts of an analyte are added to the second test

portion before analysis.
3.17
conversion factor
factor for the conversion of measurement results to a reporting unit
Note 1 to entry: The measurement results are converted into mg protein/kg food.
4 General laboratory requirements
4.1 Principle

Samples are extracted for proteins with a high-yielding, reproducible and matrix-specific procedure

including enzymatic digestion for the generation of peptides. A processing step for the reduction and

alkylation may be included. Specific marker peptides are measured by HPLC-MS/MS using targeted MS

analysis for qualitative or quantitative analysis. The signal (usually the peak area) of a marker peptide

measured at a specific chromatographic retention time is used for quantitation.
4.2 Apparatus and equipment

The laboratory shall use properly maintained equipment suitable for the method employed.

NOTE For example, according to the requirements outlined by EN ISO/IEC 17025.

In addition to standard laboratory equipment, additional apparatus are described in the specific methods.

Apparatus and equipment should be maintained according to manufacturer’s instructions. Calibration

systems shall be available and calibration shall be routinely performed for measuring equipment,

according to laboratory quality assurance programmes.

In a tandem mass spectrometer, ions are formed in the ion source and separated by mass-to-charge ratio

in the first stage of mass spectrometry (MS1). In targeted mass spectrometry, ions of a particular mass-

to-charge ratio (precursor ions) are selected and product ions are created by collision-induced

dissociation, ion-molecule reaction, photodissociation, or other processes. The resulting ions are then

separated and detected in a second stage of mass spectrometry (MS2).
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SIST EN 17644:2022
EN 17644:2022 (E)

In MRM mode, the mass spectrometer is set to scan a very small mass range in MS1, typically one mass

unit, at the expected masses of the targeted precursor ions. After fragmentation, the product ions are

detected in MS2 by successively scanning small mass ranges at their expected masses. Alternatively, some

MS instruments (e.g. high-resolution instruments (HRMS) with fast switching MS to MS/MS capacity)

allow the simultaneous detection of all precursor and product ions (Parallel Reaction Monitoring (PRM)).

In MS-based ion fragmentation analysis, the precursor ion selected for analysis shall be clearly specified,

e.g. as the molecular ion, a characteristic ion adduct of the molecular ion, a characteristic fragment ion or

a typical isotope ion. The signal-to-noise ratio for each product ion should be ≥ 3:1.

4.3 Material and reagents

Analytes shall be clearly described, including the type of standard (e.g. synthetic peptides, purified

protein, protein extracts), and information on purity, protein profiles, storage conditions and shelf-life.

Only reagents of MS quality grade and only de-ionized or distilled water or water that has been purified

should be applied for HPLC-MS analysis, unless otherwise stated in specific method descriptions. Other

reagents, such as enzymes, reducing and alkylating agents, should be of MS-grade. Buffer components,

organic solvents standards, analyte, reference material, controls, and samples are method-specific.

Storage conditions and shelf-life of reagents and samples should be determined in method validation

(method robustness) and clearly specified in the method protocol.
5 Method development
5.1 General

For the use of this document, general requirements of quality assurance for laboratories shall be

observed (e.g. concerning calibration of apparatus, extraction of samples and measurement of replicates,

blanks, use of reference materials, preparation of calibration curves, etc.). The scope of the method,

including applicability to certain food matrices, shall be clearly specified.

Before conducting food allergen analysis, special considerations should be made regarding:

a) the laboratory lay-out (e.g. ideally, extraction working area should be spatially separated from

detection working area);

b) the current workflow (e.g. other activities in the laboratories that can increase the potential for cross-

contamination should be separated);

c) sample types handled (because cross-contamination issues can undermine the capability to perform

analysis reliably);

d) equipment in the laboratories (e.g. the risk of cross-contamination should be considered if

equipment is shared; use dedicated equipment if appropriate);

e) containers (e.g. disposable consumables are preferable, those that exhibit low protein binding - not

polystyrene);

f) general house-keeping tasks (e.g. effective cleaning of items and surfaces is important and should be

considered). Specific cleaning routines for rooms housing mass spectrometry instruments can be

required because cleaning chemicals can disturb the analysis, depending on the MS instrument type.

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SIST EN 17644:2022
EN 17644:2022 (E)
5.2 Sample preparation
5.2.1 Grinding

To ensure reliable results, sample preparation is a critical step in food allergen analysis. Its objective shall

be to provide a homogenous and representative portion of the original sample material. Equipment

should be available in the laboratory for sample homogenization - including apparatus for milling,

chopping, grinding and/or blending – the exact items depend on the sample types to be routinely

analysed.

A larger test portion will usually improve the potential for detection, as will reducing the particle size to

improve sample homogeneity.
5.2.2 Extraction/purification

The protein is extracted according to the procedure described in the specific method protocol.

Considerations should be given to:

a) the possibility of increasing the amounts, whilst maintaining the ratio between the test portion to

extraction volume, in order to improve the extractability/recovery of the target allergen;

b) the composition of the sample matrix.
5.2.3 Enzymatic digestion

In order to obtain a complete enzymatic digestion of the proteins in the test portion extract, it is important

to ensure that all specific cleavage sites are accessible to the respective enzymes. The following steps are

usually performed:

Denaturation step: unfolds the proteins (e.g. through sonication, addition of detergents or chaotropic

agents such as urea, denaturation with high temperature, etc.).

Reduction step: cleaves any disulfide bond present in the protein, resulting in free thiol groups (mainly

in cysteines).

Alkylation step: prevents disulfide bond reformation by irreversible capping of the free thiols obtained

during the reduction step. Typically, alkylation of cysteine is carried out by using iodoacetamide,

yielding S-carboxyamidomethylcysteine. Disulfide bond regeneration is known to be problematic before

enzymatic digestion is performed, but can also occur in the resulting peptide mixture. The omission of

the alkylation step would lead to random dimerization of cysteine-containing peptides, which would

significantly complicate the mass spectrometry analysis.

A number of enzymes are commonly used for the digestion of proteins in MS sample preparation. Pepsin

cleaves unspecifically the amino acid (AA) chain, whereas trypsin has very specific cleavage sites

(carboxyl side of lysines and arginines). Proteins that are enzymatically digested can differ widely in their

AA compositions and suitability for MS analysis. Therefore, digestion shall be optimized during method

development, and a digestion protocol including the choice of enzyme is part of the sample preparation

procedure.

After enzymatic digestion, protein extracts should immediately be processed; protein extracts could be

stored if stability data are available.
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SIST EN 17644:2022
EN 17644:2022 (E)
5.3 Detection
5.3.1 General
General information can be found in EN 15842.
5.3.2 Selection of the target proteins/peptides

The full sequence of the protein should be available in a reference protein database. Protein expression

variability should be characterized, e.g. by exploring the literature, and ideally should be minimal. If

different isoforms are known, they should be characterized and their sequences should be available.

Preferably, the most abundant isoform should be used.

The sequences of the selected marker peptides should be specific for the allergenic ingredients to be

detected.

In theory, any protein unique to the allergenic ingredient may be used to indicate the presence of the

ingredient itself. In practice, several considerations will guide the selection of individual proteins and

peptides as targeted markers to guarantee specificity and robustness of the method.

During peptide selection, special attention should be paid to select marker peptides that are present in

all known isoforms of a protein. If not possible, different marker peptides should be selected to detect all

isoforms.

Attention should be paid with regard to the analysis of collective allergens such as fish, crustaceans,

molluscs, egg and milk. When selecting suitable marker peptides, it shall be considered whether all

species belonging to the allergen group or one distinct species are detected. When selecting suitable

marker peptides, e.g. milk, it has to be considered whether the selected marker peptides are specific only

for cow’s milk or are also present in e.g. buffalo, goat, sheep or horse milk. For egg, it has to be considered

whether the selected marker peptides are specific only for hen’s egg or are also present in e.g. goose,

duck, turkey or quail eggs.

Preferably, a peptide with reproducible yield in sample preparation, low matrix background and high

signal intensity is selected as marker peptide. Optimally, a marker peptide should produce at least two

measurable product ions. Mass spectrometry-based methods should include one peptide for quantitation

(“quantifier”) and one peptide for quality control (“qualifier”) per targeted protein. The selection criteria

for proteins and peptides are summarized in Table 1.
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SIST EN 17644:2022
EN 17644:2022 (E)

Table 1 — Proteins and peptides selection criteria for allergen analysis by mass spectrometry

[3], [13]
Protein selection criteria Peptide selection criteria

Full protein sequence should be available, Sequence unique to the allergenic ingredient (BLAST

including isoforms identification details provided together with details about used

database and querying date/time)
Protein should be abundant in the appropriate endoprotease selection
ingredient, sufficient extract
...

SLOVENSKI STANDARD
oSIST prEN 17644:2021
01-april-2021
Živila - Odkrivanje prisotnosti alergenov v živilih z metodo tekočinske
kromatografije z masno spektrometrijsko detekcijo (LC-MS) - Splošne ugotovitve

Foodstuffs - Detection of food allergens by liquid chromatography - mass spectrometry

(LC-MS) methods - General considerations

Lebensmittel - Nachweis von Lebensmittelallergenen mit flüssigkeitschromatographisch-

massenspektrometrischen (LC-MS) Verfahren - Allgemeine Betrachtungen

Produits alimentaires - Détection des allergènes alimentaires par des méthodes de

chromatographie en phase liquide couplée à la spectrométrie de masse (CL-SM) -
Considérations générales
Ta slovenski standard je istoveten z: prEN 17644
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
oSIST prEN 17644:2021 en,fr,de

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

---------------------- Page: 1 ----------------------
oSIST prEN 17644:2021
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oSIST prEN 17644:2021
DRAFT
EUROPEAN STANDARD
prEN 17644
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2021
ICS 67.050
English Version
Foodstuffs - Detection of food allergens by liquid
chromatography - mass spectrometry (LC-MS) methods -
General considerations

Produits alimentaires - Détection des allergènes Lebensmittel - Nachweis von Lebensmittelallergenen

alimentaires par des méthodes de chromatographie en mit flüssigkeitschromatographisch-

phase liquide couplée à la spectrométrie de masse (CL- massenspektrometrischen (LC-MS) Verfahren -

SM) - Considérations générales Allgemeine Betrachtungen

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 275.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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.

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.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
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© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17644:2021 E

worldwide for CEN national Members.
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Contents Page

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

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 General laboratory requirements ............................................................................................................. 8

4.1 Principle ............................................................................................................................................................. 8

4.2 Apparatus and equipment ........................................................................................................................... 8

4.3 Material and reagents .................................................................................................................................... 9

5 Method development ..................................................................................................................................... 9

5.1 General ................................................................................................................................................................ 9

5.2 Sample preparation ..................................................................................................................................... 10

5.2.1 Grinding ........................................................................................................................................................... 10

5.2.2 Extraction/purification .............................................................................................................................. 10

5.2.3 Enzymatic digestion .................................................................................................................................... 10

5.3 Detection ......................................................................................................................................................... 11

5.3.1 Selection of the target proteins/peptides ........................................................................................... 11

5.3.2 Selection of measured MRM transitions .............................................................................................. 12

5.3.3 Internal standard (IS) ................................................................................................................................. 13

6 Method validation procedure .................................................................................................................. 13

6.1 General ............................................................................................................................................................. 13

6.2 Method validation parameters ................................................................................................................ 14

6.2.1 Measurand ...................................................................................................................................................... 14

6.2.2 Limit of detection and limit of quantification .................................................................................... 14

6.2.3 Limit of detection (LOD) ............................................................................................................................ 14

6.2.4 Limit of quantification (LOQ) ................................................................................................................... 14

6.3 Selectivity ........................................................................................................................................................ 14

6.4 Calibration curves ........................................................................................................................................ 14

6.5 Trueness .......................................................................................................................................................... 15

6.6 Precision .......................................................................................................................................................... 15

6.7 Measurement uncertainty ........................................................................................................................ 15

6.8 Robustness...................................................................................................................................................... 15

6.9 Fit-for-purpose/applicability .................................................................................................................. 16

7 Routine analysis of allergenic food ingredients................................................................................ 16

7.1 General ............................................................................................................................................................. 16

7.2 Validation of an analytical run ................................................................................................................ 16

7.3 Test report ...................................................................................................................................................... 17

Bibliography ................................................................................................................................................................. 19

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European foreword

This document (prEN 17644:2021) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - Horizontal methods”, the secretariat of which is held by DIN.
This document is currently submitted to CEN Enquiry.
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Introduction

Specific peptides or groups of peptides deriving from specific proteins can serve as markers for the

presence of food or food ingredients provoking allergic reactions. This document describes the

procedure for the development of qualitative and/or quantitative mass spectrometry-based methods

for the determination of protein-derived peptides as markers for potentially allergenic food ingredients

or constituents by analysing the protein/s extracted from a sample. Appropriate procedures for the

extraction of the targeted proteins are an essential part of each method. This document describes

general considerations for the application of liquid chromatography mass spectrometry-based methods

in qualitative or quantitative targeted analysis of specific peptides (derived from specific proteins) that

are representative for a food allergen. The document includes recommendations for method validation

and for the conversion of the analytical results to units of mg protein/kg food.
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1 Scope

This document establishes an overall framework covering qualitative and quantitative methods for the

determination of food allergens and allergenic ingredients using mass spectrometry-based methods for

the determination of specific peptides/proteins. This document provides general guidelines and

performance criteria applicable to this methodology. Guidelines, minimum requirements and

performance criteria laid down in this document are intended to ensure that comparable and

reproducible results are obtained by different analysts, instrumentation and laboratories.

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.

EN 15842, Foodstuffs - Detection of food allergens - General considerations and validation of methods

3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 15842 and the following 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
high-performance liquid chromatography–mass spectrometry
HPLC-MS

analytical chemistry technique that combines the separation capabilities of high-performance liquid

chromatography with the detection capabilities of mass spectrometry (MS)
Note 1 to entry: The abbreviation LC-MS is also used.
3.2
tandem mass spectrometry
MS/MS
sequential combination of two mass analyses

Note 1 to entry: Different mass spectrometer instrument types exist, combining different principles of mass

detection, e.g. quadrupole, time-of-flight, ion trap, Fourier-Transform mass spectrometer.

3.3
multi-stage mass spectrometry
sequential combination of more than two mass analyses
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3.4
targeted mass spectrometry

mass spectrometry application analysing only specific ions (m/z) at specific times (RT)

Note 1 to entry: The targets are defined in an inclusion list.
Note 2 to entry: The opposite is untargeted MS measuring any ion present.
Note 3 to entry: In general targeted MS increases method sensitivity.
3.5
peptide
amide that consists of two or more amino acids

Note 1 to entry: Peptides are formed by amide bonding of the amino group of one amino acid (AA) with the

carboxyl group of another AA.

Note 2 to entry: Peptides are usually obtained by enzymatic hydrolysis of proteins during sample preparation

for mass spectrometry-based methods.
3.6
marker peptide

peptide that is specific/unique for a definite protein and used as analyte in mass spectrometry-based

methods

Note 1 to entry: Portion of a protein used for its identification, recovery and purification.

[SOURCE: ISO 20418-1]
3.7
analyte
substance or chemical constituent that is subjected to measurement
[SOURCE: CEN/TS 15968:2010, 3.1]
3.8
measurand
quantity intended to be measured

Note 1 to entry: The specification of a measurand requires knowledge of the kind of quantity, description of the

state of the phenomenon, body, or substance carrying the quantity, including any relevant component, and the

chemical entities involved.

Note 2 to entry: In the second edition of the VIM and in IEC 60050-300:2001, the measurand is defined as the

“particular quantity subject to measurement”.

Note 3 to entry: The measurement, including the measuring system and the conditions under which the

measurement is carried out, might change the phenomenon, body, or substance such that the quantity being

measured may differ from the measurand as defined. In this case, adequate correction is necessary.

[SOURCE: ISO/IEC Guide 99:2007, 2.3, modified – removed Examples and note 4 to entry]

3.9
incurred samples

material produced by adding a specific amount of allergenic ingredient to a relevant matrix before it is

processed by food manufacturing techniques
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3.10
mass-to-charge ratio
m/z

dimensionless quantity formed by dividing the ratio of the mass (m) of an ion to the unified atomic

mass unit by its charge number (z)

[SOURCE: IUPAC Recommendations 2013: Definitions of terms relating to mass spectrometry, Pure

Appl. Chem., Vol. 85, No. 7, pp. 1515–1609]
3.11
retention time

time taken for an analyte to pass through a chromatography column from injection to detection

3.12
selected reaction monitoring
SRM

determination of a targeted analyte by measuring the precursor-ion to product-ion transition in MS/MS

fragmentation

Note 1 to entry: The mass filters in the mass spectrometer are adjusted to the ion masses of, respectively, the

precursor ion and the product ion (“mass windows”), increasing the selectivity and sensitivity of the

measurement.
Note 2 to entry: SRM is monitoring only a single fixed mass window.
Note 3 to entry: SRM is also called single reaction monitoring.
Note 4 to entry: Parent ion is another expression for precursor ion.
Note 5 to entry: Fragment ion is another expression for product ion.
3.13
multiple reaction monitoring
MRM
application of SRM to multiple product ions from one or more precursor ions

Note 1 to entry: MRM scans rapidly over multiple (narrow) mass windows and thus captures traces of multiple

product/fragment ion masses in parallel.
3.14
basic local alignment search tool
BLAST

bioinformatic algorithm for the comparison of primary sequence information such as amino acid

sequences of peptides or/and proteins or nucleotide sequences of DNA and/or RNA molecules

National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine, Bethesda MD, USA

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3.15
internal standard
ISTD

substance, which is similar in the chemical behaviour (chemical structure, polarity) and analytical

response to a certain target analyte

Note 1 to entry: Stable-isotope labelled ISTDs are preferred in mass spectrometry-based methods, which are

then sometimes called absolute quantitation (AQUA)-methods.
[SOURCE: IEC 62697-1:2012, 3.12]
3.16
standard addition
procedure in which a known amount of an analyte is added to a test sample

Note 1 to entry: To perform standard addition procedure, the test sample is divided in two (or more) test

portions. One test portion is analysed as such, whereas known amounts of an analyte are added to the second test

portion before analysis.
3.17
conversion factor
factor for the conversion of measurement results to a reporting unit

Note 1 to entry: The measurement results are converted into e.g. mg protein/kg food.

4 General laboratory requirements
4.1 Principle

Samples are extracted for proteins with a high-yielding, reproducible and matrix-specific procedure

including enzymatic digestion for the generation of peptides. A processing step for the reduction and

alkylation may be included. Specific marker peptides are measured by HPLC-MS/MS using targeted

MRM for qualitative or quantitative analysis. The signal (usually the peak area) of a marker peptide

measured at a specific chromatographic retention time is used for quantitation.
4.2 Apparatus and equipment

The laboratory shall use properly maintained equipment suitable for the method employed, e.g.

according to the requirements outlined by EN ISO/IEC 17025. In addition to standard laboratory

equipment, additional apparatus are described in the specific methods.

Apparatus and equipment should be maintained according to manufacturer’s instructions. Calibration

systems shall be available and calibration shall be routinely performed for measuring equipment,

according to laboratory quality assurance programmes.

In a tandem mass spectrometer, ions are formed in the ion source and separated by mass-to-charge

ratio in the first stage of mass spectrometry (MS1). In targeted mass spectrometry, ions of a particular

mass-to-charge ratio (precursor ions) are selected and product ions are created by collision-induced

dissociation, ion-molecule reaction, photodissociation, or other processes. The resulting ions are then

separated and detected in a second stage of mass spectrometry (MS2).

In MRM mode, the mass spectrometer is set to scan a very small mass range in MS1, typically one mass

unit, at the expected masses of the targeted precursor ions. After fragmentation, the product ions are

detected in MS2 by successively scanning small mass ranges at their expected masses. Alternatively,

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some MS instruments (e.g. high-resolution instruments (HRMS) with fast switching MS to MS/MS

capacity) allow the simultaneous detection of all product ions (Parallel Reaction Monitoring (PRM)).

In MS-based ion fragmentation analysis, the precursor ion selected for analysis shall be clearly defined,

e.g. as the molecular ion, a characteristic ion adduct of the molecular ion, a characteristic fragment ion

or a typical isotope ion. The selected product ions should not exclusively originate from the same part in

the molecule of the marker peptide that is to be analysed. The signal-to-noise ratio for each product ion

should be ≥ 3:1.
4.3 Material and reagents

Analytes shall be clearly described, including the type of standard (e.g. synthetic peptides, purified

protein, protein extracts), and information on purity, protein profiles …), storage conditions and shelf-

life.

Only reagents of MS quality grade and only de-ionized or distilled water or water that has been purified

should be applied for HPLC-MS analysis, unless otherwise stated in specific method descriptions. Other

reagents, such as enzymes, reducing and alkylating agents, should be of MS-grade. Buffer components,

organic solvents standards, analyte, reference material, controls, and samples are method-specific.

Storage conditions and shelf-life of reagents and samples should be determined in method validation

(method robustness) and clearly specified in the method protocol.
5 Method development
5.1 General

For the use of this document, general requirements of quality assurance for laboratories shall be

observed (e.g. concerning calibration of apparatus, extraction of samples and measurement of

replicates, blanks, use of reference materials, preparation of calibration curves, etc.). The scope of the

method, including applicability to certain food matrices, needs to be clearly defined.

Before conducting food allergen analysis, special considerations should be made regarding:

a) the laboratory lay-out (e.g. ideally, extraction working area should be spatially separated from

detection working area);

b) the current workflow (e.g. other activities in the laboratories that can increase the potential for

cross-contamination should be separated);

c) sample types handled (because cross-contamination issues can undermine the capability to

perform analysis reliably);

d) equipment in the laboratories (e.g. the risk of cross-contamination should be considered if

equipment is shared; use dedicated equipment when appropriate);

e) containers (e.g. disposable consumables are preferable, those that exhibit low protein binding - not

polystyrene);

f) general house-keeping tasks (e.g. effective cleaning of items and surfaces is important and should

be considered). Specific cleaning routines for rooms housing mass spectrometry instruments might

be required because cleaning chemicals might disturb the analysis, depending on the MS

instrument type.
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5.2 Sample preparation
5.2.1 Grinding

To ensure reliable results, sample preparation is a critical step in food allergen analysis. Its objective

shall be to provide a homogenous and representative portion of the original sample material.

Equipment should be available in the laboratory for sample homogenization - including apparatus for

milling, chopping, grinding and/or blending – the exact items depend on the sample types to be

routinely analysed.

A larger test portion will usually improve the potential for detection, as will reducing the particle size to

improve sample homogeneity.
5.2.2 Extraction/purification

The protein is extracted according to the procedure described in the specific method protocol.

Considerations should be given to:

a) the possibility of increasing the amounts, whilst maintaining the ratio between the test portion to

extraction volume, in order to improve the extractability/recovery of the target allergen;

b) the composition of the sample matrix;
5.2.3 Enzymatic digestion

In order to obtain a complete enzymatic digestion of the proteins in the test portion extract, it is

important to ensure that all specific cleavage sites are accessible to the respective enzymes. The

following steps are usually performed:

Denaturation step: unfolds the proteins (e.g. through sonication, addition of detergents or chaotropic

agents such as urea, denaturation with high temperature, etc.).

Reduction step: cleaves any disulfide bond present in the protein, resulting in free thiol groups (mainly

in cysteines).

Alkylation step: prevents disulfide bond reformation by irreversible capping of the free thiols obtained

during the reduction step. Typically, alkylation of cysteine is carried out by using iodoacetamide,

yielding S-carboxyamidomethylcysteine. Disulfide bond regeneration is known to be problematic before

enzymatic digestion is performed, but can also occur in the resulting peptide mixture. The omission of

the alkylation step would lead to random dimerization of cysteine-containing peptides, which would

significantly complicate the mass spectrometry analysis.

A number of enzymes are commonly used for the digestion of proteins in MS sample preparation.

Pepsin cleaves unspecifically the amino acid (AA) chain, whereas trypsin has very specific cleavage sites

(carboxyl side of lysines and arginines). Proteins that are enzymatically digested may differ widely in

their AA compositions and suitability for MS analysis. Therefore, digestion shall be optimized during

method development, and a digestion protocol including the choice of enzyme is part of the sample

preparation procedure.

After enzymatic digestion, protein extracts should immediately be proceed; protein extracts could be

stored if stability data are available.
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5.3 Detection
5.3.1 General
General information can be found in EN 15842.
5.3.2 Selection of the target proteins/peptides

The full sequence of the protein should be available in a reference protein database. Protein expression

variability should be characterized and ideally should be minimal. If different isoforms are known, they

should be characterized and their sequences should be available.

The sequences of the selected marker peptides should be specific for the allergenic ingredients to be

detected.

In theory, any protein unique to the allergenic ingredient may be used to indicate the presence of the

ingredient itself. In practice, several considerations will guide the selection of individual proteins and

peptides as targeted markers to guarantee specificity and robustness of the method.

During peptide selection, special attention should be paid to select marker peptides that are present in

all known isoforms of a protein. If not possible, different marker peptides should be selected to detect

all isoforms.

Attention should be paid with regard to the analysis of collective allergens such as fish, crustaceans,

molluscs, egg and milk. When selecting suitable marker peptides, it shall be considered whether all

species belonging to the allergen group or one distinct species are detected. When selecting suitable

marker peptides, e.g.milk, it has to be considered whether the selected marker peptides are specific

only for cow’s milk or are also present in buffalo, goat, sheep, horse,… milk. For egg, it has to be

considered whether the selected marker peptides are specific only for hen’s egg or are also present in

goose, duck, turkey, quail … eggs.

Preferably, a peptide with reproducible yield in sample preparation and high signal intensity is selected

as marker peptide. Optimally, a marker peptide should produce at least two measurable product ions.

Mass spectrometry-based methods should include one peptide for quantitation (“quantifier”) and one

peptide for quality control (“qualifier”) per targeted protein. The selection criteria for proteins and

peptides are summarized in Table 1.
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Table 1 — Proteins and peptides selection criteria for allergen analysis by mass spectrometry

[3,13]
Protein selection criteria Peptide selection criteria

Full protein sequence should be available, Sequence unique to the allergenic ingredient

including isoforms identification (BLAST details provided together with details
about used database and querying date/time)
Protein should be abundant in the appropriate endoprotease selection
ingredient, sufficient extraction recovery,
— peptide length: preferably 7-20 amino acids
relatively stable
— preferably no cysteines (oxidation)
— preferably no glutamine (deamidation)
— preferably no miscleavage
Protein expression variability (cultivar, Sequence ideally conserved among known
environmental/post-harvest treatment,…) isoforms
should be minimal
If not possible, 1 peptide should be selected per
isoform
Protein should be reproducible digested Minimize peptide modification:
by the selected endoprotease
Exclude sequence containing methionine (M),
glutamine (Q)in N-terminus of the peptide, and
asparagine (N)-glycine (G) motif;
Sequences containing cysteine (C), asparagine
and glutamine residues should preferably be
excluded
5.3.3 Selection of measured MRM transitions
Each pe
...

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