SIST EN 17644:2022
(Main)Foodstuffs - Detection of food allergens by liquid chromatography - mass spectrometry (LC-MS) methods - General considerations
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
Standards Content (Sample)
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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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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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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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
2
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
n
MS
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 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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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
RT
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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3.15
internal standard
IS
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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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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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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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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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.
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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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 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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oSIST prEN 17644:2021
prEN 17644:2021 (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 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
2
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
n
MS
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
RT
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
1
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
1
National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine, Bethesda MD, USA
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3.15
internal standard
IS
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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