SIST EN 16274:2022
(Main)Method for analysis of allergens - Quantification of an extended list of 57 suspected allergens in ready to inject fragrance materials by gas chromatography mass spectrometry
Method for analysis of allergens - Quantification of an extended list of 57 suspected allergens in ready to inject fragrance materials by gas chromatography mass spectrometry
The present method permits the identification and quantification of the volatile compounds suspected as allergens, which are present in the fragrance compounds and fragrance raw materials used in cosmetic products. The analysis is performed by gas chromatography and mass spectrometry (GC-MS) on matrix samples which are "ready to be injected" and which are compatible with gas chromatography.
The analytes covered by this procedure are based on the contents of Tables 13.1 and 13.2 in the SCCS 1459/11 opinion document (1) and as listed in the legislation proposed by the European Commission. The rationale behind the final choice of procedure analytes is given in the table found in Annex J.
The method was validated at IFRA and CEN level.
Analyseverfahren für Allergene - Quantifizierung einer erweiterten Liste von 57 zu vermutenden Allergenen in einspritzfertigen Duftstoffen mittels Gaschromatographie/Massenspektrometrie
Das vorliegende Verfahren ermöglicht die Identifizierung und Quantifizierung der als Allergene vermuteten flüchtigen Verbindungen, die in den in kosmetischen Mitteln verwendeten Verbindungen von Duftstoffen und Rohstoffen von Duftstoffen enthalten sind. Die Analyse wird mithilfe der Gaschromatographie und Massenspektrometrie (GC MS) an Matrixproben durchgeführt, die „einspritzfertig“ und mit der Gaschromatographie kompatibel sind.
Die von diesem Verfahren erfassten Analyten basieren auf den Inhalten der in der Stellungnahme des SCCS 1459/11 (1) enthaltenen Tabelle 13.1 und Tabelle 13.2 und den von der Europäischen Kommission vorgeschlagenen aufgeführten Rechtsvorschriften. Die Begründung für die endgültige Auswahl der während des Verfahrens angewendeten Analyten ist in der in Anhang J enthaltenen Tabelle aufgeführt.
Das Verfahren wurde auf IFRA und CEN Ebene validiert.
Méthode d’analyse des allergènes - Quantification de la liste étendue des 57 allergènes suspectés dans les matières premières de parfumerie et les compositions parfumantes prêtes à être injectées, par chromatographie en phase gazeuse/spectrométrie de masse
La présente méthode permet l’identification et la quantification des composés volatils suspectés allergènes, qui sont présents dans les matières premières de parfumerie et les compositions parfumantes utilisées dans les produits cosmétiques. L’analyse est réalisée par chromatographie en phase gazeuse couplée à la spectrométrie de masse (CG-SM) sur des échantillons de matrice qui sont « prêts à être injectés » et qui sont compatibles avec la chromatographie en phase gazeuse.
Les analytes couverts par ce mode opératoire sont basés sur le contenu des Tableaux 13.1 et 13.2 du document d’opinion CSSC 1459/11 (1) et sont mentionnés dans la législation proposée par la Commission européenne. Les raisons qui justifient le choix final des analytes pour ce mode opératoire sont données dans le tableau figurant à l’Annexe J.
La méthode a été validée au niveau de l’IFRA et du CEN.
Analizne metode za alergene - Kvantitativno določevanje 57 domnevnih alergenov z razširjenega seznama v dišavnih izdelkih s plinsko kromatografsko analizo vzorcev, ki so pripravljeni za injeciranje, in masno spektrometrijo
Namen predlaganega standarda je opisati metodo za analizo 57 kemično določenih domnevnih alergenov (nekateri od njih obstajajo v več izomernih oblikah ali kot mešanice) v vzorcih dišav in surovin, ki so pripravljeni za injiciranje v skladu z mnenjem SCCS (SCCS/1459/11).
Ta nova analitična metoda uporablja plinsko kromatografijo in masno spektrometrijo (GC-MS) za odkrivanje in kvantitativno določevanje 57 dišavnih snovi in njihovih pomembnih izomerov s koncentracijo, ki presega 0,0002 % (2 mg/kg), v vzorcih dišav in surovin, ki so pripravljeni za injiciranje. Z zagotavljanjem te metode bo omogočeno presejanje (kompleksnih) vzorcev dišav in surovin, ki so pripravljeni za injiciranje, s čimer se ugotavlja prisotnost katerega koli od teh kemično določenih domnevnih alergenov. To bo torej osnova za izračun ustreznih informacij, na podlagi katerih bo lahko kozmetična industrija zagotovila ustrezno obveščanje potrošnikov. Ta analitična metoda uporablja plinsko kromatografijo in masno spektrometrijo s kombinacijo dveh kolon GC različne polarnosti s posebno metodologijo za kvantitativno določevanje.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-januar-2022
Nadomešča:
SIST EN 16274:2012
Analizne metode za alergene - Kvantitativno določevanje 57 domnevnih alergenov
z razširjenega seznama v dišavnih izdelkih s plinsko kromatografsko analizo
vzorcev, ki so pripravljeni za injeciranje, in masno spektrometrijo
Method for analysis of allergens - Quantification of an extended list of 57 suspected
allergens in ready to inject fragrance materials by gas chromatography mass
spectrometry
Analyseverfahren für Allergene - Quantifizierung einer erweiterten Liste von 57 zu
vermutenden Allergenen in einspritzfertigen Duftstoffen mittels
Gaschromatographie/Massenspektrometrie
Méthode d’analyse des allergènes - Quantification de la liste étendue des 57 allergènes
suspectés dans les matières premières de parfumerie et les compositions parfumantes
prêtes à être injectées, par chromatographie en phase gazeuse/spectrométrie de masse
Ta slovenski standard je istoveten z: EN 16274:2021
ICS:
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
71.100.70 Kozmetika. Toaletni Cosmetics. Toiletries
pripomočki
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 16274
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2021
EUROPÄISCHE NORM
ICS 71.100.60 Supersedes EN 16274:2012
English Version
Method for analysis of allergens - Quantification of an
extended list of 57 suspected allergens in ready to inject
fragrance materials by gas chromatography mass
spectrometry
Méthode d'analyse des allergènes - Quantification de la Analyseverfahren für Allergene - Quantifizierung einer
liste étendue des 57 allergènes suspectés dans les erweiterten Liste von 57 zu vermutenden Allergenen
matières premières de parfumerie et les compositions in einspritzfertigen Duftstoffen mittels
parfumantes prêtes à être injectées, par Gaschromatographie/Massenspektrometrie
chromatographie en phase gazeuse/spectrométrie de
masse
This European Standard was approved by CEN on 8 February 2021.
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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16274:2021 E
worldwide for CEN national Members.
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 6
5 Reagents . 7
5.1 Solvents . 7
5.1.1 Methyl pivalate . 7
5.1.2 Tert-Butyl Methyl Ether (MTBE) . 7
5.2 Reference samples (suspected allergens) . 7
5.2.1 General. 7
6 Apparatus . 11
6.1 Gas chromatograph equipped with flame ionization detector (GC-FID) . 11
6.2 Gas chromatograph coupled to a mass spectrometer (GC-MS) . 11
6.2.1 General. 11
6.2.2 GC-MS system. 11
6.3 Capillary columns for GC . 11
6.4 Analytical balance . 12
7 Stock and sample solutions - preparation and storage . 12
7.1 General information . 12
7.1.1 Choice of the solvent . 12
7.1.2 Miscellaneous . 13
7.2 Preparation of stock solutions from reference samples . 13
7.3 Preparation of internal standard solutions and calibration solutions . 13
7.3.1 General. 13
7.3.2 Stock Solutions of Internal Standards (1g/kg equivalent) . 13
7.3.3 Calibration Solutions Concentrations . 13
7.3.4 Preparation of Calibration solutions . 14
8 Mass spectrometer acquisition conditions . 16
8.1 Establishment of retention times and SIM chromatograph windows . 16
8.2 SIM Window – Set up and criteria . 17
8.3 GC-MS scan mode verification . 17
9 Sample analysis . 17
9.1 General. 17
9.2 Sample preparation for analysis . 17
9.3 Sequence . 18
9.3.1 Blank . 18
9.3.2 Calibration solutions . 18
9.3.3 Check solution . 18
9.3.4 Samples . 19
10 Data validation and treatment . 19
10.1 General. 19
10.2 Examination of Q values . 19
10.3 Relative intensity of characteristic ions in SIM . 20
10.4 Relative ion intensity using scan data . 20
10.5 Data verification scheme and reporting of final concentration . 21
10.6 GC-MS scan mode verification . 21
10.7 Mass spectrum evaluation in SCAN mode . 21
11 Test report . 21
Annex A (informative) Determination of calibrant and reference sample purity . 22
Annex B (informative) GC capillary column parameters . 29
Annex C (informative) SIM Ions for SIM or SIM-SCAN Use. 30
Annex D (informative) Example of SIM windows . 37
Annex E (informative) Example of chromatograms . 46
Annex F (normative) Decisional tree for quantification of suspected allergens . 48
Annex G (informative) Preparation of stock solutions from reference samples . 49
Annex H (informative) Calibration methods and approach . 54
Annex I (informative) Quantification of allergens . 56
Annex J (informative) Suspected allergens selected as analytes targets – Rationale . 59
Annex K (informative) Other general information. 69
Bibliography . 70
European foreword
This document (EN 16274:2021) has been prepared by Technical Committee CEN/TC 347 “Methods for
analysis of allergens”, the secretariat of which is held by SNV.
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 November 2021, and conflicting national standards shall
be withdrawn at the latest by November 2021.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 16274:2012.
The most significant technical changes made in comparison to the previous version are as follows:
— Number of allergens has been extended from 26 to 57 chemically defined molecules.
— Solvents: moves from methyl pivalate and ortho fluorotoluene and acetone to methyl pivalate and
MTBE and other solvents provided they are tested prior being used.
— Sample preparation: allows to use weight/volume approach rather than only weight/weight.
— Data processing: provides explanations for a calibration curve using a forced through zero approach.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive(s).
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.
Introduction
Directive 2003/15/EC amending Council Directive 76/768/EEC, relating to cosmetic products, regulates
the obligation to inform consumers of the presence of 24 chemically-defined fragrance substances
identified as potential allergens in cosmetic products. Following the publication of the Scientific
Committee on Consumer Safety’s document (SCCS/1459/11), it was proposed to extend that to 57
fragrance substances, some of them existing under several isomeric forms or as mixtures. This required
the development of a new quantification method in response to the evolution of regulatory requirements.
The new analytical method has been developed using gas chromatography and mass spectrometry (GC-
MS), to detect and to quantify the 57 fragrance substances and their relevant isomers in ready to inject
fragrance compounds and fragrance raw materials.
The method described in this document does not include requirements for the preparation of samples in
matrices for which direct injection in GC is not feasible.
The present document describes a working analytical method based on IFRA Analytical Working Group
developments. The analytical method was validated based on a ring test performed by the CEN working
group using the accuracy profile approach.
1 Scope
The present method permits the identification and quantification of the volatile compounds suspected as
allergens, which are present in the fragrance compounds and fragrance raw materials used in cosmetic
products. The analysis is performed by gas chromatography and mass spectrometry (GC-MS) on matrix
samples which are “ready to be injected” and which are compatible with gas chromatography.
The analytes covered by this procedure are based on the contents of Tables 13.1 and 13.2 in the
SCCS 1459/11 opinion document (1) and as listed in the legislation proposed by the European
Commission. The rationale behind the final choice of procedure analytes is given in the table found in
Annex J.
The method was validated at IFRA and CEN level.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
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
4 Principle
This procedure has a calibration range between 2 ppm and 240 ppm, this permits the quantification of
suspected allergens in diluted matrices in the range 20 ppm to 2 400 ppm per analyte. Beyond the upper
concentration level the recommendation is that the sample should be diluted further, or that GC-FID (GC
with Flame Ionization Detection) is used preferably in combination with internal standard and response
factors.
The matrix samples are analysed for suspected allergens by GC-MS in a total of 4 runs, using 2 analyte
sets, both injected on two separate columns of differing polarity. Where necessary the matrix samples
should first be diluted in an appropriate solvent.
Their identification and quantification are achieved by selected ion monitoring (SIM; SIM-SCAN) mode
via the relative abundance of 3 characteristic fragment ions. The calculation and use of the corresponding
Q value or similar data evaluation factor can be applied and a ‘Decisional Tree’ (see Annex F, Figure F.1)
for the final inspection and validation of the data by a trained and experienced analyst is described. An
additional full-SCAN analysis is recommended to confirm the presence of the allergen in matrix samples
if only SIM methodology was used.
Their quantification is achieved in all modes by calibration using standard solutions and the internal
standards 1,4-dibromobenzene and 4,4’-dibromobiphenyl. The ‘Decisional Tree’ is employed to
determine the final concentration taking into account the different concentration values obtained from
analysis on both columns.
5 Reagents
5.1 Solvents
5.1.1 Methyl pivalate
CAS [598-98-1]
Purity ≥ 99 %
Distillation to obtain a purity > 99,9 % is recommended to eliminate impurities, which are likely to
interfere with the signal of analytes such as terpenes. If a commercial grade is used then that shall be
analysed and confirmed as not giving rise to interfering artefacts.
5.1.2 Tert-Butyl Methyl Ether (MTBE)
CAS [1634-04-4]
Purity ≥ 99 %
Distillation to obtain a purity > 99,9 % is recommended to eliminate impurities, which are likely to
interfere with the signal of analytes such as terpenes. If a commercial grade is used then that shall be
analysed and confirmed as not giving rise to interfering artefacts.
Alternative solvents are available. If these are used for this method, then the Operator shall undertake
sufficient evaluation to verify that the solvent contains no constituents that would interfere with the
analytes in this method.
5.2 Reference samples (suspected allergens)
5.2.1 General
Initially, the purity of all standards should be measured by GC-FID (Annex A) if not certified by the
supplier. The precise CAS number of the target analyte and the rationale behind this is given in Annex J.
5.2.1.1 Acetyl cedrene alpha (main isomer in Vertofix® referred to in CAS number), CAS No.
[32388-55-9]
Highly variable composition with at least twelve constituents of molecular weight equal to 246 atomic
mass units (hereafter u).
5.2.1.2 Acetyl isoeugenol / Isoeugenyl acetate, CAS No. [93-29-8]
5.2.1.3 Amyl salicylate, pentyl salicylate CAS No. [2050-08-0]
This can contain isoamyl salicylate (CAS No. [87-20-7]), which should not be included in this assay.
5.2.1.4 Alpha amyl cinnamaldehyde (Flosal®), CAS No. [122-40-7, 78605-96-6]
2 possible isomers (E, Z); only E is quantified.
5.2.1.5 Alpha amylcinnamyl alcohol, CAS No. [101-85-9, 184900-07-0]
2 possible isomers (E, Z); only E is quantified.
5.2.1.6 Anethole, CAS No. [104-46-1, 4180-23-8]
2 possible isomers (E, Z); only E is quantified.
5.2.1.7 Anise alcohol, CAS No. [105-13-5]
5.2.1.8 Benzaldehyde, CAS No. [100-52-7]
5.2.1.9 Benzyl alcohol, CAS No. [100-51-6]
5.2.1.10 Benzyl benzoate, CAS No. [120-51-4]
5.2.1.11 Benzyl cinnamate, CAS No. [103-41-3]
2 possible isomers (E,Z), only E is quantified.
5.2.1.12 Benzyl salicylate, CAS No. [118-58-1]
5.2.1.13 Camphor, CAS No. [76-22-2]
5.2.1.14 Carvone, CAS No. [99-49-0]
5.2.1.15 Caryophyllene beta, CAS No. [87-44-5]
5.2.1.16 Cinnamaldehyde, CAS No. [104-55-2, 14371-10-9]
2 possible isomers (E, Z), only E is quantified.
5.2.1.17 Cinnamyl alcohol, CAS No. [104-54-1, 4407-36-7]
2 possible isomers (E, Z), only E is quantified.
5.2.1.18 Citral, CAS No. [5392-40-5]
5.2.1.18.1 Neral
5.2.1.18.2 Geranial
Both Neral (Z isomer, CAS No. [106-26-3]) and Geranial (E isomer, CAS No. [141-27-5]) are quantified.
5.2.1.19 Citronellol, CAS No. [106-22-9]
5.2.1.20 Coumarin, CAS No. [91-64-5]
5.2.1.21 Damascenone beta (Rose Ketone-4), CAS No. [23696-85-7]
5.2.1.22 Damascone alpha, CAS No. [43052-87-5] covers both E and Z isomers
2 possible isomers (E, Z); only the E main isomer CAS No. [24720-09-0] (92 to 99) % is quantified.
5.2.1.23 Damascone beta (E), CAS No. [23726-91-2]
5.2.1.24 Damascone delta (Rose Ketone-3), CAS No. [57378-68-4]
3 possible isomers (trans/trans, cis/trans, trans/cis) only the major isomer (trans/trans, up to 90) % is
quantified.
5.2.1.25 Dimethylbenzylcarbinyl acetate (DMBCA), CAS No. [151-05-3]
5.2.1.26 Ebanol, CAS No. [67801-20-1]
5.2.1.26.1 Ebanol Isomer 1
5.2.1.26.2 Ebanol Isomer 2
The 2 isomers (E, Z) are quantified.
5.2.1.27 Eugenol, CAS No. [97-53-0]
5.2.1.28 Eugenyl acetate, CAS No. [93-28-7]
5.2.1.29 Farnesol, CAS No. [4602-84-0]
Recommendation: Use E,E calibration curve to quantify other isomers.
4 possible isomers: (E,E) isomer is CAS No. [106-28-5] and the (Z,E) isomer is CAS No. [3790-71-4], and
two additional minor isomers.
5.2.1.30 Galaxolide (Hexamethylindanopyran), CAS No. [1222-05-5]
Only the two main isomers are quantified.
5.2.1.31 Geraniol, CAS No. [106-24-1]
5.2.1.32 Geranyl acetate, CAS No. [105-87-3]
5.2.1.33 Hexadecanolactone / Dihydroambrettolide, CAS No. [109-29-5]
5.2.1.34 Hexylcinnamaldehyde alpha, CAS No. [101-86-0, 165184-98-5]
2 isomers at least; only the E is quantified.
5.2.1.35 Hydroxycitronellal, CAS No. [107-75-5]
5.2.1.36 Tetramethylacetyloctahydronaphthalene (ISO E® Super)
NOTE This consists of several isomers including the alpha, (CAS No. [68155-66-8]); the major isomer, beta
(CAS No. [54464-57-2]); the gamma (CAS No. [68155-67-9]) and a minor (CAS No. [54464-59-4]).
5.2.1.37 Isoeugenol, CAS No. [97-54-1]
2 possible isomers (E, Z), only E- isoeugenol is quantified.
5.2.1.38 Isomethylionone alpha, CAS No. [127-51-5]
Can contain beta isomethylionone (CAS No. [79-89-0]), alpha methylionone (CAS No. [7779-30-8]), beta
methylionone (CAS No. [127-43-5]), pseudo isomethylionone (CAS No. [1117-41-5]).
Other grades may contain other isomers including the major one. These constituents should not be
assayed.
5.2.1.39 Butylphenyl methylpropional (Lilial®), CAS No. [80-54-6]
5.2.1.40 Limonene, CAS No. [138-86-3]
5.2.1.41 Linalool, CAS No. [78-70-6]
5.2.1.42 Linalyl acetate, CAS No. [115-95-7]
5.2.1.43 Hydroxyisohexyl 3-cyclohexene carboxaldehyde (Lyral®), CAS No. [31906-04-4]
Contains Hydroxyisohexyl 4-cyclohexene carboxaldehyde (CAS No. 51414-25-6) (noted Lyral minor) that
has to be quantified.
5.2.1.44 Trimethyl-benzenepropanol (Majantol®), CAS No. [103694-68-4]
5.2.1.45 Menthol, CAS No. [1490-04-6, 89-78-1]
5.2.1.46 Methyl salicylate, CAS No. [119-36-8]
5.2.1.47 Methyl-2-octynoate (Folione®), CAS No. [111-12-6]
5.2.1.48 Pinene alpha, CAS No. [80-56-8]
5.2.1.49 Pinene beta, CAS No. [127-91-3]
5.2.1.50 3-Propylidene phthalide, CAS No. [17369-59-4]
The 2 isomers (E,Z) (E-isomer: CAS No. [56014-72-3] and Z-isomer: CAS No.[94704-89-9]) are quantified.
5.2.1.51 Salicylaldehyde, CAS No. [90-02-8]
5.2.1.52 Santalol, CAS No. [11031-45-1]
NOTE The 2 isomers, alpha-santalol (CAS No. [115-71-9]) and beta-santalol (CAS No. [77-42-9]), are quantified.
5.2.1.53 Sclareol, CAS No. [515-03-7]
5.2.1.54 Terpinene alpha, CAS No. [99-86-5]
5.2.1.55 Terpineol-alpha, CAS No. [98-55-5]
5.2.1.55.1 for terpineol gamma CAS No. [586-81-2]
5.2.1.55.2 for Terpineol cis-beta CAS No. [138-87-4]
5.2.1.55.3 for Terpineol trans-beta CAS No. [7299-41-4]
Recommendation: use alpha-terpineol calibration curve to quantify other isomers.
5.2.1.56 Terpinolene, CAS No. [586-62-9]
5.2.1.57 Vanillin, CAS No. [121-33-5]
5.3 Internal standards (ISTD)
5.3.1 1,4-Dibromobenzene (IS ), CAS No. [106-37-6], purity ≥ 98 %
A
5.3.2 4,4'-dibromobiphenyl (IS ), CAS No. [92-86-4], purity ≥ 98 %
B
6 Apparatus
6.1 Gas chromatograph equipped with flame ionization detector (GC-FID)
This apparatus is only used to determine the purity of reference samples intended to be used for
calibration purposes and the ISTDs before quantitative analysis if required, or for the quantification of
high concentration analytes (above 2,4 % as found in essential oils for example). It is recommended to
perform the purity study using the procedure described in Annex A.
[3]
A GC-FID method is available for the measurement of analytes at concentrations above 2,4 %. Note
that this method does not implicitly cover all the allergens mentioned in this procedure: however, the
principle covered in that method can be applied to all the analytes contained within the proposed
legislation and this procedure.
6.2 Gas chromatograph coupled to a mass spectrometer (GC-MS)
6.2.1 General
This apparatus is used for quantitative analysis, to check for the presence and measure the concentration
of the suspected allergens. The system shall be able to comply with the following recommendation.
6.2.2 GC-MS system
a) Equipped with electronic control of carrier gas pressures and/or flows;
b) it is recommended that an autosampler, fitted with a syringe of suitable size, be used for the injection
of the calibration and sample solutions;
c) the glass injection liner shall be inert with an interior volume compatible with the expansion volume
of the dilution solvent;
d) two capillary columns of different phase types are to be used for quantitative analysis;
e) mass spectrometer tuning and the levels of air and water should be checked regularly to ensure
optimal sensitivity, as well as the injector cleanliness to maintain analytical performance.
The sensitivity of the mass spectrometer should also be optimized through maximizing the detector
signal to noise ratio. One option could be to increase the detector (electron multiplier) voltage according
to the instrument capabilities and manufacturers recommendations.
6.3 Capillary columns for GC
Whilst the retention times and SIM windows have been specified for the columns mentioned, these are
for guidance only and the user shall verify all compound retention times and the associated SIM windows
for their own installations.
The column lengths are those used for the initial development and validation by the AWG. In practice,
additional separation of the target analyte may be achieved by moving to a longer column (50 m or 60 m).
If such columns are used, then it is the user’s responsibility to validate the performance of that column
type/length in the context of this method (resolution; SIM windows) and validate that length for the
analytes(s) being investigated (see Annex B).
Table 1 — Recommended GC Column Phase/Types and Analytical Parameters
Dimensions Carrier MS Mean
Recommended
(Length × gas conditions resolution
oven Injection
Phase internal
temperature conditions R
diameter × thic
program
kness)
Helium Source SIM method
Constant Temperature 1
80 °C for 4 min,
flow at at 250 °C –
1.43
then 15 °C/min
1,2 mL/m Quadrupole
to 105 °C for 2
in temp. at
100 % min,
(velocity 150 °C –
30 m × 0,25 mm
Split/
Polydimethylsiloxane then 4 °C/min to
approx. Scanning
× 0,25 µm
SIM method
(PDMS) 150 °C, then splitless
40 cm/se mass range
10 °C/min to injector at
c) (m/z) from
280 °C – hold 5 250 °C -
1.40
35 Da to 350
min. Deactivated
Da
liner-
-
Inject 1 µL
Ionization
SIM method
with split
energy:
1:20 – Inj. 1
70 eV
Temp.
1.40
80 °C for 1 min,
250 °C
then 10 °C/min
50 %
(same for
to 135 °C for 2
Phenylmethylpolysiloxane
both
min,
calibration
50 % 30 m × 0,25 mm
then 3 °C/min to
and
Polydimethylsiloxane × 0,25 µm
SIM method
170 °C for 1 min,
samples)
(PDMS)
then 10 °C/min
to 280 °C – hold 1.42
5 min
6.4 Analytical balance
All weighing of reference samples and internal standards shall be carried out on an analytical balance
with 0,000 1 g readability.
7 Stock and sample solutions - preparation and storage
7.1 General information
7.1.1 Choice of the solvent
Methyl pivalate and MTBE are suitable for the preparation of stock and calibration solutions as well as
the dilution of samples prior to analysis as they have been proven to satisfy the following requirements:
1) inertness to components and allergens in fragrance matrices;
2) medium volatility to ensure solution’s stability and concentration;
3) expansion volume compatible with the interior volume of the injector insert.
Recommendation: For information related to the solvent, see 5.1.
The use of any other solvent requires the user to perform preliminary tests to demonstrate, in particular,
inertness towards the analytes and to determine its purity. The use of solvents possessing hydroxyl or
carbonyl functions (ethanol, methanol, acetone…) is not recommended in order to avoid degradation or
reaction with the analytes (e.g. acetal formation).
7.1.2 Miscellaneous
The same solvent shall be used to prepare the calibration solutions, the system blank and to dilute
samples.
7.2 Preparation of stock solutions from reference samples
The preparation of the Calibration Stock Solutions and the subsequent Calibration Solutions can be made
from two sources – from the single reference materials themselves (after purity
determination/confirmation), or from commercially obtained Calibration Solutions of known constituent
concentrations.
The following description covers making the calibration solutions from commercial Calibration Solutions
of known concentration. If the user wishes to follow the procedure for making these solutions from single
reference materials, the procedure for this can be found in Annex G.
The following description suggests working using a weight/weight approach when preparing the
different solutions. It is also possible to use volumes when preparing the diluted solutions without any
drastic loss of accuracy in the final results.
7.3 Preparation of internal standard solutions and calibration solutions
7.3.1 General
All below explanations are provided for a weight/weight approach. In case of weight/volume, the only
difference is that the analyst does not have to weigh the solvent but rather make up with the solvent to
the mark in the flask. Operators can refer to Annex G if externally purchased solutions are not used.
7.3.2 Stock Solutions of Internal Standards (1g/kg equivalent)
Prepare a stock solution (S-ISTD) containing 1 g of 1,4-dibromobenzene (5.3.1) and 1 g 4,4’-
dibromobiphenyl (5.3.2) made up to 1 kg with methyl pivalate or MTBE. This solution can be prepared
as follows:
— Prepare a solution (S-ISTD) by weighing accurately 0,1 g of each compound (5.3.1 and 5.3.2) in a
suitable flask and make up to 100 g with methyl pivalate or MTBE.
— In case of weight/weight concentrations, note the weight of each compound and the final weight of
solvent used, to determine the final solution concentration of each internal standard.
— Pay attention to the differences in density between the solvents. For instance, for MTBE, relative
density is 0,740 4, thus only weigh 74 mg of each of the standards and complete with MTBE in a
100 ml flask (the volume of added MTBE will be 74 g).
7.3.3 Calibration Solutions Concentrations
To limit chemical reactions between, and degradation of analytes, two separated stock solutions are
prepared in the dilution solvent:
— One stock solution contains alcohols and terpenes without carbonyl function;
— The other solution contains the remaining suspected allergens, in particular aldehydes and ketones.
These 2 solutions are called A1 and A2 respectively.
The calibration solutions are prepared from these two sets of ‘stock’ solutions, A1 and A2, containing the
analytes at a nominal concentration of 2 g/kg. The final calibration solutions are arrived at by a dilution
of these solutions (or a further derivative of these; A3 and A4) to a known weight with methyl pivalate or
MTBE.
For Commercial Standards of alternative concentrations, the dilution volumes used may need to be
adjusted to arrive at the final concentrations as shown in Table 2.
To avoid dispensing very small (i.e. < 10µL) volumes for the lower calibration concentrations from the
stock solutions A1 and A2, if required, two further solutions A3 and A4 can be prepared using the
following schemes (Table 2 and Table 3).
Note the weight of all standard solutions as well as the solvent weight, in case you are using w/w
concentrations.
Table 2 — Preparation volume for Solution A3
Final Volume of Final
Primary Solution Volume of Primary
Diluted Solution Concentration of
(2 g/kg) Solution
(A3) Allergens (mg/kg)
A1 1 mL 10 mL 200
Table 3 — Preparation volume for Solution A4
Final Volume of Final
Primary Solution Volume of Primary
Diluted Solution Concentration of
(2 g/kg) Solution
(A4) Allergens (mg/kg)
A2 1 mL 10 mL 200
Ensure they are adequately stoppered to minimize any potential evaporation of the solvent.
The stability of substances in the stock solutions, prepared according to the protocol described below,
was investigated in glass bottle in darkness and in a freezer at a temperature lower than −18 °C. One
month stability period was ascertained under these conditions for the two stock solutions A1 and A2 as
well as for A3 and A4.
If the storage conditions differ from those described above, investigations shall be performed to
demonstrate the substances stability in the new defined conditions.
7.3.4 Preparation of Calibration solutions
Prepare the calibration solutions C1, C2, C3 by diluting the two allergen primary solutions, A1 and A2 to
10mL with methyl pivalate or MTBE after the addition of the internal standard solution, (S-ISTD), (8.3)
according to the following instructions, and referring to the dilution scheme in Table 4. The weight of the
individual combined solutions shall be recorded.
Similarly, prepare the calibration solutions C4, C5, C6, C7 by diluting solutions A3 and A4 (8.3) to 10 mL
with methyl pivalate after the addition of the internal standard solution, (S-ISTD), (8.3) according to the
dilution scheme in Table 4. The weight of the individual combined solutions shall be recorded.
To create the Calibration Solutions C1 through C3, the following process should be adopted:
— For Solution C1: Dispense 1,2 mL of Solution A1 and 1,2 mL of solution A2 into a 10 mL tared
volumetric flask, noting the weights of A1 and A2 (mA1 and mA2 respectively) taken at each step.
Add 1mL of the Internal Standard solution, note the weight, and make up to the 10 mL mark with
solvent.
Note the final weight = mA1+mA2+mISTD+msolvent.
— For Solution C2: Dispense 0,6 mL of Solution A1 and 0,6mL of solution A2 into a 10 mL tared
volumetric flask, noting the weights of A1 and A2 (mA1 and mA2 respectively) taken at each step.
Add 1 mL of the Internal Standard solution, note the weight, and make up to the 10 mL mark with
solvent.
Note the final weight= mA1+mA2+mISTD+msolvent.
— For Solution C3: Dispense 0,25 mL of Solution A1 and 0,25 mL of solution A2 into a 10 mL tared
volumetric flask, noting the weights of A1 and A2 (mA1 and mA2 respectively) taken at each step.
Add 1 mL of the Internal Standard solution, note the weight, and make up to the 10 mL mark with
solvent.
Note the final weight= mA1+mA2+mISTD+msolvent.
For Solutions C4 to C7, the solutions A3 and A4 should be used to avoid potential errors in dispensing
small weights of A1 and A2:
— For Solution C4: Dispense 1 mL of Solution A3 and 1 mL of solution A4 into a 10 mL tared volumetric
flask, noting the weights of A3 and A4 taken at each step.
Add 1 mL of the Internal Standard solution, note the weight, and make up to the 10 mL mark with
solvent.
Note the final weight= mA3+mA4+mISTD+msolvent.
— For Solution C5: Dispense 0,5 mL of Solution A3 and 0,5 mL of solution A4 into a 10 mL tared
volumetric flask, noting the weights of A3 and A4 taken at each step.
Add 1 mL of the Internal Standard solution, note the weight, and make up to the 10 mL mark with
solvent.
Note the final weight= mA3+mA4+mISTD+msolvent.
— For Solution C6: Dispense 0,25 mL of Solution A3 and 0,25 mL of solution A4 into a 10 mL tared
volumetric flask, noting the weights of A3 and A4 taken at each step.
Add 1 mL of the Internal Standard solution, note the weight, and make up to the 10 mL mark with
solvent.
Note the final weight= mA3+mA4+mISTD+msolvent.
— For Solution C7: Dispense 0,1 mL of Solution A3 and 0,1 mL of solution A4 into a 10 mL tared
volumetric flask, noting the weights of A3 and A4 taken at each step.
Add 1 mL of the Internal Standard solution, note the weight, and make up to the 10 mL mark with
solvent.
Note the final weight= mA3+mA4+mISTD+msolvent.
The 7 points calibration curve has been used to validate this method. It is also possible to work with a 6
point calibration curve keeping only C1(240 mg/kg), C2 (120 mg/kg), C3 (50 mg/kg), C4 (20 mg/kg), C5
(10 mg/kg), C7 (2 mg/kg).
These instructions can be summarized and found in Table 4 below.
Table 4 — Individual Allergen Calibration Standard Solution concentrations
Allergens Volume Volume Volume Concentration Final
Concentration of of of of Internal Volume of
(mg/kg) Allergen Allergen Internal Standards
Calibration
Standard (mg/kg)
Solution Solution Solution
Solution
(mL) (mL) (mL)
added
(mL)
Primary A1 A2
(2 g/kg) (2 g/kg)
Solutions (1 g/kg)
Used
C1 240 1,200 1,200 1,000 100 10
C2 120 0,600 0,600 1,000 100 10
C3 50 0,250 0,250 1,000 100 10
Secondary A3 A4
(0,2 g/kg) (0,2 g/kg)
Solutions (1 g/kg)
C4 20 1,000 1,000 1,000 100 10
C5 10 0,500 0,500 1,000 100 10
C6 5 0,250 0,250 1,000 100 10
C7 2 0,100 0,100 1,000 100 10
General notes and information:
In the absence of stability data, freshly prepare these solutions and do not store. Do not use these
calibration solutions after 48 h.
8 Mass spectrometer acquisition conditions
8.1 Establishment of retention times and SIM chromatograph windows
Operating in SCAN mode, inject one of the standard solutions (High to mid calibration concentrations for
example) to determine the retention times and suitable SIM windows for each analyte.
It is recommended that a concentration in the mid-range of the calibration curve be selected.
For each column, the user will create two SIM or SIM-SCAN methods (SIM1 and SIM2) in order to analyse
the 57 potentially allergenic substances in two sets of analytes.
Typical SIM ions (I1, I2 and I3) and their associated Internal Standard are detailed in Table C.1, Table C.2,
Table C.3 as given in Annex C. In rare cases, the selected ions for one given allergen on a polar column can
be different from the selected ions for a non-polar column.
8.2 SIM Window – Set up and criteria
The choice of internal standard to be assigned for quantification of each analyte is also provided in
Annex C, Table C.1, Table C.2 and Table C.3.
Examples of SIM windows for the two sets of analytes and the two columns are given in Annex D,
Table D.1, Table D.2, Table D.3 and Table D.4.
As far as possible, each SIM window should not include more than 6 ions. Nevertheless, in the regions of
the chromatogram where the elution of the analytes are close to one another, the SIM windows may
contain up to 9 or more ions. In each case, the dwell time should be chosen to have a minimum of 3 scan
cycles per sec, and/or at least 10 to 15 acquisition points per chromatographic peak for accurate
integration.
The width of the SIM window shall also take into account peak shape (including potential peak tailing)
and possible elution delay to facilitate further peak integration. The same acquisition time shall be
applied for all allergens ions in the same SIM window.
8.3 GC-MS scan mode verification
A full SCAN mode is required to identify the presence of peaks that are thought to have moved out of their
usual SIM window or been hidden by interfering peaks (see Annex E, including Figures E.1, E.2, E.3 and
E.4).
For complementary information, see Annex K.
9 Sample analysis
9.1 General
It is recommended to perform a 10-fold dilution of the sample prior to analysis, which implies weighing
exactly about 1 g of sample in a 10 mL volumetric flask. If analyte concentrations are found to be outside
of the calibration range, then dilution of another sample up to a maximum dilution of 240-fold can be
undertaken with re-analysis.
9.2 Sample preparation for analysis
— Accurately weigh (to the nearest milligram) a sample amount (typically 1 g), in a 10 mL volumetric
flask.
— Add 1,0 mL of the internal standard solution (S-ISTD), 8.3, and note the weight. The final
concentration of internal standard added should be the same as that used in the calibration solutions.
— Adjust to the 10 mL mark with methyl pivalate (or the same dilution solvent as that used to prepare
calibration solutions).
— Note the final weight (mDIL) of this solution.
Homogenize and transfer an aliquot of the prepared sample into a GC vial for GC-MS analysis.
9.3 Sequence
9.3.1 Blank
If the GC-MS system has been left ‘idle’ for any length of time, material can build up in the injection system
and create interference with the analysis. It is therefore recommended that an initial blank solvent run
be undertaken to clean the inlet of any residues prior to the start of the calibration/analysis sequence.
Adequate blank values should be obtained before analysis commences. No analyte should be detected in
the blank analysis above 75 % of the lower calibration level. Blank corrections are not applied to this
procedure. A blank analysis is used within the analytical sequence to determine carry-over or
contamination of the system. If analyte concentrations above 75 % of the lower level of calibration are
seen in these samples, then the inlet system requires
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