SIST EN 16274:2021

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 proposed standard aims at describing a method to analyze 57 chemically defined suspected allergens (some of them existing under several isomeric forms or as mixtures) in ready to inject fragrance and raw material samples according to the SCCS opinion. (SCCS/1459/11).
This new analytical method uses gas chromatography and mass spectrometry (GC-MS) to detect and to quantify the 57 fragrance substances and their relevant isomers at a concentration higher than 0.0002% (2 mg/kg) in ready to inject fragrance and raw material samples. Making this method available will allow the screening of (complex) ready to inject fragrance and raw material samples to be undertaken for the presence of any of those chemically defined suspected allergens. It will therefore be a basis for the calculation of adequate information to the cosmetics industry in order to provide adequate consumer information. The present analytical method uses GC-MS by combination of two GC columns of different polarity with a dedicated methodology for quantitation.

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

General Information

• Status: Not Published
• Public Enquiry End Date: 02-Apr-2020
• ICS: 71.040.50 - Physicochemical methods of analysis
• ICS: 71.100.70 - Cosmetics. Toiletries
• Technical Committee: KDS - Cosmetics, chemical disinfectants and surface active agents
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 12-Aug-2021
• Due Date: 17-Oct-2021
• Ref Project: EN 16274:2021 - 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

RELATIONS

• Replaces: SIST EN 16274:2012 - Methods for analysis of allergens - Quantification of suspected fragrance allergens in consumer products - Step 1: GC analysis of ready-to-inject sample
• Effective Date: 01-Nov-2021

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN 16274:2020
01-marec-2020
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

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 zu
vermutenden Allergenen in einspritzfertigen Duftstoffen mittels
Gaschromatographie/Massenspektrometrie
Méthodes d'analyse des allergènes - Quantification des fragrances allergènes
suspectées dans les produits de consommation - Étape 1 : Analyse par GC
d'échantillons prêts à être injectés
Ta slovenski standard je istoveten z: prEN 16274
ICS:
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
71.100.70 Kozmetika. Toaletni Cosmetics. Toiletries
pripomočki
oSIST prEN 16274:2020 en,fr,de

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

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oSIST prEN 16274:2020
DRAFT
EUROPEAN STANDARD
prEN 16274
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2020
ICS 71.100.60 Will supersede 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

Méthodes d'analyse des allergènes - Quantification des Analyseverfahren für Allergene - Quantifizierung einer

fragrances allergènes suspectées dans les produits de erweiterten Liste von zu vermutenden Allergenen in

consommation - Étape 1 : Analyse par GC einspritzfertigen Duftstoffen mittels
d'échantillons prêts à être injectés Gaschromatographie/Massenspektrometrie

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

CEN/TC 347.

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

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16274:2020 E

worldwide for CEN national Members.
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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

5.2 Reference samples (suspected allergens) ............................................................................................. 7

5.3 Internal standards (ISTD) .......................................................................................................................... 11

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

6.3 Capillary columns for GC ............................................................................................................................ 11

6.4 Analytical Balance ........................................................................................................................................ 12

7 Mass Spectrometer Acquisition Conditions ........................................................................................ 12

7.1 Establishment of Retention Times and SIM chromatograph Windows ..................................... 12

7.2 SIM Window – Set up and Criteria ........................................................................................................... 13

7.3 GC-MS Scan Mode Verification .................................................................................................................. 13

8 Stock and Sample solutions - preparation and storage .................................................................. 13

8.1 General information .................................................................................................................................... 13

8.2 Preparation of stock solutions from reference samples ................................................................ 14

8.3 Preparation of internal standard solutions and calibration solutions ..................................... 14

9 Sample Analysis ............................................................................................................................................. 17

9.1 General.............................................................................................................................................................. 17

9.2 Sample Preparation for Analysis............................................................................................................. 17

9.3 Sequence .......................................................................................................................................................... 18

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 ................................................... 20

10.6 GC-MS scan mode verification .................................................................................................................. 20

10.7 Mass spectrum evaluation in SCAN mode ............................................................................................ 20

11 Assay report .................................................................................................................................................... 21

12 References: ...................................................................................................................................................... 21

Annex A (informative) Determination of calibrant and reference sample purity ............................. 22

Annex B (informative) GC capillary column parameters............................................................................. 28

Annex C (informative) SIM Ions for SIM or SIM-SCAN Use ........................................................................... 29

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Annex D (informative) Example of SIM Windows .......................................................................................... 38

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

Tables

Table 1 — Recommended GC Column Phase/Types & Analytical Parameters .................................................... 12

Table 2 — Preparation volume for Solution A3 ................................................................................................................ 15

Table 3 — Preparation volume for Solution A4 ................................................................................................................ 15

Table 4 — Individual Allergen Calibration Standard Solution concentrations .................................................... 17

Table 5 — Maximum allowable tolerances for the relative ion intensities (expressed in percentage of

intensity of the most abundant ion i.e. the base peak) ................................................................................................... 20

Table C.1 — SIM ions (m/z) of suspected allergens listed in the elution order on 100%PDMS-type

column, corresponding SIM methods (1 or 2) and internal standard (ISA or ISB) used for quantification

............................................................................................................................................................................................................... 29

Table C.2 — SIM ions (m/z) of allergens listed in the elution order on 50%Polydimethylsiloxane

50%Phenylmethylpolysiloxane -type column, corresponding SIM methods (1 or 2) and internal

standard (ISA or ISB) used for quantification .................................................................................................................... 32

Table C.3 — Alternative SIM ions (m/z) of allergens listed in the elution order on 100% PDMS or 00%

PDMS type columns, corresponding SIM methods (1 or 2) and internal standard (ISA or ISB) used for

quantification ................................................................................................................................................................................... 35

Table D.1 — Example of windows for SIM1 method on 100% PDMS type-column .......................................... 38

Table D.2 — Example of windows for SIM2 method on 100% PDMS type-column .......................................... 40

Table D.3 — Example of windows for SIM1 method on 50%Polydimethylsiloxane

50%Phenylmethylpolysiloxane type-column .................................................................................................................... 42

Table D.4 — Example of windows for SIM2 method on 50%Polydimethylsiloxane

50%Phenylmethylpolysiloxane type-column .................................................................................................................... 44

Table G.1 — Required mass for the preparation of Solution A1 ................................................................................ 51

Table G.2 — Required mass for the preparation of Solution A2 ................................................................................ 52

Table I.1 — Resolution of ∆ for the determination of C ............................................................................................... 58

Table J.1 — Allergens Selected as Analysis Targets – Rationale ................................................................................ 59

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European foreword

This document (prEN 16274:2020) has been prepared by Technical Committee CEN/TC 347 “Methods

for analysis of allergens”, the secretariat of which is held by SNV.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 16274:2012.

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).

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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 raw materials and oils and dilutions thereof.

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.
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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 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 with two solvents, namely methyl pivalate and tert-

methyl butyl ether.

Many of the Allergens in the SCCS ‘Opinion’ documentation submitted to the EU Commission consist of

multiple isomers of the same molecules and/or complex mixtures.

In many cases, this complexity can be resolved by this Procedure but that leaves a number of target

analytes where this isn’t the case.

In addition, the SCCS Opinion also documents a number of CAS Numbers related to the same parent

molecule. These are related to the optically active forms of the similar parent structure. For this

procedure, the optically active forms are not considered as these require special (and in some cases, very

different) GC phases to achieve the required separation.
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 http://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 Ionisation 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) 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.
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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.

This Procedure is written as a methodology to be applied in a working laboratory (QC and R&D type).

Particular attention shall be paid by the user to the details contained in the Annexes and to specific

comments (‘Notes’) in the text regarding the application of this methodology.
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)

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 Acetyl cedrene alpha (main isomer in Vertofix® referred to in CAS number), CAS No.

[32388-55-9]

NOTE Highly variable composition with at least twelve constituents of molecular weight equal to 246 amu.

5.2.2 Acetyl isoeugenol / Isoeugenyl acetate, CAS No. [93-29-8]
5.2.3 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.

5.2.4 Alpha amyl cinnamaldehyde (Flosal®), CAS No. [122-40-7, 78605-96-6]
NOTE 2 possible isomers (E, Z); only E is quantified.
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5.2.5 Alpha amylcinnamyl alcohol, CAS No. [101-85-9, 184900-07-0]
NOTE 2 possible isomers (E, Z); only E is quantified.
5.2.6 Anethole, CAS No. [104-46-1, 4180-23-8]
NOTE 2 possible isomers (E, Z); only E is quantified.
5.2.7 Anise alcohol, CAS No. [105-13-5]
5.2.8 Benzaldehyde, CAS No. [100-52-7]
5.2.9 Benzyl alcohol, CAS No. [100-51-6]
5.2.10 Benzyl benzoate, CAS No. [120-51-4]
5.2.11 Benzyl cinnamate, CAS No. [103-41-3];
NOTE Only E is quantified.
5.2.12 Benzyl salicylate, CAS No. [118-58-1]
5.2.13 Camphor, CAS No. [76-22-2]
5.2.14 Carvone, CAS No. [99-49-0]
5.2.15 Caryophyllene beta, CAS No. [87-44-5]
5.2.16 Cinnamaldehyde, CAS No. [104-55-2, 14371-10-9]
NOTE 2 possible isomers (E, Z), only E is quantified.
5.2.17 Cinnamyl alcohol, CAS No. [104-54-1, 4407-36-7]
NOTE 2 possible isomers (E, Z), only E is quantified.
5.2.18 Citral, CAS No. [5392-40-5]

NOTE Both Neral (Z isomer, CAS No. [106-26-3]) and Geranial (E isomer, CAS No. [141-27-5]) are quantified.

5.2.19 Citronellol, CAS No. [106-22-9]
5.2.20 Coumarin, CAS No. [91-64-5]
5.2.21 Damascenone beta (Rose Ketone-4), n° CAS [23696-85-7]
5.2.22 Damascone alpha, CAS No. [43052-87-5]

NOTE 2 possible isomers (E, Z); only the E main isomer (92 to 99%) is quantified.

5.2.23 Damascone beta (E), CAS No. [23726-91-2]
5.2.24 Damascone delta (Rose Ketone-3), CAS No. [57378-68-4]

NOTE 3 possible isomers (trans/trans, cis/trans, trans/cis) only the major isomer (trans/trans, up to 90%) is

quantified.
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5.2.25 Dimethylbenzylcarbinyl acetate (DMBCA), CAS No. [151-05-3]
5.2.26 Ebanol, CAS No. [67801-20-1]
NOTE The 2 isomers (E, Z) are quantified.
5.2.27 Eugenol, CAS No. [97-53-0]
5.2.28 Eugenyl acetate, CAS No. [93-28-7]
5.2.29 Farnesol, CAS No. [4602-84-0]

NOTE 4 possible isomers: (E,E) isomer is (n° CAS [106-28-5]) and the (Z,E) isomer is (n° CAS [3790-71-4]).

Recommendation: Use E,E calibration curve to quantify other isomers.
5.2.30 Galaxolide (Hexamethylindanopyran), CAS No. [1222-05-5]
NOTE Only the two main isomers are quantified.
5.2.31 Geraniol, CAS No. [106-24-1]
5.2.32 Geranyl acetate, CAS No. [105-87-3]
5.2.33 Hexadecanolactone / Dihydroambrettolide, CAS No. [109-29-5]
5.2.34 Hexylcinnamaldehyde alpha, CAS No. [101-86-0, 165184-98-5]
NOTE 2 isomers at least; only the E is quantified.
5.2.35 Hydroxycitronellal, CAS No. [107-75-5]
5.2.36 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]).

5.2.37 Isoeugenol, CAS No. [97-54-1]
NOTE 2 possible isomers (E, Z), only E- iso eugenol is quantified.
5.2.38 Isomethylionone alpha, CAS No. [127-51-5]

NOTE 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. These constituents should not be assayed.

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5.2.39 Butylphenyl methylpropional (Lilial®), CAS No. [80-54-6]
5.2.40 Limonene, CAS No. [138-86-3]
5.2.41 Linalool, CAS No. [78-70-6]
5.2.42 Linalyl acetate, CAS No. [115-95-7]

5.2.43 Hydroxyisohexyl 3-cyclohexene carboxaldehyde (Lyral®), CAS No. [31906-04-4]

NOTE Contains Hydroxyisohexyl 4-cyclohexene carboxaldehyde (CAS No.51414-25-6) (noted Lyral minor),

has to be quantified.
5.2.44 Trimethyl-benzenepropanol (Majantol®), CAS No. [103694-68-4]
5.2.45 Menthol, CAS No. [1490-04-6, 89-78-1]
5.2.46 Methyl salicylate, CAS No. [119-36-8]
5.2.47 Methyl-2-octynoate (Folione®), CAS No. [111-12-6]
5.2.48 Pinene alpha, CAS No. [80-56-8]
5.2.49 Pinene beta, CAS No. [127-91-3]
5.2.50 3-Propylidene phthalide, CAS No. [17369-59-4]

NOTE The 2 isomers (E,Z) (E-isomer: CAS No.56014-72-3 and Z- isomer: CAS No.94704-89-9) are quantified

(isomers ratio near 1:6).
5.2.51 Salicylaldehyde, CAS No. [90-02-8]
5.2.52 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.

5.2.53 Sclareol, CAS No. [515-03-7]
5.2.54 Terpinene alpha, CAS No. [99-86-5]

5.2.55 Terpineol-alpha, CAS No. [98-55-5] for alpha-terpineol; 5.2.55a for gamma-terpineol CAS

No. [586-81-2]; 5.2.55b for Terpineol cis-beta CAS No. [138-87-4]; 5.2.55c for Terpineol trans-

beta CAS No. [7299-41-4]
Recommendation: use alpha-terpineol calibration curve to quantify other isomers.
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5.2.56 Terpinolene, CAS No. [586-62-9]
5.2.57 Vanillin, CAS No. [121-33-5]
5.3 Internal standards (ISTD)
5.3.1 1,4-Dibromobenzene (ISA), CAS No. [106-37-6], purity ≥ 98%.
5.3.2 4,4'-dibromobiphenyl (IS ), v [92-86-4], purity ≥ 98%.
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 (>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 >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 requirements:

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 weekly, as well as the

injector cleanliness to maintain analytical performance.

The sensitivity of the mass spectrometer should also be optimized through maximising 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.
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oSIST prEN 16274:2020
prEN 16274:2020 (E)

NOTE 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 or 60m). 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 me
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