1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16274:2012Methods for analysis of allergens Quantification of suspected fragrance allergens in consumer products Step 1: GC analysis of r e a d y - t o - i n j e c t s a m
2、p l eBS EN 16274:2012 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16274:2012. The UK participation in its preparation was entrusted to TechnicalCommittee CW/217, Cosmetics.A list of organizations represented on this committee can beobtained on request to its
3、 secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012 ISBN 978 0 580 74898 1 ICS 71.100.60 Compliance with a British Standard
4、cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 October 2012. Amendments issued since publicationDate T e x t a f f e c t e dBS EN 16274:2012EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 1
5、6274 September 2012 ICS 71.100.60 English Version Methods for analysis of allergens - Quantification of suspected fragrance allergens in consumer products - Step 1: GC analysis of ready-to-inject sample Mthodes danalyse des allergnes - Quantification des fragrances allergnes suspectes dans les produ
6、its de consommation - tape 1 : Analyse par GC dchantillons prts tre injects Analyseverfahren fr Allergene - Quantifizierung von mutmalichen Allergie auslsenden Duftstoffen in Verbrauchsgtern - Stufe 1: GC-Analyse von einspritzfertigen Proben This European Standard was approved by CEN on 4 August 201
7、2. 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 o
8、n 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-CENE
9、LEC 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Ital
10、y, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17
11、, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16274:2012: EBS EN 16274:2012EN 16274:2012 (E) 2 Contents Page Foreword 3Introduction .41 Scope 52 Principle 53 Reagents . 5 3.1 Solvents 5 3.2 Fragrance (suspe
12、cted allergen) standards . 5 3.3 Internal standards (ISTD) 7 4 Apparatus . 7 4.1 Analytical Balance . 7 4.2 GC-FID (for solvent or standard purity only) 7 4.3 GC-MS . 7 4.4 GC capillary columns 7 5 Standard preparation and preservation 85.1 General 85.2 Standard preparation 85.2.1 General 85.2.2 Sto
13、ck solution of all allergens (5 g/l) .85.2.3 Separate stock solutions (carbonyl / non carbonyl compounds) (10 g/l) 95.2.4 Internal standard solution .95.2.5 Working solutions95.2.6 Calibration solution .96 Procedure 106.1 General . 106.2 Chromatographic conditions . 106.3 MS conditions . 106.3.1 Gen
14、eral . 106.3.2 SCAN mode . 106.3.3 SIM mode . 116.4 Calibration . 137 Sample analysis 138 Data treatment and calculation of results 148.1 Identification of allergens 148.2 Quantification of allergens 148.3 Assessment of the analytical measurement 158.3.1 General . 158.3.2 Examination of the Q-values
15、 . 158.3.3 Maximum permitted tolerances . 159 Test report . 16Annex A (informative) Column performances . 17Annex B (informative) SIM windows . 19Annex C (informative) Example of chromatographic separation 20Annex D (normative) Decisional tree 22Bibliography . 23BS EN 16274:2012EN 16274:2012 (E) 3 F
16、oreword This document (EN 16274:2012) has been prepared by Technical Committee CEN/TC 347 “Methods for analysis of allergens”, the secretariat of which is held by DS. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement
17、, at the latest by March 2013, and conflicting national standards shall be withdrawn at the latest by March 2013. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any
18、 or all such patent rights. 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, Former Yugoslav Republ
19、ic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16274:2012EN 16274:2012 (E) 4 Introduction Human skin
20、 exposure to suspected allergenic fragrances can occur through diverse sources such as detergents and cosmetics intended to be rinsed or not. As a result of their possible effect, 26 fragrance substances have been restricted under Council Directives with labelling requirements in order to insure a h
21、igh level of protection of consumers, particularly for sensitive population. In this context, several analytical methods have been developed to detect and determine their presence in cosmetics such as Gas Chromatography/Flame Ionisation Detector (GC-FID), Gas Chromatography/Mass Spectrometry (GC-MS)
22、, comprehensive GC or MS-MS in raw materials and finished products. The present analytical method uses GC-MS by combination of two GC columns of different polarity with a dedicated methodology for quantification 1. This allows separation and quantification of the 24 volatile suspected allergens abov
23、e 0,001 % (10 mg/kg) of each, in ready-to-inject sample from a cosmetic ingredient or product matrix. The present protocol has been validated thanks to a ring test 2. BS EN 16274:2012EN 16274:2012 (E) 5 1 Scope This European Standard describes a method for the identification and determination of 24
24、volatile suspected allergens from ready-to-inject cosmetics and raw materials used in cosmetic products and are compatible with GC analysis. This analysis uses GC-MS after sample preparation. The 24 suspected allergens are restricted under Council Directives (7thamendment to the Cosmetic Directive 2
25、003/15/EC). The method described in this European Standard does not include requirements for the preparation of samples in matrices for which direct injection in GC is not feasible. 2 Principle The method described in this European Standard is a comprehensive analysis of 24 volatile suspected allerg
26、ens by Gas Chromatography coupled with Mass Spectrometry after dilution of the sample in an inert solvent. Two assays are performed for the chromatographic separation of the 24 suspected allergens using two GC capillary columns of different polarities. Suspected allergen identification is achieved w
27、hen possible using GC-MS in scan mode. Quantification is performed by single ion monitoring (SIM) using 1,4-dibromobenzene and 4,4-dibromobiphenyl as internal standards. The final result depends on the agreement of the different ion ratios obtained for both injections according to specific requireme
28、nts. 3 Reagents Unless otherwise stated, use only reagents of recognised analytical grade. The solvent shall be of quality for GC-MS analysis. 3.1 Solvents 3.1.1 Methyl pivalate, CAS no: 598-98-1, analytical grade or higher. 3.1.2 Ortho-fluorotoluene, CAS no: 95-52-3, analytical grade or higher. 3.1
29、.3 Acetone, CAS no: 67-64-1, analytical grade or higher. IMPORTANT if other solvents are used, their inertness with the analytes shall be demonstrated. In any case, the same solvent shall be used both for calibration and determination. 3.2 Fragrance (suspected allergen) standards 3.2.1 Amylcinnamic
30、alcohol, CAS no: 101-85-9, with known purity. NOTE Possibly two isomers. 3.2.2 Amylcinnamic aldehyde (flosal), CAS no: 122-40-7, with known purity. NOTE Possibly two isomers. 3.2.3 Anisyl alcohol, CAS no: 105-13-5, with known purity. 3.2.4 Benzyl alcohol, CAS no: 100-51-6, with known purity. 3.2.5 B
31、enzyl benzoate, CAS no: 120-51-4, with known purity. BS EN 16274:2012EN 16274:2012 (E) 6 3.2.6 Benzyl cinnamate, CAS no: 103-41-3, with known purity. NOTE Possibly two isomers. 3.2.7 Benzyl salicylate, CAS no: 118-58-1, with known purity. 3.2.8 Butylphenyl methylpropional (lilial), CAS no: 80-54-6,
32、with known purity. 3.2.9 Cinnamic alcohol, CAS no: 104-54-1, with known purity. NOTE Possibly two isomers. 3.2.10 Cinnamic aldehyde, CAS no: 104-55-2, with known purity. NOTE Possibly two isomers. 3.2.11 Citral, CAS no: 5392-40-5, with known purity. NOTE Two isomers, neral and geranial, which are de
33、termined separately. 3.2.12 Citronellol, CAS no: 106-22-9, with known purity. 3.2.13 Coumarine, CAS no: 91-64-5, with known purity. 3.2.14 Eugenol, CAS no: 97-53-0, with known purity. 3.2.15 Farnesol, CAS no: 4602-84-0, with known purity. NOTE Possibly four isomers. The two major isomers are (E,E)-f
34、arnesol (CAS no 106-28-5) and (Z,E)-farnesol (CAS no 3790-71-4). 3.2.16 Geraniol, CAS no: 106-24-1, with known purity. 3.2.17 Hexylcinnamic aldehyde (jasmonal), CAS no: 101-86-0 with known purity. NOTE At least two isomers. 3.2.18 Hydroxycitronellal, CAS no: 107-75-5, with known purity. 3.2.19 Hydro
35、xyisohexyl-3-cyclohexene carboxaldehyde (lyral), CAS no: 31906-04-4, with known purity. WARNING This fragrance standard also contains hydroxyisohexyl-4-cyclohexene carboxaldehyde which shall not be quantified. 3.2.20 Isoeugenol, CAS no: 97-54-1, with known purity. NOTE Possibly two isomers (cis, tra
36、ns). 3.2.21 -Isomethyl ionone, CAS no: 127-51-5, with known purity. 3.2.22 Limonene, CAS no: 5989-27-5, with known purity. 3.2.23 Linalool, CAS no: 78-70-6, with known purity. 3.2.24 Methyl-2-octynoate (folione), CAS no 111-12-6, with known purity. BS EN 16274:2012EN 16274:2012 (E) 7 3.3 Internal st
37、andards (ISTD) 3.3.1 1,4-dibromobenzene, analytical grade or higher. 3.3.2 4,4-dibromobiphenyl, analytical grade or higher. 4 Apparatus Use standard laboratory glassware and equipment. 4.1 Analytical Balance Capable of weighing to the nearest 0,000 1 g. 4.2 GC-FID (for solvent or standard purity onl
38、y) GC-FID equipped with a split/splitless injector with a glass insert maintained at 250 C. The glass insert shall have an inner volume compatible with the expansion volume of the analytical solvent. A GC capillary column shall be connected to the FID. 4.3 GC-MS GC-MS equipped with a split/splitless
39、 injector with a glass insert maintained at 250 C. The glass insert shall have an inner volume compatible with the expansion volume of the analytical solvent. A GC capillary column shall be connected to the mass spectrometer, with the interface heated at least 10 C above the final oven temperature.
40、CAUTION Use of an ion trap mass spectrometer (ITD-MS) or time of flight mass spectrometer (ToF-MS) is possible if the conditions are adapted to such instruments, particularly for peak recognition and quantification. At least, the linearity of such instruments shall be checked in the calibration rang
41、e used. 4.4 GC capillary columns The two columns shall significantly differ in polarity and should be chosen according to Annex A. When a new column is used (not listed in Annex A), its characteristics shall be evaluated in terms of peak resolution during the column life. This shall be checked by ei
42、ther measuring the resolution between all analytes or by calculation of the mean resolution. The resolution between all peaks shall be 1. The mean resolution R shall be 5. 11111,1,18,1=+=nniiihihiRiRwwttR where tR,itotal retention time of the ithpeak; wh,iwidth at half height of the ithpeak; n numbe
43、r of peaks in the chromatogram. If 0=R ,the column can only be used if there is no ion listed in Table 2 in common between 2 co-eluted allergens. BS EN 16274:2012EN 16274:2012 (E) 8 5 Standard preparation and preservation 5.1 General Solvents used for analysis and storage shall comply with the follo
44、wing requirements: chemical inertia towards the allergen analytes, low volatility to ensure solution stability and concentration, and expansion volume compatible with the inner volume of injector insert. The volume of injection shall be compatible with the volume of the insert and with the capacity
45、of the column. Overflowing of the expanded volume of vaporised solvent shall be avoided. Methyl pivalate (3.1.1) and ortho-fluorotoluene (3.1.2) are suitable for the preparation of all standard and sample solutions. Acetone (3.1.3) may be used for the preparation of calibration solutions or final sa
46、mple dilutions. In any case, the stock solution shall not be diluted in a volatile solvent such as acetone. NOTE Ethanol is normally not suitable. If present in high concentration in the sample, ethanol can be used provided that immediate injection is performed. Iso-octane is not suitable for polar
47、fragrances. The solvent purity shall be checked to ensure that no impurities interfere with any of the 24 fragrance suspected allergens analysed under the GC conditions used in this European Standard. For ready-to-inject samples, the same solvent shall be used to prepare calibration solutions and sa
48、mple dilution, except where a volatile solvent is used. During analysis by GC-MS, a vial containing either the calibration solution or sample dilution shall be injected only once for analysis. At least, half of the sample dilution solvent should be the same as the solvent used for calibration. 5.2 S
49、tandard preparation 5.2.1 General The purity of each standard and each internal standard and the respective percentages for geometrical isomers (amylcinnamic alcohol (3.2.1), flosal (3.2.2), benzyl cinnamate (3.2.6), cinnamic alcohol (3.2.9), cinnamic aldehyde (3.2.10), citral (3.2.11), farnesol (3.2.15), jasmonal (3.2.17
