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    DIN 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 German ver.pdf

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    DIN 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 German ver.pdf

    1、December 2012 Translation by DIN-Sprachendienst.English price group 13No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).I

    2、CS 71.100.60!$f“1929067www.din.deDDIN EN 16274Methods for analysis of allergens Quantification of suspected fragrance allergens in consumer products Step 1: GC analysis of ready-to-inject sample;English version EN 16274:2012,English translation of DIN EN 16274:2012-12Analyseverfahren fr Allergene Qu

    3、antifizierung von mutmalichen Allergie auslsenden Duftstoffen in Verbrauchsgtern Stufe 1: GC-Analyse von einspritzfertigen Proben;Englische Fassung EN 16274:2012,Englische bersetzung von DIN EN 16274:2012-12Mthode danalyse des allergnes Quantification des fragrances allergnes suspectes dans les prod

    4、uits deconsommation tape 1: Analyse par GC dchantillons prts tre injects;Version anglaise EN 16274:2012,Traduction anglaise de DIN EN 16274:2012-12www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 25 pages11.12 DIN EN 16274:2012-12 2 A co

    5、mma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee CEN/TC 347 “Methods for analysis of allergens” (Secretariat: DS, Denmark). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche

    6、Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-07-01 AA Kosmetische Mittel. For the sake of legibility, some symbols have been modified in the German version of this standard: in Subclauses 6.1 “General”, 6.3.3 “SIM mode” and Table 2 “List of ions for quantif

    7、ication” the symbols “l1”, “l2” and “l3” have been replaced by “i1”, “i2” and “i3” and in Subclause 8.2 “Quantification of allergens” “lx” has been replaced by “ix”. EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16274 September 2012 ICS 71.100.60 English Version Methods for analysis of allerg

    8、ens - 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 produits de consommation - tape 1: Analyse par GC dchantillons prts tre injects Analy

    9、severfahren 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 2012. CEN members are bound to comply with the CEN/CENELEC Internal Regulations whic

    10、h 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 Eur

    11、opean 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 a

    12、re 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, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, R

    13、omania, 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, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any mean

    14、s reserved worldwide for CEN national Members. Ref. No. EN 16274:2012: EEN 16274:2012 (E) 2 Contents Page Foreword 3Introduction .41 Scope 52 Principle 53 Reagents . 5 3.1 Solvents 5 3.2 Fragrance (suspected allergen) standards . 5 3.3 Internal standards (ISTD) 7 4 Apparatus . 7 4.1 Analytical Balan

    15、ce . 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 Stock solution of all allergens (5 g/l) .85.2.3 Separate stock solutions (carbonyl / non carbonyl co

    16、mpounds) (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 General . 106.3.2 SCAN mode . 106.3.3 SIM mode . 116.4 Calibration . 137 Sample analysis 138 Data tr

    17、eatment 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 . 158.3.3 Maximum permitted tolerances . 159 Test report . 16Annex A (informative) Column perfor

    18、mances . 17Annex B (informative) SIM windows . 19Annex C (informative) Example of chromatographic separation 20Annex D (normative) Decisional tree 22Bibliography . 23DIN EN 16274:2012-12 EN 16274:2012 (E) 3 Foreword This document (EN 16274:2012) has been prepared by Technical Committee CEN/TC 347 “M

    19、ethods 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, at the latest by March 2013, and conflicting national standards shall be withdrawn at the

    20、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 or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national

    21、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 Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuani

    22、a, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16274:2012-12 EN 16274:2012 (E) 4 Introduction Human skin exposure to suspected allergenic fragrances can occur through diverse sources such as

    23、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 high level of protection of consumers, particularly for sensitive population. In this co

    24、ntext, 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), comprehensive GC or MS-MS in raw materials and finished products. The present analyti

    25、cal 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 above 0,001 % (10 mg/kg) of each, in ready-to-inject sample from a cosmetic ingredient or p

    26、roduct matrix. The present protocol has been validated thanks to a ring test 2. DIN EN 16274:2012-12 EN 16274:2012 (E) 5 1 Scope This European Standard describes a method for the identification and determination of 24 volatile suspected allergens from ready-to-inject cosmetics and raw materials used

    27、 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 2003/15/EC). The method described in this European Standard does not include requir

    28、ements 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 allergens by Gas Chromatography coupled with Mass Spectrometry after dilution of the sam

    29、ple 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 when possible using GC-MS in scan mode. Quantification is performed by single ion m

    30、onitoring (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 requirements. 3 Reagents Unless otherwise stated, use only reagents of recognised analytica

    31、l 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. 3.1.3 Acetone, CAS no: 67-64-1, analytical grade or higher. IMPORTANT if other solven

    32、ts 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 alcohol, CAS no: 101-85-9, with known purity. NOTE Possibly two isomers. 3.2.2 Amy

    33、lcinnamic 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 Benzyl benzoate, CAS no: 120-51-4, with known purity. DIN EN 16274:2012-12 EN 16274

    34、: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, with known purity. 3.2.9 Cinnamic alcohol, CAS no: 104-54-1, with known purit

    35、y. 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 determined separately. 3.2.12 Citronellol, CAS no: 106-22-9, with known purity.

    36、 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)-farnesol (CAS no 106-28-5) and (Z,E)-farnesol (CAS no 3790-71-4). 3.2.16 Geran

    37、iol, 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 Hydroxyisohexyl-3-cyclohexene carboxaldehyde (lyral), CAS no: 31906-04-4, with kno

    38、wn 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, trans). 3.2.21 -Isomethyl ionone, CAS no: 127-51-5, with known purity. 3.2.22 Li

    39、monene, 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. DIN EN 16274:2012-12 EN 16274:2012 (E) 7 3.3 Internal standards (ISTD) 3.3.1 1,4-dibromobenzene, analytical grade or higher. 3.3

    40、.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 only) GC-FID equipped with a split/splitless injector with a glass insert m

    41、aintained 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 injector with a glass insert maintained at 250 C. The glass insert shal

    42、l 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. CAUTION Use of an ion trap mass spectrometer (ITD-MS) or time of flight

    43、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 range used. 4.4 GC capillary columns The two columns shall significantly dif

    44、fer 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 either measuring the resolution between all analytes or by calculation of

    45、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 number of peaks in the chromatogram. If 0=R ,the column can only be used if t

    46、here is no ion listed in Table 2 in common between 2 co-eluted allergens. DIN EN 16274:2012-12 EN 16274:2012 (E) 8 5 Standard preparation and preservation 5.1 General Solvents used for analysis and storage shall comply with the following requirements: chemical inertia towards the allergen analytes,

    47、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 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 sample dilutions. In any case, the stock solution s


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