1、BSI Standards PublicationBS EN 16344:2013Cosmetics Analysis ofcosmetic products Screeningfor UV-filters in cosmeticproducts and quantitativedetermination of 10 UV-filtersby HPLC.BS EN 16344:2013 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16344:2013. The UK
2、participation in its preparation was entrusted to TechnicalCommittee CW/217, Cosmetics.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for
3、its correct application. The British Standards Institution 2013.Published by BSI Standards Limited 2013ISBN 978 0 580 76573 5ICS 71.100.70 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy
4、and Strategy Committee on 31 July 2013.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS EN 16344:2013EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16344 July 2013 ICS 71.100.70 English Version Cosmetics - Analysis of cosmetic products - Screening for UV-filters in
5、cosmetic products and quantitative determination of 10 UV-filters by HPLC. Cosmtiques - Analyse des produits cosmtiques - Dtection des filtres UV dans les produits cosmtiques et dtermination quantitative de 10 filtres UV par CLHP Kosmetische Mittel - Untersuchung von kosmetischen Mitteln - Screening
6、 und quantitative Bestimmung von 10 UV-Filtern in Sonnenschutzmitteln, HPLC-Verfahren This European Standard was approved by CEN on 29 May 2013. 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
7、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
8、 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, Cypr
9、us, 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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kin
10、gdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16344:2013: EBS EN
11、 16344:2013EN 16344:2013 (E) 2 Contents Page Foreword . 3 Introduction 4 1 Scope 5 2 Terms and definitions 5 3 Principle 5 4 Reagents . 6 5 Apparatus and equipment . 11 6 Sampling . 11 7 Procedure . 12 8 Evaluations . 14 9 Test report 15 Annex A (informative) Results of the inter-laboratory test 16
12、Annex B (informative) Sample Chromatogram 1 . 22 Annex C (informative) Sample chromatogram 2 24 Bibliography 25 BS EN 16344:2013EN 16344:2013 (E) 3 Foreword This document (EN 16344:2013) has been prepared by Technical Committee CEN/TC 392 “Cosmetics”, the secretariat of which is held by AFNOR. This
13、European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by January 2014, and conflicting national standards shall be withdrawn at the latest by January 2014. Attention is drawn to the possibility that some of the
14、 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 standards organisations of the following countries are bound to implement th
15、is 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, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovak
16、ia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16344:2013EN 16344:2013 (E) 4 Introduction Reference is made to the relevant annex of the Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products: Annex VI List
17、of UV-filters allowed in cosmetic products. BS EN 16344:2013EN 16344:2013 (E) 5 1 Scope This European Standard specifies a multi-screening method using reversed-phase HPLC for the detection of UV-filters listed in the cosmetic regulations. The method is applicable for the quantitative determination
18、of 10 UV-filters, which are mainly used in emulsion-based cosmetic products and sunscreen sprays particularly with regard to the maximum concentration listed in the cosmetic regulation. Other analytical methods for the qualification and quantification of UV-filters may be used if they lead to compar
19、able results. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 UV-filters organic chemical compounds that absorb ultraviolet light and inorganic substances that reflect, scatter and absorb UV light Note 1 to entry: The UV-filters and UV-absorb
20、er of this method are only organic chemical compounds and are used in sunscreen products to protect the skin against UV radiation. 3 Principle The UV-filters are extracted with an acetone/methanol mixture. For the qualitative detection of the listed UV-filters and the quantitative determination of t
21、he 10 validated UV-filter reversed phase HPLC with UV (DAD) detection is used. The method is also applicable for the quantification of the other listed UV-filters after proper validation. Quantitative determination of samples containing the following UV-filters require the use of additional extracti
22、on methods and determinations: Terephthalylidene Dicamphor Sulfonic Acid (TDSA) and Disodium Phenyl Dibenzimidazole Tetrasulfonate (DPDT) are additionally extracted with methanolic-aqueous sodium hydroxide solution. Methylene Bis-benzotriazolyl Tetramethylbutylphenol (MBBT) and Bis-Ethylhexyloxyphen
23、ol Methoxyphenyl Triazine (BEMT) are additionally extracted with a mixture of tetrahydrofuran/acetone. In the case of an unsatisfactory peak shape, Butyl Methoxydibenzoylmethane (BMDM) is additionally extracted with a mixture of acetone/methanol/EDTA. The quantitative determination is made by means
24、of RP-HPLC with UV (DAD). The UV-spectra are compared with the reference spectra in a database. The concentration of each UV-filter determined in accordance with this method is reported in g/100 g. This method has been tested in an inter-laboratory test on specific cosmetic matrix (q.v. Annex A). Th
25、e user should verify the performance of the method in their laboratory for each different matrix and pay particular attention to the recommended quality control elements. BS EN 16344:2013EN 16344:2013 (E) 6 4 Reagents 4.1 General If not otherwise specified, analytical-grade chemicals shall be used.
26、Water shall be distilled or of a corresponding purity. “Solution“ shall be understood as an aqueous solution unless otherwise specified. 4.2 Methanol (MeOH), HPLC grade. 4.3 Acetone, HPLC grade. 4.4 Tetrahydrofuran (THF), HPLC grade. 4.5 Ammonia solution, mass fraction = 25 g/100 g. 4.6 Sodium hydro
27、xide solution, molar concentration c = 1 mol/l. 4.7 Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate (Na2EDTA 2H2O, CAS 6381-92-6, purity 99 %). 4.8 EDTA solution Weigh 1,8 g of EDTA disodium salt dihydrate (4.7) into a 100 ml volumetric flask and fill up to the calibration mark with w
28、ater. 4.9 Ethanol, HPLC grade. 4.10 Lauryl Trimethyl Ammonium Bromide (LTAB, synonym: dodecyltrimethylammonium bromide. CAS 1119-94-4), if possible HPLC quality (purity 98 %). 4.11 Ammonium bromide (CAS 12124-97-9, purity 99 %). 4.12 Reference substances BS EN 16344:2013EN 16344:2013 (E) 7 Table 1 P
29、olar UV-filters (calibration solution in methanol) EUaAbbrev. INCIband other common names 4.12.1 A2 CBM Camphor Benzalkonium Methosulfate, CAS 52793-97-2 4.12.2 A6 PBSA Phenylbenzimidazole Sulfonic Acid (2-phenylbenzimidazole-5-sulfonic acid), CAS 27503-81-7 4.12.3 A7 TDSA Terephthalylidene Dicampho
30、r Sulfonic Acid, CAS 90457-82-2, present as triethanolamine salt (molecular weight m = 860 g/mol), free acid (molecular weight m = 562 g/mol) 4.12.4 A22 B-4/5 Benzophenone-4/5 (2-hydroxy-4-methoxybenzophenone-5-sulfonic acid, Sulisobenzone), CAS 4065-45-6 4.12.5 A24 DPDT Disodium Phenyl Dibenzimidaz
31、ole Tetrasulfonate, CAS 180898-37-7 4.12.6 A28 DHHB Diethylamino Hydroxybenzoyl Hexyl Benzoate, CAS 302776-68-7 aEU = serial number in accordance with Annex VI of (EC) No 1223/2009. bINCI = International Nomenclature of Cosmetic Ingredients. Table 2 Medium polar UV-filters (calibration solution in m
32、ethanol acetone (1:1) EUaAbbrev. INCIband other common names 4.12.7 A4 B-3 Benzophenone-3 (oxybenzonum, 2-hydroxy-4-methoxy-benzophenone), CAS 131-57-7 4.12.8 A10 OC Octocrylene (2-ethylhexyl-2-cyano-3,3-diphenylacrylate), CAS 6197-30-4 4.12.9 A12 EHMC Ethylhexyl Methoxycinnamate (octylmethoxycinnam
33、ate), CAS 5466-77-3 4.12.10 A14 IMC Isoamyl p-Methoxycinnamate, CAS 71617-10-2 4.12.11 A18 MBC 4-Methylbenzylidene Camphor (3-(4-methylbenzylidene)-dl-camphor), CAS 36861-47-9 4.12.12 A19 3-BC 3-Benzylidene Camphor, CAS 15087-24-8 4.12.13 A21 EHDP Ethylhexyl Dimethyl PABA (2-ethylhexyl-4-dimethylami
34、nobenzoate), CAS 21245-02-3 aEU = serial number in accordance with Annex VI of (EC) No 1223/2009. bINCI = International Nomenclature of Cosmetic Ingredients. Table 3 Non polar UV-filters (calibration solution in THF) EUaAbbrev. INCIband other common names 4.12.14 A3 HMS Homosalate (3,3,5-trimethylcy
35、clohexylsalicylate), CAS 118-56-9 4.12.15 A8 BMDM Butyl Methoxydibenzoylmethane (4-tert-butyl-4-methoxydibenzoylmethane), CAS 70356-09-1 4.12.16 A15 EHT Ethylhexyl Triazone (octyltriazone), CAS 88122-99-0 4.12.17 A16 DTS Drometrizole Trisiloxane (2-benzotriazole-2-yl-methylphenol trisiloxane), CAS 1
36、55633-54-8 4.12.18 A17 DEBT Diethylhexyl Butamido Triazone, CAS 154702-15-5 4.12.19 A20 EHS Ethylhexyl Salicylate (2-ethylhexylsalicylate), CAS 118-60-5 4.12.20 A23 MBBT Methylene Bis-Benzotriazol Tetramethylbutylphenol, CAS 103597-45-1 4.12.21 A25 BEMT Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine
37、 (anisotriazine), CAS 187393-00-6 aEU = serial number in accordance with Annex VI of (EC) No 1223/2009. bINCI = International Nomenclature of Cosmetic Ingredients. BS EN 16344:2013EN 16344:2013 (E) 8 Table 4 Other UV-filter or UV absorber (not listed in Annex VI of the (EC) No 1223/2009) Abbrev. INC
38、Iband other common names 4.12.22 MA Menthyl Anthranilate, CAS 134-09-8 4.12.23 PABA PABA (4-aminobenzoic acid), CAS 150-13-0 4.12.24 B-1 Benzophenone-1 (2,4-dihydroxybenzophenone), CAS 131-56-6 4.12.25 B-2 Benzophenone-2 (2,2,4,4-tetrahydroxybenzophenone), CAS 131-55-5 4.12.26 DPLT Dimethyl-PABAmido
39、propyl Laurdimonium Tosylate, CAS 156679-41-3 4.12.27 B-6 Benzophenone-6 (2,2-dihydroxy-4,4-dimethoxybenzophenone), CAS 131-54-4 4.12.28 B-8 Benzophenone-8 (2,2-dihydroxy-4-methoxybenzophenone, dioxybenzone),CAS 131-53-3 4.12.29 B-9 Benzophenone-9 (disodium 3,3-carbonylbis4-hydroxy-6-methoxybenzenes
40、ulphonate), CAS 76656-36-5 4.12.30 B-10 Benzophenone-10 (2-hydroxy-4-methoxy-4-methylbenzophenone), CAS 1641-17-4 4.12.31 SA Sodium Salicylate, CAS 54-21-7 bINCI = International Nomenclature of Cosmetic Ingredients. NOTE The following UV-filters listed in Annex VI of the Regulation (EC) No 1223/2009
41、 are not part of this method: A 9: 3-(4-sulfo)-benzylidene-bornan-2-one, CAS 56039-58-8 A 11: polymer of N-(2(and 4)-(2-oxoborn-3-ylidenemethyl)benzyl)acrylamid, CAS 113783-61-2 (the substances are no longer commercially available) A 13: ethoxylated ethyl-4-aminobenzoate, CAS 116242-27-4 (can be det
42、ermined only qualitatively) A 26: dimethicodiethylbenzalmalonate, CAS 207574-74-1 (cannot be determined using this standard) 4.13 Extraction solution 4.13.1 Acetone/methanol mixture (for the preparation of the reference solutions and for extraction). Mix 500 ml of acetone (4.3) and 500 ml of methano
43、l (4.2) in a 1 000 ml conical flask. 4.13.2 Acetone/tetrahydrofuran mixture (for extraction of non-polar UV-filters). Mix 500 ml of acetone (4.3) and 500 ml of tetrahydrofuran (4.4) in a 1 000 ml conical flask. 4.13.3 Methanolic-aqueous sodium hydroxide solution (for extraction of polar UV-filters).
44、 Mix 800 ml of methanol (4.2) and 200 ml of water and 10 ml of sodium hydroxide solution (4.6) in a 1 000 ml conical flask. 4.13.4 Acetone/methanol/EDTA mixture (for extraction of BMDM-containing samples with unsatisfactory peak shape). Add 1,0 ml of EDTA solution (4.8) to 200 ml of acetone/methanol
45、 mixture (4.13.1) in a 200 ml conical flask. As the solution is supersaturated with regard to EDTA it shall be used on the same day. BS EN 16344:2013EN 16344:2013 (E) 9 4.14 Reference solutions 4.14.1 Stock solutions The stock solutions are prepared in accordance with Table 5, Table 6 and Table 7. F
46、or each substance, weigh the initial weights with an accuracy of 0,1 mg to the mentioned volume. One stock solution is prepared per substance. For the initial weight, the purity of the substances shall be taken into account and if applicable be converted to an initial weight of 4 mg/ml of “active su
47、bstance“ in each case. The stock solutions can be stored for at least one month in a refrigerator between 2 C and 8 C in the absence of light. Table 5 Scheme for preparing the stock solutions of polar UV-filters Polar UV-filter Initial weight Volume Solvent CBM (29 %) (4.12.1) 140 mg 10 ml Methanol
48、(4.2) (Initial weight: 140 mg, as a 29 % standard solution is used; corresponds to 40 mg of “active substance“) PBSA (4.12.2) 40 mg 10 ml Add 2 ml of methanol (4.2) and 3 drops of ammonia (4.5). Fill up to the calibration mark with water. TDSA (4.12.3) 60 mg 10 ml Add 2 ml of methanol (4.2) and 3 dr
49、ops of ammonia (4.5). Fill up to the calibration mark with water. (Initial weight: 60 mg because of triethanolamine salt; corresponds to 40 mg of free acid, see limit value) B-4/5 (4.12.4) 40 mg 10 ml Methanol (4.2) DPDT (4.12.5) 40 mg 10 ml Add 2 ml of methanol (4.2) and 3 drops of ammonia (4.5). Fill up to the calibration mark with water. DHHB (4.12.6) 40 mg 10 ml Methanol (4.2) Table 6 Scheme for preparing the stock solutions of medium polar UV-filters Medium-polar UV-filter Initial weight Volume Solvent B-3 (4.12.7) 40 mg 10
