SIST EN 16344:2013

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.NURPDWRJUDILMRKosmetische Mittel - Untersuchung von kosmetischen Mitteln - Screening und quantitative Bestimmung von 10 UV-Filtern in Sonnenschutzmitteln, HPLC-VerfahrenCosmétiques - Analyse des produits cosmétiques - Détection des filtres UV dans les produits cosmétiques et détermination quantitative de 10 filtres UV par CLHPCosmetics - Analysis of cosmetic products - Screening for UV-filters in cosmetic products and quantitative determination of 10 UV-filters by HPLC71.100.70SULSRPRþNLCosmetics. ToiletriesICS:Ta slovenski standard je istoveten z:EN 16344:2013SIST EN 16344:2013en,fr,de01-november-2013SIST EN 16344:2013SLOVENSKI
STANDARD



SIST EN 16344:2013



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16344
July 2013 ICS 71.100.70 English Version
Cosmetics - Analysis of cosmetic products - Screening for UV-filters in cosmetic products and quantitative determination of 10 UV-filters by HPLC.
Cosmétiques - Analyse des produits cosmétiques - Détection des filtres UV dans les produits cosmétiques et détermination quantitative de 10 filtres UV par CLHP
Kosmetische Mittel - Untersuchung von kosmetischen Mitteln - Screening 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 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, 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 Kingdom.
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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: ESIST EN 16344:2013



EN 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 Annex B (informative)
Sample Chromatogram 1 . 22 Annex C (informative)
Sample chromatogram 2 . 24 Bibliography. 25
SIST EN 16344:2013



EN 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 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 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 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, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 16344:2013



EN 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 of UV-filters allowed in cosmetic products. SIST EN 16344:2013



EN 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 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 comparable 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-absorber 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 the 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 extraction 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-Ethylhexyloxyphenol 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 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). The 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.
SIST EN 16344:2013



EN 16344:2013 (E) 6 4 Reagents 4.1 General If not otherwise specified, analytical-grade chemicals shall be used. 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 ω = 25 g/100 g.
4.6 Sodium hydroxide 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 water. 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 SIST EN 16344:2013



EN 16344:2013 (E) 7 Table 1 — Polar UV-filters (calibration solution in methanol)
EUa Abbrev. INCIb
and 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 Dicamphor 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 Dibenzimidazole Tetrasulfonate, CAS 180898-37-7 4.12.6 A28 DHHB Diethylamino Hydroxybenzoyl Hexyl Benzoate, CAS 302776-68-7 a EU = serial number in accordance with Annex VI of (EC) No 1223/2009. b INCI = International Nomenclature of Cosmetic Ingredients. Table 2 — Medium polar UV-filters (calibration solution in methanol acetone (1:1))
EUa
Abbrev. INCIb
and 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 (octylmethoxycinnamate), 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-dimethylaminobenzoate), CAS 21245-02-3 a EU = serial number in accordance with Annex VI of (EC) No 1223/2009. b INCI = International Nomenclature of Cosmetic Ingredients. Table 3 — Non polar UV-filters (calibration solution in THF)
EUa
Abbrev. INCIb
and other common names 4.12.14 A3 HMS Homosalate (3,3,5-trimethylcyclohexylsalicylate), 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 155633-54-8
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 (anisotriazine), CAS 187393-00-6 a EU = serial number in accordance with Annex VI of (EC) No 1223/2009. b INCI = International Nomenclature of Cosmetic Ingredients. SIST EN 16344:2013



EN 16344:2013 (E) 8
Table 4 — Other UV-filter or UV absorber (not listed in Annex VI of the (EC) No 1223/2009)
Abbrev. INCIb
and 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-PABAmidopropyl 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'-carbonylbis[4-hydroxy-6-methoxybenzenesulphonate]), 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 b INCI = International Nomenclature of Cosmetic Ingredients. NOTE The following UV-filters listed in Annex VI of the Regulation (EC) No 1223/2009 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 determined 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 methanol (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). 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 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. SIST EN 16344:2013



EN 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. For 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 substance" 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 (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 drops 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 ml Acetone/methanol mixture (4.13.1) OC (4.12.8) 40 mg 10 ml Acetone/methanol mixture (4.13.1) EHMC (4.12.9) 40 mg 10 ml Acetone/methanol mixture (4.13.1) MBC (4.12.11) 40 mg 10 ml Acetone/methanol mixture (4.13.1) IMC (4.12.10) 40 mg 10 ml Acetone/methanol mixture (4.13.1) 3-BC (4.12.12) 40 mg 10 ml Acetone/methanol mixture (4.13.1) EHDP (4.12.13) 40 mg 10 ml Acetone/methanol mixture (4.13.1)
SIST EN 16344:2013



EN 16344:2013 (E) 10
Table 7 — Scheme for preparing the stock solutions of non polar UV-filters Non-polar UV-filter Initial weight Volume Solvent HMS (4.12.14) 40 mg 10 ml Tetrahydrofuran (4.4) BMDM (4.12.15) 40 mg 10 ml Tetrahydrofuran (4.4) EHT (4.12.16) 40 mg 10 ml Tetrahydrofuran (4.4) DTS (4.12.17) 40 mg 10 ml Tetrahydrofuran (4.4) DEBT (4.12.18) 40 mg 10 ml Tetrahydrofuran (4.4) EHS (4.12.19) 40 mg 10 ml Tetrahydrofuran (4.4) MBBT (4.12.20) 40 mg 10 ml Tetrahydrofuran (4.4) BEMT (4.12.21) 40 mg 10 ml Tetrahydrofuran (4.4)
4.14.2 Preparing the stock solutions for the UV-filters of Table 4 (not permitted UV-filters) The stock solutions of UV-Filter B-1, B-2, B-6, B-8, B-10, MA and DPLT can be prepared using the acetone/methanol mixture (4.13.1) as a solvent. PABA and SA can be prepared using methanol as solvent. B-9 can be prepared according to the procedure for PBSA (4.12.2). The B-9 solution colours yellow but experiments showed that no degradation of the substance took place and the colouring of the solution did not influence the result. 4.14.3 Calibration solutions The calibration solutions are prepared according to the scheme given in Table 8. The stock solution volumes of all UV-filters for polar (see Table 5), medium polar (see Table 6) and non-polar (see Table 7) are mixed in each case in calibration solution 1 (Cal 1) and made to the volumetric graduation mark with either methanol (MeOH), acetone/methanol mixture (MeOH/Acetone) or tetrahydrofuran (THF). Follow up calibration solutions are prepared from Cal 1. This results in a calibration within the range of 10 ng and 400 ng. A 1 µl volume is injected for the analysis. The calibration solutions may be stored for at least one week in a refrigerator between 2 °C and 8 °C in the absence of light. Table 8 — Scheme for preparing the calibration solutions Calibration solution Volume Solution ad to Solvent Concentration Polar Medium-polar Non-polar Solution Samplea
ml
ml
ng/µl g/100 g 1 2 Stock 20 MeOH MeOH/Acetone THF
400 10,0 2 5 Cal 1 10 MeOH MeOH/Acetone THF
200 5,0 3 2,5 Cal 1 10 MeOH MeOH/Acetone THF
100 2,5 4 1 Cal 1 10 MeOH MeOH/Acetone THF
40 1,0 5 0,5 Cal 1 10 MeOH MeOH/Acetone THF
20 0,5 6 0,5 Cal 1 20 MeOH MeOH/Acetone THF
10 0,25 a Referring to a sample initial weight of 100 mg. All standard solutions shall be protected from light during the entire analysis. SIST EN 16344:2013



EN 16344:2013 (E) 11 4.15 Mobile phase for HPLC 4.15.1 Mobile phase A: aqueous phase Weigh 3,0 g of LTAB (4.10), 1,0 g of ammonium bromide (4.11) and 2,5 ml of EDTA solution (4.8) into a 1 000 ml volumetric flask and fill up to the calibration mark with water. 4.15.2 Mobile phase B: organic phase Weigh 3,0 g of LTAB (4.10) and 1,0 g of ammonium bromide (4.11) into a 1 000 ml volumetri
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