EN 16956-2017 en Cosmetics - Analytical methods - HPLC UV method for the identification and assay of hydroquinone ethers of hydroquinone and corticosteroids in skin whitening cosme.pdf

1、Cosmetics - Analytical methods - HPLC/UV method for the identification and assay of hydroquinone, ethers of hydroquinone and corticosteroids in skin whitening cosmetic productsBS EN 16956:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06EUROPEAN STANDARD NORME EUR

2、OPENNE EUROPISCHE NORM EN 16956 September 2017 ICS 71.100.70 English Version Cosmetics - Analytical methods - HPLC/UV method for the identification and assay of hydroquinone, ethers of hydroquinone and corticosteroids in skin whitening cosmetic products Cosmtiques - Mthodes analytiques - Mthode de C

3、LHP couple la dtection UV pour lidentification et lanalyse de lhydroquinone, de ses thers et des corticostrodes dans les produits cosmtiques claircissants de la peau Kosmetische Mittel - Untersuchungsverfahren - HPLC/UV Verfahren fr die Identifizierung und Bestimmung von Hydrochinon, Hydrochinonethe

4、rn und Kortikosteroiden in hautaufhellenden kosmetischen Mitteln This European Standard was approved by CEN on 19 June 2017. 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 wi

5、thout 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 othe

6、r 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,

7、Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPE

8、AN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16956:2017 ENation

9、al forewordThis British Standard is the UK implementation of EN 16956:2017.The UK participation in its preparation was entrusted to Technical Committee CW/217, Cosmetics.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purpor

10、t to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2017 Published by BSI Standards Limited 2017ISBN 978 0 580 89040 6ICS 71.100.70Compliance with a British Standard cannot confer immunity from legal obligation

11、s.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 October 2017.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN 16956:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16956 September 2017 ICS 71

12、100.70 English Version Cosmetics - Analytical methods - HPLC/UV method for the identification and assay of hydroquinone, ethers of hydroquinone and corticosteroids in skin whitening cosmetic products Cosmtiques - Mthodes analytiques - Mthode de CLHP couple la dtection UV pour lidentification et lan

13、alyse de lhydroquinone, de ses thers et des corticostrodes dans les produits cosmtiques claircissants de la peau Kosmetische Mittel - Untersuchungsverfahren - HPLC/UV Verfahren fr die Identifizierung und Bestimmung von Hydrochinon, Hydrochinonethern und Kortikosteroiden in hautaufhellenden kosmetisc

14、hen Mitteln This European Standard was approved by CEN on 19 June 2017. 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 bibliograp

15、hical 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 responsibili

16、ty 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 o

17、f Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NOR

18、MALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16956:2017 EBS EN 16956:2017EN 16956:2017 (E) 2 Contents Page European

19、foreword . 3 Introduction 4 1 Scope 5 2 Principle . 5 3 Reagents . 5 4 Apparatus and equipment . 8 5 Procedure. 9 5.1 Sample preparation 9 5.2 Liquid chromatography measurement conditions . 9 5.3 Detection . 10 6 Evaluation . 10 6.1 Identification . 10 6.2 Quantitative determination . 10 6.3 Result

20、expression . 11 7 Test report 11 Annex A (informative) Example of chromatograms obtained 12 Annex B (informative) Validation Data for the quantitative method hydroquinone and its three ethers 13 Annex C (informative) Validation data for the quantitative method for the 4 most frequently found cortico

21、steroids . 18 Annex D (normative) Screening methods for the identification of hydroquinone, 3 ethers of hydroquinone and 38 corticosteroids 24 Bibliography . 37 BS EN 16956:2017EN 16956:2017 (E) 3 European foreword This document (EN 16956:2017) has been prepared by Technical Committee CEN/TC 392 “Co

22、smetics”, 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 March 2018, and conflicting national standards shall be withdrawn at the latest by March 2018.

23、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. The existing peer review validation data for hydroquinone are preliminary and will be

24、supplemented by inter-laboratory test data if available. 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, Fi

25、nland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16956:2017EN 169

26、56:2017 (E) 4 Introduction Hydroquinone is not allowed for use in cosmetic products for skin whitening and depigmentation of dermal spots or imperfections. Due to its cytotoxic effects its use has been regulated. Hydroquinone and 3 of its ethers (hydroquinone monomethylether (MME), hydroquinone mono

27、ethylether (MEE) and hydroquinone monobenzylether (MBE) are regulated by the cosmetic regulation 1223/2009. Nowadays the use of these substances is prohibited in skin whitening cosmetic products. Depigmentation is a side effect of topical steroids, in this way corticosteroids might be used as compou

28、nds in products illegally sold as cosmetics. Corticosteroids most commonly found in these products are clobetasol propionate, fluocinonide, betamethasone dipropionate, and fluocinolone acetonide (see Figure 1). Corticosteroids are listed in Regulation 1223/2009 Annex II “List of substances prohibite

29、d in cosmetic products” (reference number 300), and their use is also prohibited in cosmetic products. a) Clobetasol propionate b) Fluocinonide c) Betamethasone dipropionate d) Fluocinolone Acetonide Figure 1 Corticosteroids most commonly found in illegal cosmetics All these substances work on the s

30、ame principle as hydroquinone which mainly consists of inhibition of melanin synthesis. The cosmetic directive 95/32/EC 2 gives an analytical method for the assay of hydroquinone and 3 of its ethers (hydroquinone monomethylether (MME), hydroquinone monoethylether (MEE) and hydroquinone monobenzyleth

31、er (MBE) in cosmetic products for lightening the skin. In order to update and extend this official method to the identification and assay of corticosteroids in cosmetic products, this standard describes an HPLC/UV method for the identification and assay of hydroquinone, ethers of hydroquinone and co

32、rticosteroids in cosmetic products. BS EN 16956:2017EN 16956:2017 (E) 5 1 Scope This European Standard specifies a HPLC/UV method for the identification and quantification of hydroquinone, 3 ethers of hydroquinone and 4 corticosteroids most frequently found in illegally sold skin whitening cosmetic

33、products: clobetasol propionate, betamethasone dipropionate, fluocinonide and fluocinolone acetonide. This standard also gives HPLC/UV methods for the identification of 38 corticosteroids that may be found in skin whitening cosmetic products (see Annex D). This standard is not dedicated to artificia

34、l nail products or soaps. 2 Principle The sample is extracted by a mixture of water/methanol and gently warmed in order to extract compounds present in the product. The obtained mixture is filtered. The quantitation of present compounds in solution is made by reversed phase HPLC with DAD (Diode Arra

35、y Detector) detection. 3 Reagents If not otherwise specified, analytical-grade chemicals shall be used; the water shall be distilled or of a corresponding purity. “Solution” shall be understood as an aqueous solution unless otherwise specified. 3.1 Methanol, HPLC grade. 3.2 Water, HPLC grade. 3.3 Ex

36、traction solution, methanol/water (1/1). Mix 500 ml of methanol (3.1) and 500 ml of water (3.2) in a 1 000 ml conical flask. 3.4 Compounds considered, see Table 1. Table 1 Compounds considered Compound CAS ManufactureraPurity % Used method clause Alclometasone dipropionate (ACD) 66734132 USP 99,2 An

37、nex D Amcinonide (AMC) 51022696 Sigma 97,9 Annex D Beclomethasone dipropionate (BCD) 5534-09-8 Sigma 99,0 Annex D Betamethasone acetate (BMA) 987246 Sigma 98,6 Annex D Betamethasone (BM) 378449 Sigma 98,4 Annex D Betamethasone dipropionate (BMD) 5593204 Sigma 98,6 2/ Annex D Betamethasone valerate (

38、BMV) 2152445 Sigma 98,1 Annex D Budesonide (BUD) 51333223 Ph. Eur. 99,7 Annex D Clobetasol propionate (CP) 25122467 Sigma 98,8 2/ Annex D Clocortolone pivalate (CLP) 34097160 USP 98,9 Annex D Cortisone (CS) 53065 Sigma 98,3 Annex D Desonide (DSN) 638948 Cil 98,0 Annex D BS EN 16956:2017EN 16956:2017

39、 (E) 6 Compound CAS ManufactureraPurity % Used method clause Desoximetasone (DXM) 382672 Sigma 99,0 Annex D Dexamethasone phosphate (DMPS) 2392394 Sigma 100,2 Annex D Dexamethasone acetate (DMA) 1177873 Sigma 99,0 Annex D Diflorasone diacetate (DFD) 33564317 USP 99,8 Annex D Diflucortolone valerate

40、DFCV) 59198708 Schering / Annex D Difluprednate (DFP) 23674864 Sigma 99,5 Annex D Flumethasone pivalate (FMP) 2002291 Farmabios 100,4 Annex D Fluocinolone acetonide (FCA) 67732 Sigma 99,6 2/ Annex D Fluocinonide (FCAA) 356127 Sigma 99,0 2/ Annex D Fluocortolone hexanoate (FCH) 303402 Schering / Ann

41、ex D Fluocortolone pivalate (FCP) 29205069 Ph. Eur. / Annex D Flurandrenolide (FDL) 1524885 USP / Annex D Halcinonide (HAL) 3093354 Sigma 99,0 Annex D Hydrocortisone (HC) 3093254 Sigma 98,0 Annex D Hydrocortisone aceponate (HCAP) 74050207 Toronto Research Chemicals 98,0 Annex D Hydrocortisone butyra

42、te (HCB) 13609671 Sigma 99,1 Annex D Hydrocortisone valerate (HCV) 57524897 Sigma 99,0 Annex D Methylprednisolone acetate (MPLA) 53361 USP 99,8 Annex D Mometasone furoate (MMF) 83919237 USP 99,8 Annex D Prednicarbate (PCN) 73771047 Ph. Eur. / Annex D Prednisolone (PL) 50248 Aventis Pharma 98,0 Annex

43、 D Prednisolone acetate (PLA) 52211 Dr Ehrenstrofer 99,4 Annex D Prednisolone hexanoate (PLH) 69164698 Schering / Annex D Prednisolone sulfobenzoate Na (PLSB) 630671 Pharmaceutical drug / Annex D Triamcinolone (TRI) 124947 Sigma 98,2 Annex D Triamcinolone acetonide (TRA) 76255 Sigma 99,6 Annex D Hyd

44、roquinone (HQ) 123319 Sigma 99,9 2/ Annex D Hydroquinone monomethylether (MME) 150765 Sigma 99,7 2/ Annex D Hydroquinone monoethylether (MEE) 622628 Sigma 99,9 2/ Annex D Hydroquinone monobenzylether (MBE) 103162 Sigma 99,5 2/ Annex D aExamples of manufacturer. BS EN 16956:2017EN 16956:2017 (E) 7 3.

45、5 Mobile phase for HPLC, mobile phase A: methanol (3.1); mobile phase B: water (3.2). 3.6 Reference solutions. Methanol (3.1) is used as a solvent for the preparation of stock solutions. Standard solutions are prepared by dilution in a methanol / water solution (3.3). 3.6.1 Standard Stock Solution o

46、f hydroquinone and its 3 ethers (between 1,2 mg/ml and 2,4 mg/ml). Weigh approximately into a 25 ml volumetric flask: 0,03 g of hydroquinone; 0,04 g of monomethylether of hydroquinone; 0,05 g of monoethylether of hydroquinone; 0,06 g of monobenzylether of hydroquinone. Firstly, dissolve in 15 ml of

47、methanol (3.1), if necessary use a shaker and then fill up to the calibration mark with methanol. This solution shall be prepared daily. 3.6.2 Standard Stock Solutions (1 mg/ml) and Standard Working Solution of 4 most frequently found corticosteroids (10 g/ml). Weigh separately and approximately int

48、o a 10 ml volumetric flask: 10 mg of betamethasone dipropionate; 10 mg of clobetasol propionate; 10 mg of fluocinonide; 10 mg of fluocinolone acetonide. Firstly, dissolve in 5 ml of methanol (3.1), if necessary use a shaker and then fill up to the calibration mark with methanol. Theses 4 solutions c

49、an be stored during 6 months at 4 C. Then prepare a daughter solution of 4 corticosteroids by introducing 1 ml of each standard stock solution with a glass pipette in a 100 ml volumetric flask. Fill up to the mark with mixture methanol/water (3.3). These solution can be stored during 6 weeks at 4 C. 3.6.3 Calibration Solutions of hydroquinone and its 3 ethers The calibration solutions shall be freshly prepared (see Table 2). With