EN 16956:2017

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Kosmetische Mittel - Untersuchungsverfahren - HPLC/UV Verfahren für die Identifizierung und Bestimmung von Hydrochinon, Hydrochinonethern und Kortikosteroiden in hautaufhellenden kosmetischen MittelnCosmétiques - Méthodes analytiques - Méthode de CLHP couplée à la détection UV pour l'identification et l'analyse de l'hydroquinone, de ses éthers et des corticostéroïdes dans les produits cosmétiques éclaircissants de la peauCosmetics - Analytical methods - HPLC/UV method for the identification and assay of hydroquinone, ethers of hydroquinone and corticosteroids in skin whitening cosmetic products71.100.70SULSRPRþNLCosmetics. ToiletriesICS:Ta slovenski standard je istoveten z:EN 16956:2017SIST EN 16956:2017en,fr,de01-november-2017SIST EN 16956:2017SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16956
September
t r s y ICS
y sä s r rä y r English Version
Cosmetics æ Analytical methoidentification and assay of hydroquinoneá ethers of hydroquinone and corticosteroids in skin whitening cosmetic products Cosmétiques æ Méthodes analytiques æ Méthode de CLHP couplée à la détection UV pour l 5identification et l 5analyse de l 5hydroquinoneá de ses éthers et des corticostéroïdes dans les produits cosmétiques éclaircissants de la peau
Kosmetische Mittel æ Untersuchungsverfahren æ Bestimmung von Hydrochinoná Hydrochinonethern und Kortikosteroiden in hautaufhellenden kosmetischen Mitteln This European Standard was approved by CEN on
s { June
t r s yä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä
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Avenue Marnix 17,
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t r s y CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s x { w xã t r s y ESIST EN 16956:2017

Contents Page European 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 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 corticosteroids . 18 Annex D (normative)
Screening methods for the identification of hydroquinone, 3 ethers of hydroquinone and 38 corticosteroids . 24 Bibliography . 37
European foreword This document (EN 16956:2017) 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 March 2018, and conflicting national standards shall be withdrawn at the latest by March 2018. 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 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, 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 the United Kingdom. SIST EN 16956:2017

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 monoethylether (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 compounds 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 prohibited 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 same 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 monobenzylether (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 corticosteroids in cosmetic products. SIST EN 16956:2017

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 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 artificial 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 Array 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 Extraction 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
Manufacturera
Purity % Used method – clause Alclometasone dipropionate (ACD) 66734–13–2 USP 99,2 Annex D Amcinonide (AMC) 51022–69–6 Sigma 97,9 Annex D Beclomethasone dipropionate (BCD) 5534-09-8 Sigma 99,0 Annex D Betamethasone acetate (BMA) 987–24–6 Sigma 98,6 Annex D Betamethasone (BM) 378–44–9 Sigma 98,4 Annex D Betamethasone dipropionate (BMD) 5593–20–4 Sigma 98,6 2/ Annex D Betamethasone valerate (BMV) 2152–44–5 Sigma 98,1 Annex D Budesonide (BUD) 51333–22–3 Ph. Eur. 99,7 Annex D Clobetasol propionate (CP) 25122–46–7 Sigma 98,8 2/ Annex D Clocortolone pivalate (CLP) 34097–16–0 USP 98,9 Annex D Cortisone (CS) 53–06–5 Sigma 98,3 Annex D Desonide (DSN) 638–94–8 Cil 98,0 Annex D SIST EN 16956:2017

Compound
CAS
Manufacturera
Purity % Used method – clause Desoximetasone (DXM) 382–67–2 Sigma 99,0 Annex D Dexamethasone phosphate (DMPS) 2392–39–4 Sigma 100,2 Annex D Dexamethasone acetate (DMA) 1177–87–3 Sigma 99,0 Annex D Diflorasone diacetate (DFD) 33564–31–7 USP 99,8 Annex D Diflucortolone valerate (DFCV) 59198–70–8 Schering / Annex D Difluprednate (DFP) 23674–86–4 Sigma 99,5 Annex D Flumethasone pivalate (FMP) 2002–29–1 Farmabios 100,4 Annex D Fluocinolone acetonide (FCA) 67–73–2 Sigma 99,6 2/ Annex D Fluocinonide (FCAA) 356–12–7 Sigma 99,0 2/ Annex D Fluocortolone –hexanoate (FCH) 303–40–2 Schering / Annex D Fluocortolone pivalate (FCP) 29205–06–9 Ph. Eur. / Annex D Flurandrenolide (FDL) 1524–88–5 USP / Annex D Halcinonide (HAL) 3093–35–4 Sigma 99,0 Annex D Hydrocortisone (HC) 3093–25–4 Sigma 98,0 Annex D Hydrocortisone aceponate (HCAP) 74050–20–7 Toronto Research Chemicals 98,0 Annex D Hydrocortisone butyrate (HCB) 13609–67–1 Sigma 99,1 Annex D Hydrocortisone valerate (HCV) 57524–89–7 Sigma 99,0 Annex D Methylprednisolone acetate (MPLA) 53–36–1 USP 99,8 Annex D Mometasone furoate (MMF) 83919–23–7 USP 99,8 Annex D Prednicarbate (PCN) 73771–04–7 Ph. Eur. / Annex D Prednisolone (PL) 50–24–8 Aventis Pharma 98,0 Annex D Prednisolone acetate (PLA) 52–21–1 Dr Ehrenstrofer 99,4 Annex D Prednisolone hexanoate (PLH) 69164–69–8 Schering / Annex D Prednisolone sulfobenzoate Na (PLSB) 630–67–1 Pharmaceutical drug / Annex D Triamcinolone (TRI) 124–94–7 Sigma 98,2 Annex D Triamcinolone acetonide (TRA) 76–25–5 Sigma 99,6 Annex D Hydroquinone (HQ) 123–31–9 Sigma > 99,9 2/ Annex D Hydroquinone monomethylether (MME) 150–76–5 Sigma 99,7 2/ Annex D Hydroquinone monoethylether (MEE) 622–62–8 Sigma 99,9 2/ Annex D Hydroquinone monobenzylether (MBE) 103–16–2 Sigma 99,5 2/ Annex D a Examples of manufacturer. SIST EN 16956:2017

3.5 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 of 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 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 µg/ml). Weigh separately and approximately into 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 can 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 so
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