Cosmetics - Analytical methods - LC/UV method for the identification and quantitative determination in cosmetic products of the 22 organic UV filters in use in the EU

This proposal described a set of HPLC/UV procedures for the identification and quantitative determination in cosmetic products of 22 organic UV filters authorized in the EU: Camphor Benzalkonium Methosulfate (CBM), Homosalate (HS), Benzophenone-3 (BZ3), Phenylbenzimidazole Sulfonic Acid (PBS), Terephthalylidene Dicamphor Sulfonic Acid (TDS), Butyl Methoxydibenzoylmethane (BDM), Octocrylene (OCR), Ethylhexyl Methoxycinnamate (EMC), PEG-25 PABA (P25), Isoamyl p-Methoxycinnamate (IMC), Ethylhexyl Triazone (ET), Drometrizole Trisiloxane (DRT), Diethylhexyl Butamido Triazone (DBT), 4-Methylbenzylidene Camphor (MBC), Ethylhexyl Salicylate (ES), Ethylhexyl Dimethyl PABA (EDP), Benzophenone-4/Benzophenone-5 (BZ4),  Methylene Bis-Benzotriazolyl Tetramethylbutylphenol (MBP), Disodium Phenyl Dibenzimidazole Tetrasulfonate (PDT), Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine (EMT), Polysilicone-15 (P-15), Diethylamino Hydroxybenzoyl Hexyl Benzoate (DHHB). They are all the currently authorized organic UV filters in the EU, with the only exception of three of them that are protected under patents and are no longer used in cosmetics (Benzylidene Camphor Sulfonic Acid, Polyacrylamidomethyl Benzylidene Camphor and 3-Benzylidene Camphor).

Kosmetische Mittel - Untersuchungsverfahren - LC/UV Verfahren für die Identifizierung und qualitative Bestimmung von den 22 in der EU verwendeten organischen UV-Filtern in kosmetischen Produkten

Diese Europäische Norm legt ein Analyseverfahren basierend auf Flüssigchromatographie (en: liquid chromatography, LC) mit UV/VIS Spektroskopie (für Wellenlängen des ultravioletten und sichtbaren (en: visible, VIS) Lichts) für die Detektion und quantitative Bestimmung der aktuell 22 organischen und im EU Rahmen verwendeten UV Filter fest. Dieses Verfahren wurde für emulsionsbasierte kosmetische Produkte, Lippenbalsame, Lotionen und Wasser validiert.

Cosmétiques - Méthodes analytiques - Procédé CL/UV pour l’identification et la détermination quantitative des 22 filtres UV organiques utilisés dans les produits cosmétiques au sein de l’UE

Le présent document spécifie une méthode analytique basée sur la chromatographie en phase liquide (CL) couplée à la détection par spectrométrie ultraviolet/visible (UV/Vis), pour la détection et la détermination quantitative des 22 filtres UV organiques utilisés dans le cadre de l’UE. Cette méthode a été validée pour les produits cosmétiques à base d’émulsion, les baumes à lèvres, les lotions et les eaux.

Kozmetika - Analizne metode - Metoda LC/UV za identifikacijo in kvantitativno določevanje 22 organskih UV-filtrov, ki se v EU uporabljajo v kozmetičnih izdelkih

Ta predlog je opisal niz postopkov HPLC/UV za identifikacijo in kvantitativno določevanje 22 organskih UV-filtrov, ki so v EU odobreni za uporabo v kozmetičnih izdelkih: benzalkonijev metasulfat kafra (CBM), homosalat (HS), benzofenon-3 (BZ3), fenilbenzimidazol sulfonska kislina (PBS), tereftaliden dikafra sulfonska kislina (TDS), butilmetoksi dibenzoilmetan (BDM), oktokrilen (OCR), etilheksil metoksicinamat (EMC), PEG-25 PABA (P25), izoamil p-metoksicinamat (IMC), etilheksil triazon (ET), drometrizol trisiloksan (DRT), dietilheksil butamidotriazon (DBT), 4-metilbenziliden kafra (MBC), etilheksil salicilat (ES), etilheksildimetil PABA (EDP), benzofenon-4/benzofenon-5 (BZ4), metilen bisbenzotriazolil tetrametilbutilfenol (MBP), dinatrijev fenil dibenzimidazol tetrasulfonat (PDT), bisetilheksiloksifenol metoksifenil triazin (EMT), polisilikon-15 (P-15), dietilamino hidroksibenzoil heksil benzoat (DHHB). To so vsi trenutno odobreni organski UV-filtri v EU, z izjemo treh, ki so zaščiteni s patenti in se ne uporabljajo več v kozmetičnih izdelkih (benziliden kafra sulfonska kislina, poliakrilamidometil benziliden kafra in 3-benziliden kafra).

General Information

Status
Published
Public Enquiry End Date
02-Nov-2017
Publication Date
13-Jan-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
09-Jan-2019
Due Date
16-Mar-2019
Completion Date
14-Jan-2019

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.YDQWLWDWLYQRKosmetische Mittel - Untersuchungsverfahren - LC/UV Verfahren für die Identifizierung und qualitative Bestimmung von den 22 in der EU verwendeten organischen UV-Filtern in kosmetischen ProduktenCosmétiques - Méthodes analytiques - Procédé CL/UV pour l’identification et la détermination quantitative des 22 filtres UV organiques utilisés dans les produits cosmétiques au sein de l’UECosmetics - Analytical methods - LC/UV method for the identification and quantitative determination in cosmetic products of the 22 organic UV filters in use in the EU71.100.70SULSRPRþNLCosmetics. ToiletriesICS:Ta slovenski standard je istoveten z:EN 17156:2018SIST EN 17156:2019en,fr,de01-februar-2019SIST EN 17156:2019SLOVENSKI

STANDARD
SIST EN 17156:2019
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 17156
December
t r s z ICS
y sä s r rä y r English Version

Cosmetics æ Analytical methoidentification and quantitative determination in cosmetic products of the

t t organic UV filters in use in the EU pour l 5identification et la détermination quantitative des

t t filtres UV organiques utilisés dans les produits cosmétiques au sein de l 5UE Verfahren für die Identifizierung und qualitative Bestimmung von den

t t in der EU verwendeten organischen UVæFiltern in kosmetischen Produkten This European Standard was approved by CEN on

w November
t r s zä

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

CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä

EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre:
Rue de la Science 23,
B-1040 Brussels

t r s z CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN

s y s w xã t r s z ESIST EN 17156:2019

EN 17156:2018 (E) 2 Contents Page European foreword ....................................................................................................................................................... 3 Introduction .................................................................................................................................................................... 4 1 Scope .................................................................................................................................................................... 6 2 Normative references .................................................................................................................................... 6 3 Terms and definitions ................................................................................................................................... 6 4 Principles ........................................................................................................................................................... 6 5 Reagents ............................................................................................................................................................. 6 6 Apparatus and equipment ........................................................................................................................... 8 7 Procedure........................................................................................................................................................... 9 7.1 Sample preparation ........................................................................................................................................ 9 7.2 LC-UV/Vis measurement conditions ........................................................................................................ 9 8 Evaluation ....................................................................................................................................................... 13 8.1 Identification ................................................................................................................................................. 13 8.2 Quantitative determination ..................................................................................................................... 13 9 Test report ...................................................................................................................................................... 14 Annex A (informative)

Results of the intra-laboratory validation ............................................................ 15 A.1 General ............................................................................................................................................................. 15 A.2 Water-soluble UV filters ............................................................................................................................ 15 A.3 Fat-soluble UV filters .................................................................................................................................. 18 A.4 UV filter P15 ................................................................................................................................................... 23 Annex B (informative)

Results of the inter-laboratory validation (Ring trial) ..................................... 24 B.1 General ............................................................................................................................................................. 24 B.2 Water-soluble UV filters ring trial ......................................................................................................... 25 B.3 Fat-soluble UV filters ring trial................................................................................................................ 26 B.4 UV filter P15 ring trial ................................................................................................................................ 28 Bibliography ................................................................................................................................................................. 29

SIST EN 17156:2019

EN 17156:2018 (E) 3 European foreword This document (EN 17156:2018) 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 June 2019 and conflicting national standards shall be withdrawn at the latest by June 2019. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN shall not be held responsible for identifying any or all such patent rights. According to the CEN-CENELEC Internal Regulations, the national standards organizations 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 17156:2019

EN 17156:2018 (E) 4 Introduction In order to protect human skin from the deleterious UV radiation of sunlight, the so-called UV filters have been used as active ingredients in the formulation of sunscreen cosmetic products. These active compounds are of organic or inorganic nature, and have the capacity to absorb and/or reflect, respectively, this UV radiation. Nowadays, they are not only added to those cosmetics intended specifically for sun protection but also in all type of daily products such as moisturizers, after shave products, shampoos, anti-aging creams, make-up products, etc. The compounds that can be used as UV filters in cosmetics and their maximum allowed concentrations are regulated in order to ensure user’s safety. Currently, the European Union (EU) 1223/2009 Regulation permits the use of 27 compounds as UV filters [1], the names of which are listed in Table 1. Among these 27 UV filters, titanium dioxide and zinc oxide are of inorganic nature, and among the remaining 25, 7 are highly polar and can be grouped in the ‘water-soluble’ group, whereas the other 18 are low polar and can be grouped in the ‘fat-soluble’ group. All these compounds are included in the formulations of the different cosmetic products consumed in the EU framework, with the only exceptions of BCSA and PBC which are not being currently used. Besides, errors during the manufacturing process of cosmetics may cause a lower concentration in the final product than that formulated. This might affect the efficacy of the product since the real Sun Protection Factor could be lower than that labelled. Therefore, reliable and practical analytical methods are needed in order to ensure compliance with the EU Regulation and thus protect user’s safety, but also to ensure product’s efficacy. In this sense, with the aim of implementing a broad-spectrum analytical method to improve and facilitate the quality control of the cosmetic industry, this European Standard presents an analytical method for the quantification of 22 organic UV filters. They constitute all the organic UV filters allowed and in use in the EU when this standard was validated. Note that TBT is not included since it was later approved. The presented method, besides good analytical characteristics, is simple, low cost, rapid and both user- and environmentally-friendly. The method is based on different analytical methods previously published by the Research Group on Quality Control of Cosmetic Products of the University of Valencia [2], [3]. SIST EN 17156:2019

EN 17156:2018 (E) 5 Table 1 — List of the UV filters permitted in cosmetic products under the EU Regulation EU Reference number a Name of Common Ingredients Glossary b Acronym c 2 Camphor Benzalkonium Methosulfate CBM 3 Homosalate HMS 4 Benzophenone-3 BZ3 6 Phenylbenzimidazole Sulfonic Acid PBSA 7 Terephthalylidene Dicamphor Sulfonic Acid TDSA 8 Butyl Methoxydibenzoylmethane BMDM 9 Benzylidene Camphor Sulfonic Acid BCSA 10 Octocrylene OC 11 Polyacrylamidomethyl Benzylidene Camphor PBC 12 Ethylhexyl Methoxycinnamate EHMC 13 PEG-25 PABA P25 14 Isoamyl p-Methoxycinnamate IMC 15 Ethylhexyl Triazone EHT 16 Drometrizole Trisiloxane DTS 17 Diethylhexyl Butamido Triazone DEBT 18 4-Methylbenzylidene Camphor MBC 20 Ethylhexyl Salicylate EHS 21 Ethylhexyl Dimethyl PABA EHDP 22 Benzophenone-4 BZ4 23 Methylene Bis-Benzotriazolyl Tetramethylbutylphenol MBBT 24 Disodium Phenyl Dibenzimidazole Tetrasulfonate PDTA 25 Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine BEMT 26 Polysilicone-15 P15 27 Titanium Dioxide TiO2 28 Diethylamino Hydroxybenzoyl Hexyl Benzoate DHHB 29 Tris-biphenyl triazine TBT 30 Zinc Oxide ZnO a Order number given according to the EU 1223/2009 Regulation. b According to the Annex VI of the European Union (EU) 1223/2009 Regulation. c Acronyms used in this standard. SIST EN 17156:2019

EN 17156:2018 (E) 6 1 Scope This document specifies an analytical method, based on liquid-chromatography (LC) with ultraviolet/visible spectrometry (UV/Vis) detection for the detection and quantitative determination of 22 organic UV filters in use in the EU framework. This method has been validated for emulsion-based cosmetic products, lip-balms, lotions and waters. 2 Normative references There are no normative references in this document. 3 Terms and definitions No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

IEC Electropedia: available at http://www.electropedia.org/

ISO Online browsing platform: available at http://www.iso.org/obp 4 Principles The cosmetic sample is weighed and solved in ethanol. For difficult-to-solve samples, the use of ultrasound or vortex can help. After that, depending on the UV filters to be determined, aliquots are taken and diluted with a mixture of ethanol:acetate buffer, ethanol or tetrahydrofuran for the determination of the water-soluble group, the fat-soluble group or P15 (which is a polymer), respectively. Regarding the water-soluble group, the 6 UV filters are measured by employing a reversed-phase column and a mobile phase of ethanol and acetate buffer. Regarding the fat-soluble group, the 15 UV filters are determined by employing a reversed-phase column and a mobile phase of ethanol and aqueous formic acid containing hydroxypropyl-beta-cyclodextrin as mobile phase modifier. Finally, for P15 determination, a size-exclusion column and tetrahydrofuran as mobile phase are used. 5 Reagents If not otherwise specified, analytical-grade chemicals shall be used. 5.1 Ethanol (EtOH), LC-grade. 5.2 Tetrahydrofuran (THF), LC-grade. 5.3 Deionized water, 18,2 ·cm resistivity. 5.4 2-Hydroxypropyl--cyclodextrin (HP--CD), 1 309 g/mol molecular weight. 5.5 Formic acid, 98 %, d = 1,22 g/ml. 5.6 Glacial acetic acid, > 99 %, d = 1,05 g/ml. 5.7 Sodium hydroxide, pellets. 5.8 Sodium hydroxide, 10 % (w/w) aqueous solution. Weigh 10 g of sodium hydroxide (5.7) and dissolve in 100 ml of deionized water (5.3). SIST EN 17156:2019

EN 17156:2018 (E) 7 5.9 Acetate buffer, 1 % (v/v) solution, pH = 4,75. Take 10 ml of glacial acetic acid (5.6) and dilute with approximately 900 ml of deionized water (5.3). Adjust with the aid of pH-meter up to pH 4,75 by adding sodium hydroxide solution (5.8). Complete up to 1 l with deionized water (5.3). 5.10 Formic acid, 1 % (v/v) solution, containing 20 mmol HP--CD. Weigh 26,2 g of HP--CD (5.4). Add 10 ml of formic acid (5.5), and complete up to 1 l with deionized water (5.3). 5.11 UV filter standards, see Table 2. Table 2 — UV filter standards Type UV filter CAS Water-soluble UV filters Disodium Phenyl Dibenzimidazole Tetrasulfonate (PDTA) 180898–37–7 Phenylbenzimidazole Sulfonic Acid (PBSA) 27503–81–7 Terephthalylidene Dicamphor Sulfonic Acid (TDSA) (triethanolamine salt) 90457–82–2 Benzophenone-4 (BZ4) 4065–45–6 Camphor Benzalkonium Methosulfate (CBM) (29 % aqueous solution) 52793–97–2 PEG-25 PABA (P25) 116242–27–4 Fat-soluble

UV filters Benzophenone-3 (BZ3) 131–57–7 Isoamyl p-Methoxycinnamate (IMC) 71617–10–2 4-Methylbenzylidene Camphor (MBC) 36861–47–9 Diethylamino Hydroxybenzoyl Hexyl Benzoate (DHHB) 302776–68–7 Octocrylene (OC) 6197–30–4 Ethylhexyl Dimethyl PABA (EHDP) 21245–02–3 Butyl Methoxydibenzoylmethane (BMDM) 70356–09–1 Ethylhexyl Methoxycinnamate (EHMC) 5466–77–3 Ethylhexyl Salicylate (EHS) 118–60–5 Homosalate (HMS) 118–56–9 Diethylhexyl Butamido Triazone (DEBT) 154702–15–5 Ethylhexyl Triazone (EHT) 88122–99–0 Drometrizole Trisiloxane (DTS) 155633–54–8 Methylene Bis-Benzotriazolyl Tetramethylbutylphenol (MBBT) 103597–45–1 Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine (BEMT) 187393–00–6 Polymeric

UV filter Polysilicone-15 (P15) 207574–74–1 SIST EN 17156:2019

EN 17156:2018 (E) 8 5.12 UV filters stock solutions Prepare a 500 µg/ml multicomponent stock solution of the 6 water-soluble UV filters in deionized water. For this purpose, weigh the appropriate amount of each standard (5.11) in a 25 ml volumetric flask and add approximately 10 ml of deionized water (5.3). In case any of the substances is not solubilized, add a few drops of 10 % sodium hydroxide solution (5.8) until complete dissolution. Finally, fill up to the mark with deionized water (5.3). This solution should be monthly prepared, stored at 4 °C and protected from light until use. Prepare a 500 µg/ml multicomponent stock solution of the fat-soluble UV filters in EtOH, except for MBBT and BEMT, which are prepared at 100 µg/ml due to their limited solubility. For this purpose, weigh the appropriate amount of each standard (5.11) in a 25 ml volumetric flask and dissolve with approximately 10 ml of EtOH (5.1). Then fill up to the mark with EtOH (5.1). This solution should be monthly prepared, stored at 4 °C and protected from light until use. Prepare a 1 000 µg/ml P15 solution in EtOH. For this purpose, weigh the appropriate amount of P15 (5.11) in a 25 ml volumetric flask and dissolve with approximately 10 ml of EtOH (5.1). Then fill up to the mark with EtOH (5.1). This solution should be monthly prepared, stored at 4 °C and protected from light until use. 5.13 Calibration standard solutions For the preparation of the water-soluble UV filters calibration solutions, transfer 0,2 ml, 0,4 ml, 0,6 ml, 0,8 ml and 1,0 ml of the 500 µg/ml multicomponent stock solution (5.12) to five 10 ml volumetric flasks, respectively. Add 3 ml of EtOH (5.1) and acetate buffer solution (5.9) to each volumetric flask up to the mark. These 30:70 EtOH:acetate buffer (v/v) solutions contain 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml and 50 µg/ml of each target compound, respectively. These solutions should be weekly prepared, stored at 4 °C and protected from light until use. For the preparation of the fat-soluble UV filters calibration solutions, transfer 0,2 ml, 0,4 ml, 0,6 ml, 0,8 ml and 1,0 ml of the 500 µg/ml multicomponent stock solution (5.12) and 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of the 100 µg/ml solution of MBBP and BEMT to five 10 ml volumetric flasks, respectively. Add EtOH (5.1) to each volumetric flask up to the mark. These ethanolic solutions contain 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml and 50 µg/ml of each target compound, respectively. These solutions should be weekly prepared, stored at 4 °C and protected from light until use. For the preparation of the P15 calibration solutions, transfer 0,2 ml, 0,4 ml, 0,6 ml, 0,8 ml and 1,0 ml of the 1 000 µg/ml P15 stock solution (5.12) to 10 ml volumetric flasks, respectively. Add either EtOH (5.1) or THF (5.2) to each volumetric flask up to the mark. These solutions contain 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml and 100 µg/ml of P15, respectively. These solutions should be weekly prepared, stored at 4 °C and protected from light until use. 5.14 Working standard solutions Calibration standard solutions (5.13) are daily transferred to 1,5 ml injection vials. Once sealed, they are ready to be injected into the LC-system. 6 Apparatus and equipment In addition to the usual laboratory equipment, the following is required. 6.1 Membrane filter, in the form of a disposable syringe filter, pore width 0,45 µm. 6.2 Analytical balance, with a precision of 0,1 mg. 6.3 Ultrasonic water bath (or vortex mixer). SIST EN 17156:2019

EN 17156:2018 (E) 9 6.4 pH-meter. 6.5 LC-UV/Vis system, equipped with pump system with gradient function, degasser, autosampler, thermostated column oven, UV/Vis detector1) and data management station. 6.6 Analytical separation column For the determination of both water-soluble and fat-soluble group Purospher® STAR RP-18 Endcapped2) (125 mm length, 4 mm i.d., 5 µm particle size) or equivalent can be used. For the determination of P15 PLgel Mixed D2) (300 mm length, 7,5 mm i.d., 5 µm particle size) or equivalent can be used. 7 Procedure 7.1 Sample preparation Homogenize the sample depending on its nature (i.e. shake creams, milks and lotions manually; cut lip balms in tiny pieces, mix and knead again). Then, in triplicate, weigh 0,02 g to 0,20 g of the sample, depending of the expected amount of UV filters, into a 25 ml volumetric flask, then dissolve and dilute with EtOH (5.1). In case of difficult-to-solve samples, an ultrasonic water bath (6.3) may be used to disperse the sample. Take different aliquots to carry out the determination of the different groups of the target compounds. For the determination of the water-soluble UV filters, transfer 1 ml to 3 ml of the above-mentioned ethanolic sample stock solution to a 10 ml volumetric flask. Add EtOH (5.1) to complete a total volume of 3 ml, and then fill up to the mark with acetate buffer solution (5.9). Transfer this solution to a 1,5 ml injection vial, previously filtered through a membrane filter (6.1) if necessary. For the determination of the fat-soluble UV filters, transfer 1 ml to 3 ml of the above-mentioned ethanolic sample stock solution to a 10 ml volumetric flask. Fill up to the mark with EtOH (5.1). Transfer this solution to a 1,5 ml injection vial previously filtered through a membrane filter (6.1) if necessary. For the determination of P15, transfer 1 ml to 3 ml of the above-mentioned ethanolic sample stock solution to a 10 ml volumetric flask. Fill up to the mark with either EtOH (5.1) or THF (5.2), as in Calibration standard solutions (5.13). Transfer this solution to a 1,5 ml injection vial, previously filtered through a membrane filter (6.1) if necessary. All these solutions should be prepared and protected from light until injection in the same working session. 7.2 LC-UV/Vis measurement conditions When using the LC-UV/Vis system (6.5) and column (6.6) for water-soluble UV-filters determination, the conditions in Table 3 have shown to be suitable. When using LC systems with different delay volumes the gradient time tables should be accordingly adjusted.

1) For identification purposes, diode-array detector is recommended. 2) Purospher® STAR RP-18 Endcapped and PLgel Mixed D are examples of suitable products available commercially. This information is given for the convenience of users of

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