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Technical Document “Cosmetics - Analytical methods – HPLC/UV method for the identification and quantitative determination in cosmetic products of all the organic UV filters authorized and in use in the EU” 1.-Introduction The compounds that can be used as UV filters are included in the Regulation (EC) No 1223/2009 [1] (Annex VI “List of UV filters allowed in cosmetic products”). Table 1 shows the 26 compounds currently authorized as UV filters in cosmetics, the abbreviations used in this draft, their maximum authorized concentration and their current use. Among these 26 compounds, only one (titanium dioxide) has an inorganic nature, whereas the other 25 are organic UV filters. Seven of the organic UV filters have a hydrophilic character (named water-soluble UV filters: CBM, PBS, TDS, BCS, P25, BZ4 and PDT, whereas the remaining eighteen present lipophilic properties (fat-soluble UV filters: HS, BZ3, BDM, OCR, PBC, EMC, IMC, ET, DRT, DBT, MBC, 3BC, ES, EDP, BZ4, MBP, EMT, DHHB). Currently all these UV filters are included in the formulations of the different cosmetic products in the EU, with the only exceptions of BCS, PBC and 3BC which are no longer used years ago. Analytical monitoring of the concentration of UV filters in the finished product is necessary because EU regulation states maximum authorized content. Moreover, errors during the manufacturing process of cosmetics may cause a lower concentration in the final product than that formulated which would affect the safety of users as the real Sun Protection Factor be lower than labelled. Thus, analytical methods to perform a quantitative determination of all the UV filters used in the different cosmetic products, either sunscreen or other daily products, are necessary. These methods should be precise and accurate and, as far as possible, simple, fast and safe for the operator and the environment, since analytical control in cosmetic industries usually require a large number of analysed samples.

2.-Reference methods In spite of the restrictions of use established for UV filters, there is only one official method [2] concerning their determination in the published Commission Directives related these methods of analysis needed to check the composition of cosmetic products. This method corresponds to the determination of glycerol 1-(4-aminobenzoate), which was banned in cosmetic products to be marketed in the EU in 1992. The analytical method was based on qualitative determination by means of thin layer chromatography and subsequent quantitative determination by reversed phase liquid chromatography with UV detection (RP-HPLC-UV). Sample preparation for quantitative determination was carried out using MeOH and Carrez reagents and the mobile phase was a phosphate buffer /MeOH mixture. Recently, a very interesting standard, describing a multi-screening RP-HPLC-UV method was proposed to the CEN/TC 392 [3] as reference candidate method. This method allows the identification of the authorized organic UV filters with the exception of BCS, PBC, P25 and P15. However, quantitative determination was proposed only for 10 of the organic UV filters (TDS, BMD, OCR, EMC, IMC, EHT, DRT, DBT, MBP, EMT). Three different stock solutions are used according to the properties of the compounds (MeOH for polar, acetone/MeOH for medium polar and tetrahydrofuran for non polar UV filters). The UV filters are extracted from samples with an acetone/MeOH mixture; for the quantitative determination of TDS, BDM, 4

Form N MBP and EMT, additional extraction methods and determinations are required: extraction with MeOH-NaOH (for TDS), extraction with tetrahydrofuran/acetone (MBP and EMT), extraction with acetone/MeOH/EDTA (BDM). Gradient elution programmes based on mixtures of an aqueous phase (dodecyltrimethylammonium bromide, ammonium bromide and EDTA in water) and an organic phase (dodecyltrimethylammonium bromide and ammonium bromide in ethanol) were proposed.

3.-Aim of the proposal With the final purpose of implementing reliable and broad spectrum analytical methods to improve and facilitate the quality control of the cosmetic industry, but using environmentally friendly analytical procedures (if possible), this draft presents a joint proposal for the analysis of cosmetic products in order to identify and quantitatively determine the concentration of the organic UV filters. The main advantages of the proposed method are: −

To the best of our knowledge this is the first proposal of a reference candidate method to the quantitative determination of all the UV filters authorized and in use.



Sample preparation and analytical procedures are fast, present a high level of safety for both, the operator and the environment, as minimizes the use of toxic organic solvents because the only organic solvent required for both sample preparation and chromatographic analysis is ethanol (except for P-15 that requires the use of tetrahydrofuran).

Moreover, the use of these procedures could be also extended to the analytical control of raw materials with some slight modifications.

4.-Scientific support of the proposed analytical procedures This draft have been elaborated and revised by the Spanish Working Group “WG-14-Methods of Analysis” of the Subcommittee SC2-Cosmetic Products of AEN/CTN84 (STANPAAENOR). The proposed procedures are scientifically supported by the previous experience and publications on analysis of cosmetic products (and particularly UV filters determination) of some of the members of this WG-14 [4-9].

5.-Summary of the proposed analytical procedures 5.1) Water-soluble UV-filters [8] Analytes: CBM, PBS, TDS, P25, BZ4, PDT Samples to be analyzed: all type of cosmetic products Sample preparation: initial solutions of samples are prepared by complete dissolution with ethanol (shaking with the aid of an ultrasonic bath, if necessary). Low solubility samples (containing titanium dioxide or other cosmetic ingredients) are also treated with ethanol to lixiviate the analytes which are separated by filtration (through a 0.45 µm nylon membrane filter). Sample solutions to be measured: initial ethanolic solutions of samples are diluted to obtain hydroethanolic solutions 30:70 ethanol:1% acetate buffer (pH 4.75) and to adjust in the working range. Calibration solutions: multi-component aqueous solution of pure UV filters are prepared and they are diluted to obtain hydroethanolic solutions 30:70 ethanol:1% acetate buffer (pH 4.75). Working range: 10-50 µg/mL of each one of the UV filters in the calibration solutions. 5

Form N Analytical technique: RP-HPLC-UV Analytical signal: area of the chromatographic peaks (see a representative chromatogram in Figure 1) Main analytical variables: −

RP-18 chromatographic column (12.5 cm x 4 mm i.d., 5 µm particle size)



flow rate 1 mL/min



temperature 45ºC



wavelength 313 nm (or wavelength of maximum absorbance of each compound if diode array or variable detector are available)



injection volume 5 µL

Mobil phase: hydroethanolic mixtures of ethanol and 1% acetate buffer (pH 4.75), according to an appropriate elution gradient program (see Table 2). Highlights: −

Simultaneous determination of all the water-soluble UV-filters without interferences among them or from fat-soluble UV-filters (which will elute after).



To the best of our knowledge this is the first proposal of a reference candidate method to the quantitative determination of all these UV filters.



The same initial solutions of samples can be used for the determination of water and fat-soluble UV filters.



Safe and environmentally green method as the only organic solvent required is ethanol.

5.2) Fat-soluble UV-filters [9] 5.2.1) General procedure Analytes: HS, BZ3, BDM, OCR, EMC, IMC, ET, DRT, DBT, MBC, ES, EDP, MBP, EMT, DHHB Samples to be analyzed: all type of cosmetic products Sample preparation: initial solutions of samples are prepared by complete dissolution with ethanol (shaking with the aid of an ultrasonic bath, if necessary). Low solubility samples (containing titanium dioxide or other cosmetic ingredients) are also treated with ethanol to lixiviate the analytes which are separated by filtration (through a 0.45 µm nylon membrane filter). Sample solutions to be measured: initial ethanolic solutions of samples are directly prepared to adjust in the working range. Calibration solutions: multi-component ethanolic solutions of pure UV filters are prepared. Working range: 10-50 µg/mL of each one of the UV filters in the calibration solutions. Analytical technique: RP-HPLC-UV Analytical signal: area of the chromatographic peaks (see a representative chromatogram in Figure 2) Main analytical variables: −

RP-18 chromatographic column (12.5 cm x 4 mm i.d., 5 µm particle size)



flow rate 1 mL/min



temperature 60ºC

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Form N −

wavelength 313 nm, except for DHHB and BDM that were monitored at 360 nm (or wavelength of maximum absorbance of each compound if diode array or variable detector are available)



injection volume 5 µL

Mobil phase: hydroethanolic mixtures of ethanol and 1% formic acid containing 20 mM 2hydroxypropyl-β-cyclodextrin, according to an appropriate elution gradient program (see Table 3). Highlights: −

Simultaneous determination of all the fat-soluble UV-filters (except P-15, see 5.2.2) without interferences among them or from water-soluble UV-filters (which would elute earlier).



To the best of our knowledge this is the first proposal of a reference candidate method to the quantitative determination of all these UV filters.



The same initial solutions of samples can be used for the determination of water and fat-soluble UV filters.



The only organic solvent required is ethanol.

5.2.2) Specific procedure for P-15 Analyte: P-15 Samples: all type of cosmetic products Sample preparation: initial solutions of samples are prepared by complete dissolution with tetrahydrofuran (shaking with the aid of an ultrasonic bath, if necessary). Low solubility samples (containing titanium dioxide or other cosmetic ingredients) are also treated with tetrahydrofuran to lixiviate the analytes which are separated by filtration (through a 0.45 µm nylon membrane filter). Sample solutions to be measured: initial solutions of samples in tetrahydrofuran are diluted to adjust in the working range. Calibration solutions: solutions of pure P-15 in tetrahydrofuran are prepared. Working range: 20-100 µg/mL of P-15 in the calibration solutions. Analytical technique: Size exclusion HPLC-UV Analytical signal: area (or height) of the chromatographic peak Main analytical variables: −

Size exclusion column PL gel Mixed D (30 cm x 7.5 mm i.d., 5 µm particle size) with similar precolumn (5 cm x 7.5 mm i.d., 5 µm particle size)



flow rate 1 mL/min



wavelength 307 nm.



Mobil phase: tetrahydrofuran



injection volume 5 µL

Highlights: −

No interferences of other UV filters (they will elute after). To the best of our knowledge this is the first proposal of a reference candidate method to the quantitative determination of P-15.

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Form N 6. References [1] 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. [2] Fourth Commission Directive 85/490/EEC of 11 October 1985 on the approximation of the laws of the Member States relating to methods of analysis necessary for checking the composition of cosmetic products: Identification and determination of glycerol 1-(4-aminobenzoate). [3] Technical Committee CEN/TC 392 Cosmetics: Cosmetics — Analysis of sun screen products — Screening for UV filters in sun screen products and quantitative determination of 10 UV-filters by HPLC, TC 392 WI 00392017. [4] A. Salvador, A. Chisvert (Eds.), Analysis of Cosmetic Products, Elsevier, Amsterdam, ISBN: 0-444-52260-3, 2007: Section 3.1. A. Salvador, A. Chisvert, UV filters in sunscreens and other cosmetics. Regulatory aspects and analytical methods, 83-120. [5] A. Chisvert, M.C. Pascual-Marti, A. Salvador, Determination of UV-filters in sunscreens by HPLC, Fresenius J. Anal. Chem., 369, 638-641, 2001. [6] A. Chisvert, M.C. Pascual-Martí, A. Salvador, Determination of the UV-filters worlwide authorised in sunscreens by HPLC. Use of cyclodextrines as mobile phase modifier, J. Chromatogr. A, 921, 207-215, 2001 [7] A. Chisvert, A. Salvador, Determination of water-soluble UV-filters in sunscreen sprays by liquid chromatography, J. Chromatogr. A, 977, 277-280, 2002 [8] A. Salvador, A. Chisvert, An environmentally friendly (“green”) reversed-phase liquid chromatography method for UV filters determination in cosmetics, Anal. Chim. Acta, 537, 15-24, 2005. [9] A. Chisvert, I. Tarazona, A. Salvador, A reliable and environmentally friendly liquidchromatographic method for multi-class determination of fat-soluble UV filters in cosmetic products, Anal. Chim. Acta, 790, 61-67, 2013.

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Form N Table 1.-UV listed authorized in the EU Order

INCI name

(Annex VI)

2 3 4 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Camphor benzalkonium methosulfate Homosalate Benzophenone-3 Phenylbenzimidazole sulfonic acid Terephthalydene dicamphor sulfonic acid Buthyl methoxydibenzoyl methane Benzylidene camphor sulfonic acid Octocrylene Polyacrylamidomethyl benzylidene camphor Ethylhexyl methoxycinnamate PEG-25 PABA Isoamyl p-methoxycinnamate Ethylhexyl triazone Drometrizole trisiloxane Diethylhexyl butamido triazone 4-Methylbenzylidene camphor 3-Benzylidene camphor Ethylhexyl salicylate Ethylhexyl dimethyl PABA Benzophenone-4 Methylene bis-benzotriazolyl tetramethylbutylphenol Disodium phenyl dibenzimidazole tetrasulfonate Bis-ethylhexyloxyphenol methoxyphenyl triazine Polysilicone-15 Titanium dioxide Diethylamino hydroxbenzoyl hexyl benzoate

Maximum authorized Currently Abbreviation concentration in use (% m/m) CBM 6 yes HS 10 yes BZ3 10 yes PBS 8 yes TDS 10 yes BDM 5 yes BCS 6 no OCR 10 yes PBC 6 no EMC 10 yes P25 10 yes IMC 10 yes ET 5 yes DRT 15 yes DBT 10 yes MBC 4 yes 3BC 2 no ES 5 yes EDP 8 yes BZ4 5 yes MBP 10 yes PDT 10 yes EMT 10 yes P15 10 yes TiO2 25 yes DHHB 10 yes

Table 2.- Elution gradient program for water-soluble UV filters Time (min) 0 6 8.5 10 11 20

Ethanol (%) 25 25 100 100 25 25

1% acetate buffer (pH 4.75) (%) 75 75 0 0 75 75

Table 3.- Elution gradient program for fat-soluble UV filters Time (min) 0 8 8.5 13 23 30 31 40

Ethanol (%) 55 55 60 60 100 100 55 55

1% formic acid containing 20 mM HP-β-CD (%) 45 45 40 40 0 0 45 45

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Form N

Figure 1 Example of chromatogram obtained on a standard solution of water-soluble UVfilters

A b s o r b Ti

( i )

Figure 2 Example of chromatogram obtained on a standard solution of fat-soluble UV-filters (except P-15)

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