International Journal of Cosmetic Science, 2012, 34, 226–233
doi: 10.1111/j.1468-2494.2012.00703.x
Development and application of a HPLC method for eight sunscreen agents in suncare products L. M. Peruchi and S. Rath Department of Analytical Chemistry, Institute of Chemistry, University of Campinas, PO Box 6154, 13084-971 Campinas, SP, Brazil
Received 9 November 2011, Accepted 29 December 2011
Keywords: high-performance liquid chromatography method, lotions, sunscreens, UV filters, validation
Synopsis This work describes the development, validation and application of a simple and fast high-performance liquid chromatography-with diode array dectection (HPLC-DAD) method for the determination of eight sunscreen agents: benzophenone-3, octocrylene, ethylhexyl methoxycinnamate, ethylhexyl salicylate, homosalate (used in two isomeric forms), butyl methoxydibenzoylmethane, 4-methylbenzylidene camphor and ethylhexyl dimethyl PABA in sunscreen formulations. The separation of the eight sunscreen compounds was achieved using an ACE C18 column (250 · 4.6 mm, 5 lm), with a column temperature 20C, and a mobile phase of 88 : 12 (v/v) methanol-water with isocratic elution. Column temperature strongly influences the retention time and resolution of the compounds. The flow rate was 1.0 mL min)1 and quantitation was performed by external calibration at the maximum wavelength of each compound. The sample preparation was simple and consisted basically of sample dilution with methanol, centrifugation and filtration in syringe filters before quantitation. Total run time was 18 min. The method was validated according to the parameters: linear range, linearity, selectivity, intra-day and inter-day precision and accuracy. Ten samples of sunscreen emulsions commercially available in Brazil (SPF 30) from different manufacturers were analysed using the proposed method. The number of the sunscreen agents varied between one and five in a single sample. The concentrations of all compounds were in the range of 0.9–10% (w/w) and were in accordance with the current Brazilian legislation. Introduction Frequent exposure to UV radiation has pronounced harmful effects on human health. UV radiation-induced effects are manifested as acute responses, such as sunburn, hyperplasia and immunosuppression, and as chronic responses, primarily photo carcinogenesis and photo ageing [1]. The use of sunscreen products is a widely accepted way of primary prevention against the harmful effects of solar radiation. The molecules employed in cosmetic products to protect skin from the sun are classified as physical and chemical sunscreens. Physical sunscreens are represented mainly by zinc and titanium Correspondence: Susanne Rath, Department of Analytical Chemistry, Institute of Chemistry, University of Campinas, PO Box 6154, 13084971 Campinas, SP, Brazil. Tel.: +55 19 35213084; fax: +55 19 35213023; e-mail:
[email protected]
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oxides which interrupt the path of UV light by scattering or reflection. Chemical sunscreens are generally aromatic compounds conjugated with an electron-donating group in ‘ortho’ or ‘para’ position and an electron acceptor group. This chemical structure favours electron delocalization and, therefore, excitation of molecules from ground state to an excited state. The energy required for this transition corresponds to the energies of UVA and UVB radiations. The names, structures and maximum absorption of chemical sunscreens widely used in cosmetics are presented in Table I [2, 3]. To achieve protection in a wide UV-spectrum range, cosmetic industries normally employ a combination of UV filters that absorb in different regions of the spectrum. Regulatory authorities of several countries have established lists of approved UV absorbers with their maximum allowed concentrations in cosmetics. Table II provides the maximum concentration of eight UV filters allowed in the European Union [4], United States [5] and Brazil [6]. High-performance liquid chromatography (HPLC) is the most widely used technique for the determination of UV filters in cosmetics and several methods have been developed in recent years. Dencausse et al. [7] proposed a method for the determination of four sunscreens by HPLC using a C18 column and a mobile phase of acetonitrile, tetrahydrofuran and acetic acid, under gradient elution. The method was validated for synthetic formulations. Salvador and Chisvert [8] developed a ‘green’ procedure using non-toxic reagents for the determination of eighteen UV filters in cosmetics. The HPLC method employed a C18 column and mobile phase of ethanol and acetic acid for the fat soluble compounds and ethanol and sodium acetate buffer for the water soluble compounds, both using gradient elution. Twenty-seven commercial sunscreen samples (cream, lipstick, make-up, lotion) and two synthetic samples were analysed. Kedor-Hackmann et al. [9] used two C18 columns connected in series and a mobile phase containing acetonitrile and water to determine five sunscreens in synthetic formulations. Sample preparation based on a supercritical fluid extraction (SFE) procedure was employed by Scalia [10] to separate five sunscreens found in cosmetic products. The quantitation was performed on a phenyl column with a mobile phase containing methanol, acetonitrile, tetrahydrofuran and 0.5% (v/v) aqueous acid acetic. Simeoni et al. [11] reported a chromatographic method for the simultaneous assay of eight sunscreens, using a cyanopropylsilica column and methanol, acetonitrile, tetrahydrofuran and aqueous acetic acid as the mobile phase.
ª 2012 The Authors ICS ª 2012 Society of Cosmetic Scientists and the Socie´te´ Franc¸aise de Cosme´tologie
L. M. Peruchi and S. Rath
Sunscreen agents in cosmetics
Table I INCI name (abbreviation), chemical structure and maximum absorption of some commonly used sunscreens
INCI name
Chemical structure
O
Benzophenone-3 (BZ3)
Maximum absorption (nm)
O
288
CH3O
CH3 Ethylhexyl Dimethyl PABA (PABA)
O
CH3
O
N
312
CH3
CH3
4-Methyl Benzylidene Camphor (MBC)
301
O
O
O
Butyl Methoxy Dibenzoil Methane (BMDM)
359
CH3
Ethylhexyl Salicylate (SCL)
O
O
237
OH
O
Homosalate (HMS)
238
O OH Octocrylene (OCT)
305
N
O O
Ethylhexyl Methoxycinnamate (MCE)
310
O O H3CO INCI, International Nomenclature of Cosmetic Ingredients.
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Authorized concentration (%)
Other columns, all from Waters (USA): X-Bridge C18 (150 · 4.6 mm, 3,5 lm); X-Terra RP18 (250 · 4.6 mm, 5 lm) and X-Terra RP18 (150 · 2.1 mm, 3.5 lm) were also evaluated.
UV filters
EU
USA
Brazil
Standards and reagents
Benzophenone-3 Ethylhexyl dimethyl amino benzoate 4-Methyl benzylidene camphor Butyl methoxy dibenzoylmethane Ethylhexyl salicylate Homosalate Octocrylene Ethylhexyl methoxycinnamate
10 8 4 5 5 10 10 10
6 8 – 3 5 15 10 7.5
10 8 4 5 5 15 10 10
Table II Maximum allowed content of UV filters in EU, USA and Brazil
–: Not authorized.
Studies have been performed on the use of cyclodextrins to encapsulate UV filters to increase their aqueous solubility and photostability. Chisvert et al. [1] used hydroxypropyl-b-cyclodextrin as a modifier of the mobile phase to achieve separation of seven UV filters on a C18 column with an ethanol-water-acetic acid mobile phase. One of the more recent papers develops and validates a HPLC method for the simultaneous determination of twelve sunscreens in 30 minutes using a C18 column and a gradient mobile phase consisting of ethanol and acidified water [12]. Another recent study, performed by Wharton et al. [13], employed a 3 lm Hypersil BDS C18 column for the simultaneous determination of seven UV filters. The mobile phase used was a gradient consisting of ethanol and 1% acetic acid. In general, reported methods that deal with the determination of a large number of sunscreen compounds by HPLC recommend the use of ternary or quaternary solvent mixtures as mobile phase. Gradient elutions have been used to obtain adequate resolution in the separation of a large number of UV filters as it allows a wide range of solvent polarity. However, this type of elution has the disadvantage of requiring longer analysis times and higher costs. The aim of this study was the development of a fast and simple HPLC method using methanol and water under isocratic elution to determine the most widely used UV filters in cosmetics commercialized in Brazil. The method was validated and applied to the determination of eight sunscreens in formulations (lotions) commercially available in Brazil to verify if they are in conformity with the current legislation. Materials and methods Equipment The high-performance liquid chromatographic analyses were performed using a Waters 1525 Multisolvent Delivery System, equipped with a photodiode array detector model 2996 (Waters, Milford, PA, USA) and a Rheodyne 7725 manual injector (Rheodyne, Rohnert Park, CA, USA) with a sample loop of 50 lL. Data acquisition and instrument control were performed by millennium32 4.0 software (Waters). Separations were carried out on an ACE 5 C18 (4.6 · 250 mm, 5 lm) column (ACT, Scotland), using a mobile phase of methanol : water (88 : 12 v/v). The flow rate was 1.0 mL min)1 and analyses were carried out at ambient temperature. Quantification was performed by external calibration.
The standards employed were: benzophenone-3 (BZ3), octocrylene (OCT), ethylhexyl dimethylaminobenzoate (PABA), butyl methoxy dibenzoylmethane (BMDM), homosalate (HMS) and ethylhexyl salicylate (SCL), purchased from Sigma-Aldrich (St. Louis, MO, USA); 4-methylbenzylidene camphor (MBC) and ethylhexyl methoxycinnamate (MCE) purchased from Accustandard (New Haven, CT, USA). The solvent employed was HPLC grade methanol (Tedia, Fairfield, CT, USA). The water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). Standard solutions Stock solutions containing 1 mg mL)1 of each compound were prepared in methanol and stored in tightly closed amber vessels at 4C. Working solutions were prepared daily from the stock solutions in methanol : water (88 : 12 v/v) at seven concentration levels (1.0, 2.5, 5.0, 10.0, 15.0, 20.0 and 30.0 lg mL)1). These solutions were used to establish the linear range of the calibration graphs for each analyte. Method validation The method was validated by the evaluation of the following performance parameters: linear range, linearity, selectivity, intra-assay and inter-assay precision and accuracy. The linearity and linear range were established through the calibration graph obtained, by triplicate analyses, at seven concentration levels between 1.0 and 30.0 lg mL)1. The intra-day precision of the method, expressed as the relative standard deviation (RSD) of peak area measurements (n = 5), was evaluated through the analyses of two commercial sunscreen samples, in quintuplicate, with the method operating over 1 day under the same conditions. The inter-day precision was determined by the results obtained from analyses of commercial samples over 3 days under the same conditions (n = 3). The two commercial samples contained in their formulation six of the studied compounds (BZ3, OCT, MCE, HMS, BMDM and SCL). The other compounds (MBC and PABA) were added to the sample before analysis. The accuracy of the method was evaluated through recovery tests, using one fortification level (50%). All analyses were carried out in quintuplicate. Samples Ten samples of sunscreen lotions (SPF 30) from different manufacturers were purchased at local drugstores in Campinas, SP, Brazil. An accurate weighed amount of 0.5 g of the cosmetic product was transferred into a 50-mL volumetric flask and dispersed in 10-mL methanol by ultrasonication (15 min). After dilution to volume with mobile phase 88 : 12 v/v methanol : water, the sample was centrifuged (10 min, 3000 g), filtered through 0.22 lm syringe filters (Millipore, Brazil) and analysed by HPLC. All analyses were performed in triplicate.
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0.04
(A) AFS
MBC
AU
0.03
BZ3
0.02
OCT
PABA
BMDM + MCE
HMS
SCL
0.01
0.00 2.00
0.016
4.00
(B)
6.00
AFS
0.014
8.00
10.00 Minutes
12.00
14.00
18.00
MBC
BZ3
0.012 0.010 AU
16.00
OCT
0.008
PABA
MCE + HMS SCL BMDM
0.006 0.004 0.002 0.000 1.00
0.05
2.00
(C)
3.00
4.00
5.00
6.00
AFS
7.00
8.00 Minutes
9.00
OCT
12.00
13.00
14.00
15.00
PABA
AU
0.03
11.00
MCE + HMS + BMDM
MBC BZ3
0.04
10.00
SCL
0.02 0.01 0.00 1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
Minutes
1.2
(D)
MBC
1.0
AFS
AU
0.8
BZ3
0.6
PABA
OCT
MCE
SCL
BMDM HMS
0.4 0.2 0.0 1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00 9.00 Minutes
10.00
11.00
12.00
13.00
14.00
15.00
16.00
Figure 1 HPLC-DAD chromatogram obtained for 10 lg mL)1 phenylbenzimidazole sulphonic acid, benzophenone-3 (BZ3), 4-methylbenzylidene camphor (MBC), octocrylene (OCT), ethylhexyl dimethylaminobenzoate (PABA), ethylhexyl methoxycinnamate (MCE), butyl methoxy dibenzoylmethane, homosalate (HMS) and ethylhexyl salicylate (SCL). Column: (A) X-BridgeTM C18 (150 · 4.6 mm, 3.5 lm); (B) X-TerraTM RP18 (250 · 4.6 mm, 5 lm); (C) X-TerraTM RP18 (150 · 2.1 mm, 3.5 lm); and (D) ACE C18 (250 · 4.6 mm, 5 lm). Mobile phase: 85 : 15 v/v methanol:water for A, B and C and 88 : 12 v/v methanol:water for column D. Flow rate of 1.0 mL min)1. Detection at 238 nm.
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Results and discussion Chromatography The main aim of this work was to develop and validate a reversed phase HPLC method, using only two solvents (methanol and water) as mobile phase under isocratic elution, for the most commonly employed sunscreens found in sun lotions commercialized in Brazil. For this purpose, four different octadecyl columns were evaluated using methanol–water as the mobile phase. The respective chromatograms for the separation of the eight sunscreens are presented in Fig. 1. As shown in the chromatograms, only the ACE octadecyl column was able to separate all compounds under isocratic elution. Moreover, inversion of the elution order of BMDM, HMS and SCL was observed using the X-Terra and X-Bridge columns. Whereas X-Terra is a first-generation hybrid material based on tetraethylsiloxane and methyltriethylsiloxane monomers, X-Bridge is a second-generation material introduced by Waters Corporation based on tetraethylsiloxane and bis(triethoxysilyl)ethane monomers. The differences in surface chemistry and bonding den-
16
(8) (7)
14
Retention time (min)
BZ3 (1) MBC (2) OCT (3) PABA (4) MCE (5) BMDM (6) HMS (7) SCL (8)
(6) (5)
12
(4)
10 (3)
8
(2)
6 (1)
4
15
20
25
30 35 40 Temperature (°C)
45
50
Figure 2 Influence of temperature on retention time for: BZ3, MBC, OCT, PABA, MCE, BMDM, HMS and SCL. Chromatographic column: ACE C18, mobile phase: 88 : 12 v/v methanol:water. Flow rate 1.0 mL min)1.
sity are reflected in differences in solvation of the stationary phase and its retention properties [14]. The lower interaction of BMDM on the X-Terra stationary phases may be attributed to steric repulsion of the bulky compound at low volume fractions of methanol. Considering system suitability parameters (number of theoretical plates > 2000, peak asymmetry in the range 0.9–1.2 and resolution > 1.5), suitable baseline separation of all component peaks and, in particular, of BMDM, MCE and HMS was achieved on the ACE 5 C18 column, using a 88 : 12 v/v methanol : water mobile phase and a flow rate of 1 mL min)1 (Fig. 1D). Phenylbenzimidazole sulphonic acid (AFS), also employed as sunscreen agent, showed no interaction with any of the stationary phases employed, being eluted in the columns void volume (approximately in 1.9 min) (Fig. 1). In a subsequent study, the influence of temperature on the separation of the sunscreens was evaluated. It was verified that temperature strongly influences the retention time and resolution of the compounds, in particular, the more retained compounds on the ACE stationary phase (Fig. 2). At temperatures higher than 30C, MCE and MBMD loose adequate resolution and coelute at 45C. Under the experimental conditions described in Fig. 2, a complete separation of all compounds, with a resolution higher than 1.5, was only possible in the temperature range of 20–25C. Validation The method was validated in house using the following performance criteria: linear range, linearity, sensitivity, selectivity, intra- and inter-day precision and accuracy. The calibration graphs for all UV filters were linear in the concentration range of 1–30 lg mL)1, with a linearity higher than 0.99 (Table III). The linearity was tested using a pure error lack of fit test with simple regression, which was not significant at the 5% level. The sensitivity is the slope of the calibration graph (Table III). The sample preparation consisted of dilution of the sample (0.50 g) in 50 mL of methanol, centrifugation at 5000 rpm for 15 min, dilution with mobile phase and filtration (0.22 lm) before HPLC analyses. No interferences of the sample matrices in the chromatograms were observed under the established experimental conditions, conferring adequate selectivity to the method. The purity of each separated compound using the spectral data generated by the diode array detector was verified by the full spectrum at the upslope, the apex and the downslope to ensure that no coeluting impurity or compound of the matrix contributed to peak response. The intra-day precision was evaluated by quintuplicate analysis of samples of sunscreens lotions containing all active compounds on the same day by the same analyst and with the same equipment.
Table III Linear range, linearity and sensitivity values for the sunscreens BZ3, MBC, OCT, PABA, MCE, BMDM, HMS and SCL
Parameters
BZ3
MBC
OCT
PABA
MCE
BMDM
HMS
SCL
Linear range (lg mL)1) Linearity (r) Sensitivity (AU/lg mL)1)
1.0–30.0 0.9994 2.96 · 105
1.0–30.0 0.9943 1.03 · 105
1.0–30.0 0.9977 2.27 · 105
1.0–30.0 0.9997 3.66 · 105
1.0–30.0 0.9993 5.69 · 105
1.0–30.0 0.9993 3.97 · 105
1.0–30.0 0.9981 3.23 · 105
1.0–30.0 0.9981 3.65 · 105
AU, area unity. Benzophenone-3, BZ3; 4-methyl benzylidene camphor, MBC; octocrylene, OCT; ethylhexyl dimethyl amino benzoate, PABA; ethylhexyl methoxycinnamate, MCE; butyl methoxy dibenzoylmethane, BMDM; homosalate, HMS; ethylhexyl salicylate, SCL.
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The inter-day precision was determined by analysing the sample on three different days, using the same equipment and performed by the same analyst. On the first day, the samples were analysed in quintuplicate and in the two subsequent days, in triplicate. The results for precisions were expressed as the relative standard deviations (Table IV). All values were lower than 5%, and, therefore, acceptable according to the values recommended in the validation guide of analytical and bioanalytical methods from the National Agency of Sanitary Surveillance of Brazil [15]. The accuracy of the method was assessed by recovery tests, analysing fortified commercial sunscreen samples. Addition of each analyte was performed at one concentration level (50% fortification level) and all analyses were carried out in quintuplicate. The mean recovery of each UV filter was calculated and their values are shown in Table V. The recoveries varied between 95 and 105%, with the exception of OCT where a recovery of 112% was obtained. This value is not acceptable and therefore samples containing OCT
were analysed using the standard addition method. Four standard additions of OCT to the sample were performed and the analyses were carried out in triplicate. It was verified that the matrix affected the result positively, i.e. an overestimated concentration (around 10%) was obtained through external calibration. This difference explains the high recovery value obtained with the recovery test and indicates the need of quantitation of OCT by the standard addition method. Sample analysis Ten commercial sunscreen lotions (SPF 30), available in Brazil, were purchased on the retail market. Samples denominated A, B, C, E, G, I and J were manufactured in Brazil, sample D was manufactured in France and samples F and H were manufactured in the USA. The samples were analysed in triplicate following the proposed analytical procedure. Table VI shows the contents obtained of the
Table IV Intra-day and inter-day precision (RSD, %) of the developed method
Active ingredient
Precision
BZ3
MBC
OCT
PABA
MCE
BMDM
HMS
SCL
RSD (%) intra-day (n = 5) RSD (%) inter-day (n = 11)
1.4 1.5
3.7 2.7
1.3 1.0
1.7 2.6
2.0 1.6
1.4 3.7
1.1 1.8
0.6 1.4
Benzophenone-3, BZ3; 4-methyl benzylidene camphor, MBC; octocrylene, OCT; ethylhexyl dimethyl amino benzoate, PABA; ethylhexyl methoxycinnamate, MCE; butyl methoxy dibenzoylmethane, BMDM; homosalate, HMS; ethylhexyl salicylate, SCL.
Table V Accuracy results obtained through recovery test. Average results for each compound and relative standard deviations (RSD)
Recovery (%) (n = 5) RSD (%)
OCT
BMDM
BZ3
MBC
PABA
MCE
HMS
SCL
112 1.3
103 1.3
101 2.7
95 1.5
100 1.3
100 2.5
105 2.5
104 1.9
Benzophenone-3, BZ3; 4-methyl benzylidene camphor, MBC; octocrylene, OCT; ethylhexyl dimethyl amino benzoate, PABA; ethylhexyl methoxycinnamate, MCE; butyl methoxy dibenzoylmethane, BMDM; homosalate, HMS; ethylhexyl salicylate, SCL.
Table VI Average results (%, w/w; n = 3) and relative standard deviation in parenthesis of the active ingredients in commercial sunscreens
Samples
BZ3
MBC
OCT
PABA
MCE
BMDM
HMS
SCL
A B C D E F G H I J
2.0 – – – 3.1 3.0 – 3.9 4.9 4.0
– – – – – – – – –
2.3 6.7 – 9.6 – – 0.9 0.8 – –
– – – – 7.9 (0.5) – – – – –
– – 5.6 – – 5.6 7.0 – 7.4 6.0
2.6 2.1 – 2.3 1.8 1.7 – 0.9 2.8 –
– – – – – 6.8 – 10.9 11.2 5.8
3.0 – – – – 4.6 – 4.9 5.0 5.1
(0.2)
(1.8) (1.3) (0.1) (1.2) (1.5)
(0.4) (1.6) (1.3)
(1.4) (1.2)
(1.9)
(1.4) (1.3) (1.5) (2.0)
(0.4) (0.7) (2.6) (1.6) (1.5) (1.2) (0.3)
(2.3) (2.8) (1.2) (1.3)
(0.3)
(1.7) (1.0) (1.8) (0.6)
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0.3
SAMPLE B
OCT
AU
0.2
0.1
BMDM
0.0 1.0
3.0
5.0
7.0
9.0
13.0
11.0
15.0
Minutes
1.6
SAMPLE E PABA
AU
1.2 0.8
BZ3
0.4
BMDM
0.0 1.0
1.4
3.0
5.0
7.0
9.0
11.0 Minutes
13.0
SAMPLE F
15.0
17.0
MCE
1.0
AU
19.0
SCL BZ3
0.6 BMDM 0.2
HMS 1.0
3.0
5.0
7.0
9.0 Minutes
11.0
13.0
15.0
17.0
Figure 3 HPLC-DAD chromatogram obtained for sunscreen samples B, E and F. Column ACE C18; mobile phase 88 : 12 v/v methanol:water; flow rate of 1.0 mL min)1 and detection at the maximum wavelength of each compound.
organic UV filters present in the samples and Fig. 3 the chromatograms of samples B, E and F. It is important to know that all samples, in addition to the organic filters identified in their formulations, contain the physical UV filter titanium dioxide. The most widely used organic UV filter in the analysed samples was BMDM owing to the fact that this compound has a broad absorption spectrum (maximum absorption in 359 nm) and is one of the few organic filters allowed that protects against UVA radiation. UVA radiation is less damaging than UVB radiation in terms of erythematic and genetic mutations, but is primarily responsible for premature ageing, a subject of increasing concern to consumers and, therefore, the pharmaceutical industry. The compound methyl benzylidene camphor (MBC) was not present in any of the samples analysed. This filter was banned by FDA, but is still allowed and used in some countries. There are several studies showing that this compound is responsible for endocrine disruption, having oestrogenic effects [16–18]. Seidlova´Wuttke et al. [19] published a study indicating that MBC have effects on several metabolic parameters, such as fat lipid homoeostasis, as well as on thyroid hormone production.
Conclusions The HPLC-DAD method developed is simple, fast and was shown to be suitable for the simultaneous determination of eight sunscreen agents (benzophenone-3, octocrylene, ethylhexyl methoxycinnamate, ethylhexyl salicylate, HMS, butyl methoxydibenzoylmethane, 4-methylbenzylidene camphor and ethylhexyl dimethyl PABA) commonly used in sunscreen formulations. The stationary phase that showed the best results for separation of all compounds was the chromatographic column ACE 5 C18 (4.6 · 250 mm, 5 lm) with a mobile phase of 88 : 12 v/v methanol: water at a flow rate of 1.0 mL min)1. The advantage of the method is that all compounds are separated under isocratic elution. Temperature affects the separation and a complete separation of all compounds, with a resolution higher than 1.5, is only possible in the temperature range of 20–25C. The sample preparation consisted of sample dilution with methanol followed by centrifugation and filtration. The number of the organic sunscreen agents determined in the commercial samples varied from one to five in a single sample. The compounds identified in the analysis are consistent with the com-
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pounds identified on the label of the packaging of the products and their concentrations were in the range of 0.8–11% (w/w). All the identified compounds and their concentrations were in accordance with current Brazilian legislation.
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Acknowledgements The authors gratefully acknowledge the financial support of FAPESP and CAPES and thank Professor C.H. Collins for language assistance.
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ª 2012 The Authors ICS ª 2012 Society of Cosmetic Scientists and the Socie´te´ Franc¸aise de Cosme´tologie International Journal of Cosmetic Science, 34, 226–233
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