Normal-Phase TLC Analysis of UV Filters Avobenzone and Octocrylene in Sunscreen Preparations

Anna W. Sobanska* and Elzbieta Brzezinska

Key Words
Avobenzone Octocrylene TLC Spectrodensitometry

Summary
TLC separation of the UV filters avobenzone (AVO) and octocrylene (OCR) on RP-18 and silica gel 60 as stationary phases, with numerous mobile phases, revealed that these compounds have similar retention properties under almost all the chromatographic conditions investigated. Analysis of avobenzone in the presence of octocrylene may be achieved on silica gel 60 with cyclohexane diethyl ether 1:1 (v/v) as mobile phase, at the selective wavelength 380 nm, at which octocrylene does not absorb (Method A). Analysis of octocrylene in the presence of avobenzone by the same method was, however, impossible because of insufficient chromatographic separation of AVO and OCR and overlap of the absorption ranges of AVO and OCR (with OCR peaks being obscured by those of AVO). An alternative separation strategy was proposed that proved successful for OCR in the presence of AVO. This involved chromatography on silica gel 60 with cyclohexanepiperidine 15:1 (v/v) as mobile phase (Method B). When these chromatographic conditions were used, retention of avobenzone changed substantially and OCR peaks became clearly visible. Spectrodensitometric scanning for OCR was then performed at 300 nm. Both methods were validated, and gave calibration plots of good linearity.

Figure 1 The structural formulas of avobenzone and octocrylene.

1 Introduction
Butyl methoxydibenzoylmethane (avobenzone, AVO; Figure 1) is the most popular UVA filter, authorized in Europe, USA, Australia, and Japan [1]. This compound affords high UVA protection but its photostability is moderate; it must, therefore, be combined with stabilizers that can act as quenchers of the avobenzone triplet state [2, 3]. Recent research has proved that one of the most efficient avobenzone stabilizers is the UVB filter octocrylene (Figure 1) [35]. Avobenzone and octocrylene are combined in sunscreen formulations either with addition of other UVB filters (e.g. ethylhexyl triazone) or on their own, depending on the level of UVB protection required for the particular cosmetic preparation.

Keeping in mind the significance of the avobenzoneoctocrylene combination for modern UV protection it is of interest to develop easy, cost-effective, and efficient methods for analysis of these filters in their binary mixture. Avobenzone and octocrylene have been simultaneously quantified by column chromatography or related techniques, for example RP HPLC [613], GC [1416], and microemulsion electrokinetic chromatography (MEEKC) [17]. There are no literature reports of TLC separation of avobenzone and octocrylene, no papers on TLC analysis of avobenzone, and only one on RP HPTLCspectrodensitometric analysis of octocrylene on RP-18 plates with methanoltetrahydrofuranwater 50:35:15 (v/v/v) as mobile phase [18]. Chromatographic separation of UV filters is, according to the literature, not an easy task, and avobenzone is chromatographically, one of the most troublesome of all authorized UV absorbers. When analyzed by HPLC avobenzone gives broad peaks and tends to migrate close to octocrylene [11], octyl salicylate [6, 10, 19], homosalate [6, 7], octyldimethyl PABA [10], or octyl methoxycinnamate [8, 10, 20]. Separation of these compounds requires gradient elution [6, 10, 12, 21, 22] or modification of the mobile phase with EDTA [6] or cyclodextrins [19]. Avobenzone peaks, because of their broad shape and the wide absorption range of this compound, obscure peaks of other similarly migrating UV filters listed above, complicating their quantification in the presence of avobenzone. In these circumstances detection by single-wavelength UV absorption is inefficient and other detectors, e.g. diode-array [9, 10] or mass spectroscopy [13] are recommended.
DOI: 10.1556/JPC.24.2011.2.14

A.W. Sobanska and E. Brzezinska, Department of Analytical Chemistry, Medical University of Lodz, 90-151 Lodz, ul. Muszyskiego 1, Poland. E-mail: a.sob@poczta.onet.pl

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TLC Analysis of Avobenzone and Octocrylene

volumetric flasks. Both UV filters were dissolved in adequate amounts of methanol and diluted to volume with the same solvent to give stock solutions of the concentration 5 mg mL1. The stock solutions of both compounds were diluted with methanol to furnish standard solutions of concentration 200 g mL1. Other compounds investigated in the course of chromatographic separation tests (ethylhexyl triazone, ethylparaben) were used as 500 g mL1 solutions in acetone and methanol, respectively. All flasks were wrapped with aluminum foil and kept refrigerated.
2.2 Sample Preparation

Figure 2 Densitograms obtained from AVO standard and the product at 380 nm (Method A).

Suncare lotion (1000 mg) was weighed accurately into a 100mL volumetric flask. Approximately 70 mL methanol was added and the flask was vigorously shaken by use of a Premed (Poland) type 327 Universal Shaker for 60 min. Methanol was then added to volume, and the flask was wrapped with aluminum foil and left to stand for 60 min.
2.3 Thin-Layer Chromatography

Figure 3 Densitograms obtained from OCR standard and the product at 300 nm (Method B).

The objective of this research was to develop a simple and costeffective method of densitometric analysis of avobenzone and octocrylene combined in a sunscreen preparation.

Thin-layer chromatography was performed on 10 cm 20 cm standard quality silica gel 60 F254 and RP-18 F254 plates (layer thickness 0.25 mm) from Merck. Plates were spotted with a Hamilton 10 L TLC microsyringe, 20 mm from the bottom edge and at 10 mm intervals, starting 10 mm from the plate edge (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 L of 200 g mL1 standard solutions of AVO and OCR) and developed with either cyclohexanediethyl ether 1:1 (v/v), Method A, or cyclohexanepiperidine 15:1 (v/v), Method B. Plates were developed in a vertical chromatographic chamber lined with filter paper and previously saturated with the appropriate mobile phase vapor for 20 min. Development distance was 80 mm from the plate bottom edge and development time was ca 20 min. After development, plates were dried at room temperature (20C), scanned, and analyzed in reflectance mode with the Desaga CD 60 densitometer at 380 nm (Method A, avobenzone) or 300 nm (Method B, octocrylene). Typical densitograms obtained from standards and from the solution of the cosmetic preparation under investigation, obtained as described above, are shown in Figures 2 and 3.
2.4 Analysis of Avobenzone and Octocrylene in the Sunscreen Lotion

2 Experimental
2.1 Chemicals, Materials, and Solutions

Eusolex 9020 (avobenzone) and Eusolex OCR (octocrylene) were kindly donated by Merck. Uvinul T 150 (ethylhexyl triazone, ET) was a free sample from BASF. Ethylparaben was purchased from SigmaAldrich. Methanol, cyclohexane, diethyl ether, isopropanol, toluene, ethyl acetate, acetic acid, tetrahydrofuran, and acetone were from Polskie Odczynniki Chemiczne (POCh), Poland. Disodium EDTA, ammonia 25% aq. solution, and pH 3 and pH 13 buffers were purchased from Chempur, Poland. Piperidine was purchased form Merck. UV filters were of cosmetic quality and all other chemicals were of analytical grade. SPF 20 water-resistant sun care lotion analyzed in the course of this study was manufactured by DAX Cosmetics, Poland. Eusolex 9020 (avobenzone), 500 mg, and Eusolex OCR (octocrylene), 500 mg, were weighed accurately into 100-mL
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The sunscreen lotion solution in methanol, prepared as described above, was spotted on silica gel 60 TLC plates. For analysis of avobenzone (Method A) the volume applied was 4 L. For analysis of octocrylene (Method B) the volume applied was 1 L. The plates were then chromatographed as described above for AVO and OCR standards (Section 2.3).

3 Results and Discussion

3.1 Method Development

The sun-care lotion analyzed in this study contained, apart from avobenzone and octocrylene, substantial amounts of other compounds that absorb within the UV range, i.e. ethylhexyl triazone, ethylparaben, and methylparaben. To separate avobenzone and octocrylene in the presence of the other components mentioned

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TLC Analysis of Avobenzone and Octocrylene Table 1 RF of AVO, OCR, ET, and ethylparaben on RP-18.

Mobile phase components MeOHH2O MeOHTHFpH 13 buffer MeOHTHFH2O MeCNH2O MeOHH2O MeOHpH 13 buffer MeOHpH 3 buffer MeOHEDTA 0.002 mol L1 MeOHNH3 2.0 mol EtOHH2OAcOH MeOHH2O MeOHMeCN i-PrOHAcOEtH2O L1

AVO Ratio (v/v) 7:3 50:35:15 50:35:15 9:1 9:1 9:1 9:1 9:1 9:1 70:29.5:0.5 4:1 9:1 6:1:2 0.02 0.40 0.37 0.32 0.25 0.21 0.20 0.23 0.22 0.08 0.10 0.50 0.44

OCR

ET

Ethylparaben

0.02 0.45 0.42 0.40 0.35 0.32 0.30 0.35 0.34 0.12 0.12 0.61 0.54

0 0.06 0.06 0 0 0 0 0 0 0 0 0.18 0.24

0.38 0.72 0.72 0.70 0.70 0.68 0.63 0.68 0.66 0.52 0.54 0.74 0.79

above two stationary phases (RP-18 and silica gel 60) and several mobile phases were tested. The results of these tests are summarized in Tables 1 and 2. On the basis of comparison of RF values obtained for AVO and OCR it was concluded that RP TLC methods reported in the lit-

erature for OCR [18] and other UV filters (benzophenone-3 [23], octyldimethyl PABA [24], octyl methoxycinnamate [25], and octyl salicylate [26]), i.e. RP-18 developed with MeOHTHFH2O 50:35:15 (v/v/v) is not particularly advantageous when avobenzone is present in the formulation. Other

Table 2 RF of AVO, OCR, ET, and ethylparaben on silica gel 60.

Mobile phase components Cyclohexaneetheri-PrOH CyclohexaneetherAcOEt Cyclohexaneether Cyclohexaneether Cyclohexaneether Cyclohexaneether Cyclohexaneetheracetone Cyclohexaneetheracetone Cyclohexaneetheracetone Cyclohexanei-PrOH CyclohexaneAcOEt CyclohexaneCHCl3 Toluene TolueneAcOEt TolueneAcOEtAcOH TolueneAcOEtAcOH Cyclohexaneetheri-PrOHAcOEt Cyclohexaneetheri-PrOH Cyclohexanepiperidine Cyclohexanepiperidine

AVO Ratio (v/v) 15:1:1 15:1:1 1:1 5:1 10:1 15:1 15:1:1 15:1:2 15:1:3 15:1 15:1 1:1 0.55 0.33 0.55 0.37 0.30 0.20 0.34 0.42 0.42 0.51 0.31 0.38 0.33 15:1 15:1:0.2 15:1:1 15:1:1:1 15:1:0.2 15:1 5:1 0.50 0.63 0.67 0.62 0.37 0.03 0.18

OCR

ET

Ethylparaben

0.55 0.36 0.60 0.40 0.34 0.15 0.34 0.44 0.43 0.51 0.34 0.38 0.27 0.68 0.67 0.72 0.67 0.41 0.29 0.56

0.23 0.02 0.23 0 0 0 0.02 0.10 0.21 0.10 0 0 0 0.08 0.12 0.36 0.29 0 0 0.30

0.22 0.07 0.30 0.08 0.02 0 0.08 0.15 0.19 0.14 0.05 0 0 0.12 0.15 0.31 0.28 0.05 0 0.03

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TLC Analysis of Avobenzone and Octocrylene

Figure 4 UV spectra obtained from AVO and OCR adsorbed on silica gel 60, by use of the Desaga CD 60.

ing the reactive -diketone moiety (Figure 1). It was established that the retention properties of avobenzone, unlike those of octocrylene, changed dramatically when the mobile phase was modified with aliphatic amines, of which piperidine was selected for further tests because of its suitable physicochemical properties (good miscibility with cyclohexane, moderate volatility, and low viscosity). Detailed separation tests were performed with two mobile phases, (1) and (2), containing cyclohexane and piperidine at different ratios (5:1 and 15:1 (v/v), respectively; Table 2). With mobile phase (1), separation of OCR and AVO was very good but the quality of the spots obtained for AVO (very broad spots with tailing) was insufficient for densitometric analysis, so simultaneous quantification of AVO and OCR under these conditions was not attempted. Attention was then turned toward mobile phase (2) containing much less piperidine. With mobile phase (2) AVO did not migrate (RF = 0.03) and OCR gave spots suitable for densitometric analysis. At this stage the suggestion was made that AVO and OCR may be quantified by use of two independent TLC methods, subsequently denoted Method A (for avobenzone) and Method B (for octocrylene).
3.2 Method A

Table 3 Linear regression data for AVO and OCR, on the basis of peak areas (n = 10).

AVO (Method A) Slope (b Sb) Y-intercept (a Sa) LOD (calculated) [ng/spot] LOQ (calculated) [ng/spot] Sxy (Residual SD) Correlation coefficient, R Linearity range [ng per spot] 1507.96 63.01 466.25 78.20 170 510 114.47 0.9931 2001800

OCR (Method B) 1956.56 94.26 304.34 106.08 180 537 146.02 0.9920 2001800

Table 4 Results from analysis of AVO and OCR in the sunscreen lotion.

Day 1 (n = 9) Day 2 (n = 9) AVO (Method A) CAVO [ng per spot] 1550 CAVO [% w/w] RSD [%] OCR (Method B) 3.87 3.20 1490 3.72 2.16 980 9.8 6.42

CAVO [ng per spot] 1010 CAVO [%] RSD [%] 10.1 4.97

Method A involved application of the standard NP mobile phase cyclohexaneether 1:1 (v/v). Under these conditions separation of common preservatives (parabens) and UV filters such as ethylhexyl triazone from co-eluting AVO and OCR was easily achieved. OCR quantification by Method A was impossible, because its densitometric peaks were obscured by those of AVO over the whole absorption range of OCR. Avobenzone, however, has a broader absorption range than OCR (Figure 4) so an analytical wavelength suitable for selective AVO analysis could be proposed. Densitometric quantification of AVO was performed at 380 nm; statistical data for the calibration plot obtained are given in Table 3. Limits of detection and quantification for AVO were estimated on the basis of the regression data for the calibration plot by using the equations: LOD = 3.3 SDa/b and LOQ = 3 LOD [27]. Results from analysis of AVO in sunscreen lotion on two different days are presented in Table 4. Method A was validated for three different concentrations of AVO; results from recovery tests are presented in Table 5. The specificity of analysis of AVO at the proposed wavelength (380 nm) was confirmed by comparing multiwavelength scans acquired for AVO and OCR (Figure 5).
3.3 Method B

mobile phases typically used in RP TLC also resulted in moderate separation. Attempts to change the migration properties of AVO by varying mobile phase pH (Table 1) were also unsuccessful. For mobile phases leading to differences between the formal RF values of AVO and OCR of ca. 0.1, attempts were made to obtain calibration plots for OCR but their quality was insufficient because of the partial overlap of broad AVO peaks with the OCR peaks. Results of separation tests were also unsatisfactory for silica gel 60 as stationary phase with most of the mobile phases under investigation (Table 2). At this stage it was decided a different approach was needed, on the basis of the relatively low reactivity of octocrylene compared with avobenzone, formally contain-

The change in the retention behavior of avobenzone compared with octocrylene in Method B made it possible to determine octocrylene densitometrically without interference from avobenzone. The analytical wavelength for OCR (300 nm) was selected on the basis of its UV absorption spectrum (Figure 4) and the multiwavelength scans (Figure 5). Statistical data for the calibration plot obtained for octocrylene are given in Table 3 and results from analysis of OCR in the sunscreen lotion on two different days are presented in Table 4. Limits of detection and quantification for OCR were estimated in the same way as for Method A [27]. Method B was validated for three different concentrations of OCR; results from recovery tests are summarized in Table 5. The purity of the OCR peaks

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TLC Analysis of Avobenzone and Octocrylene Table 5 Recovery of AVO and OCR, on the basis of peak areas (n = 3).

Spiked amount [ng per spot] 375 1000 1600

AVO (Method A) Found Recovery [ng per spot] [%] 380 1050 1603 101.3 105.0 100.0

RSD [%] 5.51 1.20 2.36

Spiked amount [ng per spot] 375 800 1600

OCR (Method B) Found Recovery [ng per spot] [%] 385 790 1620 102.7 98.8 101.2

RSD [%] 3.75 0.84 5.95

Figure 6 UV spectra acquired from the OCR sample peak (A, B, C) and from the OCR standard (D), by use of the Desaga CD 60. Figure 5 Multiwavelength scans of AVO and OCR, obtained by use of the Desaga CD 60.

Acknowledgments

was confirmed by comparing UV spectra acquired at three different points on the OCR peak obtained from the sunscreen product and the spectrum acquired for the OCR standard (Figure 6).

This work was supported by an internal grant from the Medical University of Lodz, Poland (project no. 503-3016-3). Thanks are due to Merck and BASF for supplying free samples of the sunscreens used throughout this research.

References

4 Conclusions
UV filters avobenzone and octocrylene may be effectively separated from other components of cosmetic preparations, for example parabens or ethylhexyl triazone, by normal-phase TLC on silica gel 60 with diethyl ethercyclohexane 1:1 (v/v) as mobile phase. Attempts to separate avobenzone and octocrylene on silica gel 60 were unsuccessful but avobenzone was quantified, in the presence of octocrylene, by densitometry at 380 nm, where octocrylene does not absorb. There is no analytical wavelength selective for octocrylene in the presence of avobenzone when chromatographic separation of these compounds fails. It is, however, possible to alter the retention properties of avobenzone substantially by using a different mobile phase, cyclohexanepiperidine 15:1 (v/v) and to quantify octocrylene densitometrically at 300 nm. The proposed strategy for quantification of avobenzone and octocrylene in sunscreen products is simple, quick, and relatively inexpensive, it may therefore be recommended for routine analysis of cosmetic products.

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