Simultaneous Multiple-Development HPTLC Quantification of Water- and Oil-Soluble Sunscreens

Anna W. Sobanska* and Jaroslaw Pyzowski

Key Words
Sunscreens Oil-soluble Water-soluble Quantification Multiple-development TLC Densitometry

Summary
A complex mixture of sunscreens of different lipophilicity was quantified for the first time by thin-layer chromatography (TLC) followed by densitometric scanning in absorption mode. Multipledevelopment normal-phase TLC was performed on silica gel 60 as stationary phase. Two mobile phases were used: A cyclohexane diethyl ether 5:1 (v/v) and B ethyl acetateethanolwater 70:35:30 (v/v). After development with mobile phase A, two oil-soluble sunscreens, avobenzone (AVO) and octyl salicylate (OS), were analyzed at 360 and 300 nm, respectively. Subsequent development of the same plates with mobile phase B made it possible to quantify a water-soluble sunscreen phenylbenzimidazol sulfonic acid (PBS) at 300 nm. Calibration curves were non-linear. Limits of detection (LOD) and quantification (LOQ) were LOD (OS) 0.02 g spot1, LOQ (OS) 0.06 g spot1, LOD (AVO) 0.03 g spot1, LOQ (AVO) 0.08 g spot1, LOD (PBS) 0.02 g spot1, and LOQ (PBS) 0.06 g spot1. The method was validated and applied to the analysis of a commercially available cosmetic product.

1 Introduction
Quantification of sunscreens in cosmetic products may be achieved by a number of analytical techniques, including chromatography gas chromatography (GC), thin-layer chromatography (TLC), and, much more often, high-performance liquid chromatography (HPLC) [1]. The retention behavior of compounds in different chromatographic conditions is strongly related to their lipophilicity [2, 3] and dissociation constants [4, 5]. The majority of papers published on TLC or HPLC analysis of sunscreens refer to ultraviolet (UV) filters of medium to high lipophilicity (logKo/w usually 4 to 8 or higher), known to formulators as fat soluble filters. These sunscreens are usually separated by reversed phase HPLC or TLC on RP-18 [1, 610] or, sometimes, on other stationary phases [1]; only recently have a few papers been published on normal-phase TLC separation of

fat soluble sunscreens on silica gel 60 [1113]. Analysis of another group of UV filters water-soluble, hydrophilic, and strongly anionic substances such as benzophenone-4, phenylbenzimidazole sulfonic acid, disodium phenyl dibenzimidazole tetrasulfonate, or terephthalylidene dicamphor sulfonic acid is described in the literature far less frequently. Until now no method has been reported on TLC separation and quantification of water-soluble UV filters; when in the course of our preliminary research normal-phase (NP) chromatographic procedures described in Refs. [1113] and other similar normal-phase chromatographic conditions were tested on these compounds, they remained on the start line (RF = 0) [11]. Our attempts to analyze water-soluble sunscreens by reversed phase TLC on RP-18 stationary phase with typical mobile phases (water and different concentrations of organic modifiers such as tetrahydrofurane, acetone, methanol, 1,4-dioxane, dimethylformamide, or acetonitrile) resulted in the rapid migration of investigated compounds (RF > 0.95 [11]). Similarly the RP HPLC method described in Ref. [14] applied to water-soluble UV filters gave very short retention times (tR 1 min) and poor baseline separation. Different approaches proposed to facilitate RP HPLC analysis of water-soluble UV filters included increasing the sunscreens retention times by adjusting the pH of mobile phases [1521], application of ion-pair chromatography (mobile phases containing tetraalkylammonium salts) [22, 23], or using stationary phases other then RP-18 (e.g. cyanopropyl packing [24]). Application of electrochromatographic techniques such as micellar electrokinetic chromatography or microemulsionelectrokinetic chromatography (MEEKC) to phenylbenzimidazol sulfonic acid (PBS) has been reported [25, 26]. When the sample contained both water- and oil-soluble sunscreens, their reversed phase HPLC separation resulted in the former ones migrating first followed by the latter ones eluting considerably later in this situation, the duration of each chromatographic run was a disadvantage [17]. The aim of this study was to propose the first TLC-densitometric method of separation and quantification of sunscreens of mixed lipophilicity rapid, based on the application of readily available silica gel 60 chromatographic plates and with very simple, low-toxicity mobile phases.
DOI: 10.1556/JPC.25.2012.4.11

A.W. Sobanska and J. Pyzowski, Department of Analytical Chemistry, Medical University of Lodz, ul. Muszynskiego 1, 90-151 Lodz, Poland. E-mail: a.sob@poczta.onet.pl and anna.sobanska@umed.lodz.pl

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2 Experimental
2.1 Chemicals, Materials, and Solutions

Eusolex 9020 (avobenzone, AVO), Eusolex OS (octyl salicylate, OS), and Eusolex 232 (PBS) were kindly donated by Merck, Darmstadt (Germany). Ethanol, ethyl acetate, cyclohexane, acetonitrile, methanol, and diethyl ether were from Chempur, Piekary Slaskie (Poland). UV filters were of cosmetic quality, and all solvents were of analytical grade. Moisturizing cream containing AVO, OS, and PBS analyzed in the course of this study was manufactured by Beiersdorf, Hamburg (Germany). The blank cream used in recovery tests was also from Beiersdorf. Eusolex 9020 (500 mg) and Eusolex OS (500 mg) were weighted accurately into 100-mL volumetric flasks, 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 solution of Eusolex 232 was prepared in the similar manner, but this compound (500 mg) was dissolved in water alkalized with NaOH aq (2 mol L1, 0.9 mL). The stock solutions of OS, AVO, and PBS were used to prepare standard solutions containing the following concentrations of all three sunscreens: 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 g L1. All flasks were wrapped with aluminum foil and kept refrigerated.
2.2 Sample Preparation

Figure 1 Densitograms of AVO, OS, and PBS standards after development with mobile phases A and B ( = 300 nm).

filters under investigation obtained as described above are shown in Figure 1.

2.4 Analysis of PBS, AVO, and OS in the Sunscreen Cream

The cream solution in methanol, prepared as described above, was spotted on silica gel 60 HP TLC plates (1 L spot1 for OS and PBS, 10 L spot1 for AVO). The plates were then chromatographed as described above for AVO, OS, and PBS standards (Section 2.3).

Moisturizing cream (1000 mg) was weighted accurately into a 100-mL 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

3 Results and Discussion

3.1 Method Development

TLC was performed on 10 cm 20 cm high-performance thinlayer chromatography (HPTLC) silica gel 60 plates (layer thickness 0.2 mm) or on 10 cm 20 cm TLC silica gel 60 plates (layer thickness 0.25 mm) from Merck. Plates were pre-washed with methanoldichloromethane 1:1 (v/v), dried overnight in the ambient conditions and spotted with the Desaga AS 30 sampler equipped with a 10 L syringe (1 L spot1 of standard solutions containing AVOOSPBS, standards concentrations according to 2.2.1.), spot-wise (band width set at 0 mm, initial diameter of spots ca. 1 mm) 0, 15 mm from the bottom edge, at 10 mm intervals, starting 10 mm from the plate edge. Plates were developed first with the mobile phase A (cyclohexanediethyl ether 5:1 v/v) and then with the mobile phase B (ethyl acetateethanolwater 70:35:30 v/v) in a vertical chromatographic chamber lined with filter paper and previously saturated with the appropriate mobile phase vapor for 20 min. Each development distance was 75 mm from the plate bottom edge. After development, plates were dried at room temperature (20 C), scanned, and analyzed in reflectance mode with the Desaga CD 60 densitometer (slit width 4.0 mm, slit height 0.1 mm): after the first development at 360 nm (AVO) and 300 nm (OS), and after the second development at 300 nm (PBS). Typical densitograms obtained for UV

According to our earlier research [1113] oil-soluble UV filters present in the majority of sun care products available on the European markets can be chromatographed on silica gel 60 with binary or tertiary non-aqueous mobile phases to achieve RF values suitable for densitometric scanning (typically 0.20.8). The same group of UV filters may be analyzed on RP-18 stationary phase with binary water-organic solvent mobile phases [11] or with the mobile phase proposed by Sherma et al. (methanol tetrahydrofuranewater) [610]. Both approaches were tested and failed when applied to water-soluble UV filters strongly hydrophilic compounds containing at least one ionized sulfonic group. The affinity of these UV filters to the aqueous mobile phase is so strong that during RP TLC they eluted rapidly with RF values typically 0.95 to 1 [11]. On the other hand their polarity is so high that chromatographed with organic solvents on silica gel 60, they do not migrate from the start line, so in both situations their densitometric scanning is impossible [11]. One possible approach to chromatography of water-soluble UV filters could involve RP-18 stationary phase combined with acidic mobile phases. It was expected that in such conditions, due to the reversed dissociation of the sulfonic acid moiety water-soluble sunscreens should loose some of their affinity to the aqueous mobile phase. This strategy was considered during our preliminary research (e.g. mobile phase methanolwater 9:1 (v/v) was replaced with methanolbuffer (pH = 3) 9:1 (v/v)) but with moderate success RF values for the water-soluble filter PBS were in these conditions 0.93 and 0.90, respectively. Since RF values

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tion of method specificity and toxicology aspects, the following mobile phases were selected for further investigations: mobile phase A (cyclohexanediethyl ether 5:1 (v/v)) and mobile phase B (ethyl acetateethanolwater 70:35:30 (v/v)). Analytical wavelengths suitable for UV filters investigated in the course of this study were selected on the basis of multiwavelength scanning of chromatograms from 200 to 420 nm at 20 nm intervals: 360 nm for AVO and 300 nm for OS and PBS.
3.2 Method Validation 3.2.1 Specificity

Moisturizing cream analyzed throughout this study contained no ingredients absorbing between 300 and 400 nm other than OS, AVO, and PBS. It was nevertheless important to check if the chromatographic method proposed in this paper is suitable also for formulations containing, e.g., parabens. It was established that mobile phases A and B separate parabens from the UV filters under investigation: Mobile phase A RF (OS) 0.85, RF (AVO) 0.50, RF (propylparaben) 0.08 Mobile phase B RF (PBS) 0.80, RF (OS) 0.95, RF (AVO) 0.95, RF (propylparaben) 0.95. Purity of OS, AVO, and PBS peaks obtained during the analysis of the cream sample was confirmed by UV/vis spectra of sunscreens acquired directly from chromatographic plates in reflectance mode. Spectra collected at two different points of particular peaks obtained for the sample solution were compared with spectra acquired for the standards (Figure 2).
Figure 2 UV spectra of OS, AVO, and PBS acquired from chromatograms of the cream sample (2, 3) and from the standards (1).

3.2.2 Calibration

obtained on RP-18 stationary phase for oil-soluble filters under investigation (AVO and OS) were too close for their effective quantification [11], this idea was discontinued. Attention was then turned towards NP chromatography on silica gel 60 with two alternative mobile phases ethyl acetateethanol water 70:35:30 (v/v) or acetonitrilewater 9:1 (v/v). Although these mobile phases were unsuitable for AVO and OS (RF 0.95), retention behavior of PBS was in these conditions far more promising (RF 0.80 and 0.82, respectively). At this stage it was decided that chromatographic plates should be developed twice at first with the non-aqueous mobile phase A suitable for oil-soluble filters and then with the aqueous mobile phase B (ethyl acetateethanolwater 70:35:30 (v/v) or acetonitrilewater 9:1 (v/v)) to elute PBS. Finally, after due considera-

Calibration plots were obtained by plotting peak areas against amount of substances in the range 0.061 g spot1. In all cases, non-linearity of calibrating plots was clearly visible. Calibration plots were finally generated for all sunscreens in the form of second degree polynomials (Table 1), and their quality was assessed by means of R values and non-numerical analysis of residues according to Ref. [27] (Figure 3).
3.2.3 Precision

Repeatability of the method was tested according to [27] by replicating the entire method on the same day, using the same cosmetic preparation, batches of solvents, and chromatographic plates, by the same analyst (Day 1, Analysis A and B). Intermediate precision was verified according to Ref. [27] by repeating the procedure on the same cosmetic preparation but on a different day, by a different analyst, using other batches of solvents

Table 1 Calibration parameters for OS, AVO, and PBS (peak areas in arbitrary units).

OS Equation R LOD (g LOQ (g spot1) spot1) 522.9x2 + 1499.7x + 11.3 0.9973 0.02 0.06

AVO 2745.4x2 + 6980.4x 175.5 R = 0.9996 0.03 0.08

PBS 1419.6x2 + 3246.4x + 145.5 R = 0.9988 0.02 0.06

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Figure 3 Residues for AVO, OS, and PBS calibration plots.

Table 2 Results of repeatability, intermediate precision, and robustness tests.

HP TLC plates, automatic spotting (n = 3) Day 1, Analyst 1 Analysis A Analysis B OS g spot1 % in formulation CV% AVO g spot1 % in formulation CV% PBS g spot1 0.50 5.00 2.15 0.40 0.40 2.46 0.40 4.0 1.30 0.50 5.00 1.70 0.41 0.41 2.63 0.41 4.1 2.24

Day 2, Analyst 2

TLC plates, manual spotting (n = 3)

0.49 4.90 2.42 0.40 0.40 1.02 0.41 4.1 1.43

0.48 4.8 6.57 0.38 0.38 4.55 0.43 4.3 5.80

% in formulation CV%

and chromatographic plates (Day 2). The results of these experiments (Table 2) prove that the method precision is sufficient for routine product analysis.
3.2.4 Limits of Detection and Quantification

The limits of detection and quantification for AVO, OS, and PBS determined experimentally by calculating the signal-to-noise ratio according to [28] are given in Table 1.
3.2.5 Robustness

chromatographic plates (HPTLC vs. TLC) and the method of spotting. The same cosmetic preparation was analyzed on HPTLC silica gel 60 chromatographic plates with automatic spotting and on standard TLC silica gel 60 plates with manual spotting with a Hamilton 10-L TLC microsyringe. The results of these analyses (Table 2) are similar with coefficients of variations being slightly worse for manual spotting.
3.2.6 Accuracy

After due consideration of factors that can influence the analysis results, it was concluded that the critical points are the quality of
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Blank cosmetic cream was spiked with AVO, PBS, and OS at three concentrations 1%, 2%, and 3% (v/v) of each sunscreen corresponding to 0.30, 0.60, and 0.90 g spot1 (3 L spot1 of

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HPTLC Quantification of Sunscreens Table 3 Recovery tests (n = 3).

References [1] A. Salvador, A. Chisvert, Anal. Chim. Acta 537 (2005) 114. OS AVO 0.29 96.6 2.80 0.59 98.3 2.85 0.94 104.4 2.13 PBS 0.31 103.3 2.05 0.58 96.7 3.83 0.88 97.8 2.74 [2] S.K. Poole, C.F. Poole, J. Chromatogr. B 797 (2003) 319. [3] K. Valko, J. Chromatogr. A 1037(2004) 299310. [4] K. Kulig, K. Rybicka, B. Malawska, Biomed. Chromatogr. 22 (2008) 12251229. [5] S. Espinosa, E. Bosch, M. Roses, J. Chromatogr. A 947 (2002) 4758. [6] E. Westgate, J. Sherma, J. Liq. Chrom. Rel. Technol. 23 (2000) 609615. [7] B. Musial, J. Sherma, J. Planar Chromatogr. 10 (1997) 368371. [8] B. Musial, J. Sherma, Acta Chromatogr. 8 (1998) 512. [9] E. Westgate, J. Sherma, American Lab. (2000) 1318. [10] J. Fisher, J. Sherma, J. Planar Chromatogr. 13 (2000) 388390 [11] A.W. Sobanska, E. Brzezinska, Acta Pol. Pharm. (2012) (in press).

0.30 g spot1

Received % recovery CV%

0.30 100.0 3.35 0.63 105.0 2.56 0.91 101.1 3.48

0.60 g

spot1

Received % recovery CV%

0.90 g spot1

Received % recovery CV%

the cream solution prepared according to Section 2.2). The analytical procedure described in Section 2 was performed on the samples and the recoveries are presented in Table 3.
3.2.7 Storage and Stability of Standard Solutions

[12] A.W. Sobanska, E. Brzezinska, J. Planar Chromatogr. 24 (2011) 154159. [13] A.W. Sobanska, E. Brzezinska, J. Planar Chromatogr. 24 (2011) 227231. [14] H. Konig, R. Ryschka, Frasenius Z. Anal Chim. 315 (1983) 434437. [15] M. Nyeborg, M. Pissavini, Y. Lemasson, O. Doucet, Int. J. Cosmet. Sci. 32 (2010) 4753. [16] A. Chisvert, A. Salvador, J. Chromatogr. A 977 (2002) 277280. [17] L. Gagliardi, G. Cavazzutti, L. Montanarella, D. Tonelli, J. Chromatogr. 464 (1989) 428433. [18] T. Liu, D. Wu, Int. J. Cosmet. Sci. (2011) 17 (early view).

Mixed standard solutions of AVO, PBS, and OS, as well as solutions of individual sunscreens were refrigerated between the experiments and not exposed to light except for time needed for plates spotting. The stability of all solutions was in these conditions excellent as tested by UV/vis spectroscopy over the period of 2 weeks.

4 Concluding Remarks
Oil- and water-soluble UV filters may be effectively separated from each other and from other components of cosmetic preparations by normal-phase, multiple-development HPTLC on silica gel 60. Chromatographic plates were first developed with the non-aqueous mobile phase that was suitable for analysis of oilsoluble UV filters (e.g. AVO and OS) and, after densitometric scanning, developed again with the more polar mobile phase containing water (in these conditions water-soluble UV filters such as PBS elute). The proposed strategy for simultaneous quantification of oil- and water-soluble sunscreens is simple, quick and relatively inexpensive, requires no toxic solvents, and may be therefore recommended for routine analysis of cosmetic products. This method can be applied with acceptable results with both automatic and manual spotting, on HPTLC and standard TLC plates.
Acknowledgments

[19] D. De Orsi, G. Giannini, L. Gagliardi, R. Porra, S. Berri, A. Bolasco, I. Carpani, D. Tonelli, Chromatographia 64 (2006) 509515. [20] D.J. Schakel, D. Kalsbeek, K. Boer, J. Chromatogr. A 1049 (2004) 127130. [21] Determination of Ten Active Ingredients in Sunscreen-Containing Product in a Single Injection, Dionex Application Note 223. [22] Z. Leon, A. Balaguer, A. Chisvert, A. Salvador, M. Herraez, O. Diez, Anal. Bioanal. Chem. 391 (2008) 859866. [23] S.C. Rastogi, G.H. Jensen, J. Chromatogr. A 828 (1998) 311 316. [24] S. Simeoni, R. Tursilli, A. Bianchi, S. Scalia, J. Pharm. Biomed. Anal. 38 (2005) 250255. [25] P.G. Pietta, A. Bruno, P.L. Mauri, C. Gardana, R. Maffei-Facino, M. Carini, J. Pharm. Biomed. Anal. 13 (1995) 229235. [26] C.W. Kampfl, T. Leitner, J. Sep. Sci. 26 (2003) 12591262. [27] K. Ferenczi-Fodor, B. Renger, Z. Vgh, J. Planar Chromatogr. 23 (2010) 173179.

This work was supported by an internal grant from the Medical University of Lodz, Poland (no. 503/3-016-03)503-01. Thanks are due to Merck for free samples of sunscreens used through this investigation.

[28] P. Koneczka, J. Namiesnik (eds.), Ocena i kontrola jakoci wynikw pomiarw analitycznych, WNT Warsaw, 2007, pp. 269288 (in Polish). Ms received: September 27, 2011 Accepted: January 3, 2012

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