pbs avo os publikacja jpc2012

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SummaryA complex mixture of sunscreens of different lipophilicity was quan-tified for the first time by thin-layer chromatography (TLC) fol-lowed by densitometric scanning in absorption mode. Multiple-development normal-phase TLC was performed on silica gel 60 asstationary 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 sun-screens, avobenzone (AVO) and octyl salicylate (OS), were analyzedat 360 and 300 nm, respectively. Subsequent development of thesame 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 spot 1 ,LOQ (OS) 0.06 g spot 1 , LOD (AVO) 0.03 g spot 1 , LOQ (AVO)0.08 g spot 1 , LOD (PBS) 0.02 g spot 1 , and LOQ (PBS) 0.06 gspot 1 . The method was validated and applied to the analysis of acommercially available cosmetic product.

1 Introduction

Quantification of sunscreens in cosmetic products may beachieved by a number of analytical techniques, including chro-matography gas chromatography (GC), thin-layer chromatog-raphy (TLC), and, much more often, high-performance liquidchromatography (HPLC) [1]. The retention behavior of com-pounds in different chromatographic conditions is strongly relat-ed 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 highlipophilicity (log K o/w usually 4 to 8 or higher), known to formu-lators as fat soluble filters. These sunscreens are usually sepa-rated 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, andstrongly anionic substances such as benzophenone-4, phenyl-benzimidazole sulfonic acid, disodium phenyl dibenzimidazoletetrasulfonate, or terephthalylidene dicamphor sulfonic acid isdescribed in the literature far less frequently. Until now nomethod has been reported on TLC separation and quantificationof water-soluble UV filters; when in the course of our prelimi-nary research normal-phase (NP) chromatographic proceduresdescribed in Refs. [1113] and other similar normal-phase chro-matographic conditions were tested on these compounds, theyremained on the start line ( R

F= 0) [11]. Our attempts to analyze

water-soluble sunscreens by reversed phase TLC on RP-18 sta-tionary phase with typical mobile phases (water and differentconcentrations of organic modifiers such as tetrahydrofurane,acetone, methanol, 1,4-dioxane, dimethylformamide, or ace-tonitrile) resulted in the rapid migration of investigated com-pounds ( RF > 0.95 [11]). Similarly the RP HPLC methoddescribed in Ref. [14] applied to water-soluble UV filters gavevery short retention times ( t R 1 min) and poor baseline separa-tion. Different approaches proposed to facilitate RP HPLCanalysis of water-soluble UV filters included increasing the sun-screens retention times by adjusting the pH of mobile phases[1521], application of ion-pair chromatography (mobile phasescontaining tetraalkylammonium salts) [22, 23], or using station-ary phases other then RP-18 (e.g. cyanopropyl packing [24]).Application of electrochromatographic techniques such asmicellar electrokinetic chromatography or microemulsionelec-trokinetic chromatography (MEEKC) to phenylbenzimidazolsulfonic acid (PBS) has been reported [25, 26]. When the sam-ple contained both water- and oil-soluble sunscreens, theirreversed phase HPLC separation resulted in the former onesmigrating first followed by the latter ones eluting considerablylater in this situation, the duration of each chromatographicrun was a disadvantage [17].

The aim of this study was to propose the first TLC-densitomet-ric method of separation and quantification of sunscreens of mixed lipophilicity rapid, based on the application of readilyavailable silica gel 60 chromatographic plates and with verysimple, low-toxicity mobile phases.

Simultaneous Multiple-Development HPTLC Quantificationof Water- and Oil-Soluble Sunscreens

Anna W. Sobanska* and Jaroslaw Pyzowski

Key Words

SunscreensOil-solubleWater-solubleQuantificationMultiple-development TLCDensitometry

A.W. Sobanska and J. Pyzowski, Department of Analytical Chemistry, MedicalUniversity of Lodz, ul. Muszynskiego 1, 90-151 Lodz, Poland.E-mail: [email protected] and [email protected]

Journal of Planar Chromatography 25 (2012) 4, 344348 DOI: 10.1556/JPC.25.2012.4.110933-4173/$ 20.00 Akadmiai Kiad, Budapest


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HPTLC Quantification of Sunscreens

Journal of Planar Chromatography 25 (2012) 4 345

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, ace-tonitrile, methanol, and diethyl ether were from Chempur,Piekary Slaskie (Poland). UV filters were of cosmetic quality,and all solvents were of analytical grade. Moisturizing creamcontaining AVO, OS, and PBS analyzed in the course of thisstudy was manufactured by Beiersdorf, Hamburg (Germany).The blank cream used in recovery tests was also from Beiers-dorf.

Eusolex 9020 (500 mg) and Eusolex OS (500 mg) were weight-ed accurately into 100-mL volumetric flasks, dissolved in ade-quate amounts of methanol, and diluted to volume with the samesolvent to give stock solutions of the concentration 5 mg mL 1 .The stock solution of Eusolex 232 was prepared in the similar

manner, but this compound (500 mg) was dissolved in wateralkalized with NaOH aq (2 mol L 1 , 0.9 mL).

The stock solutions of OS, AVO, and PBS were used to preparestandard solutions containing the following concentrations of allthree sunscreens: 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6,0.8, and 1 g L 1 . All flasks were wrapped with aluminum foiland kept refrigerated.

2.2 Sample Preparation

Moisturizing cream (1000 mg) was weighted accurately into a 100-mL volumetric flask. Approximately 70 mL methanol wasadded and the flask was vigorously shaken by use of a Premed(Poland) type 327 Universal Shaker for 60 min. Methanol wasthen added to volume, and the flask was wrapped with alu-minum foil and left to stand for 60 min.

2.3 Thin-Layer Chromatography

TLC was performed on 10 cm 20 cm high-performance thin-layer chromatography (HPTLC) silica gel 60 plates (layer thick-ness 0.2 mm) or on 10 cm 20 cm TLC silica gel 60 plates(layer thickness 0.25 mm) from Merck. Plates were pre-washedwith methanoldichloromethane 1:1 ( v/v), dried overnight in theambient conditions and spotted with the Desaga AS 30 samplerequipped with a 10 L syringe (1 L spot 1 of standard solutions

containing AVOOSPBS, standards concentrations accordingto 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 inter-vals, starting 10 mm from the plate edge. Plates were developedfirst with the mobile phase A(cyclohexanediethyl ether 5:1 v/v)and then with the mobile phase B (ethyl acetateethanolwater70:35:30 v/v) in a vertical chromatographic chamber lined withfilter paper and previously saturated with the appropriate mobilephase vapor for 20 min. Each development distance was 75 mmfrom the plate bottom edge. After development, plates weredried at room temperature (20 C), scanned, and analyzed inreflectance mode with the Desaga CD 60 densitometer (slitwidth 4.0 mm, slit height 0.1 mm): after the first development at

360 nm (AVO) and 300 nm (OS), and after the second develop-ment at 300 nm (PBS). Typical densitograms obtained for UV

filters under investigation obtained as described above areshown 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 spot 1 for OSand PBS, 10 L spot 1 for AVO). The plates were then chro-matographed as described above for AVO, OS, and PBS stan-dards (Section 2.3).

3 Results and Discussion

3.1 Method Development

According to our earlier research [1113] oil-soluble UV filterspresent in the majority of sun care products available on theEuropean markets can be chromatographed on silica gel 60 withbinary or tertiary non-aqueous mobile phases to achieve RF val-ues suitable for densitometric scanning (typically 0.20.8). Thesame group of UV filters may be analyzed on RP-18 stationaryphase with binary water-organic solvent mobile phases [11] orwith the mobile phase proposed by Sherma et al. (methanol tetrahydrofuranewater) [610]. Both approaches were testedand failed when applied to water-soluble UV filters stronglyhydrophilic compounds containing at least one ionized sulfonicgroup. The affinity of these UV filters to the aqueous mobilephase is so strong that during RP TLC they eluted rapidly with

RF values typically 0.95 to 1 [11]. On the other hand their polar-ity is so high that chromatographed with organic solvents on sil-ica gel 60, they do not migrate from the start line, so in both sit-uations their densitometric scanning is impossible [11]. Onepossible approach to chromatography of water-soluble UV fil-ters could involve RP-18 stationary phase combined with acidicmobile phases. It was expected that in such conditions, due tothe reversed dissociation of the sulfonic acid moiety water-solu-ble sunscreens should loose some of their affinity to the aqueousmobile phase. This strategy was considered during our prelimi-nary research (e.g. mobile phase methanolwater 9:1 ( v/v) wasreplaced with methanolbuffer (pH = 3) 9:1 ( v/v)) but with mod-

erate success RF values for the water-soluble filter PBS were inthese conditions 0.93 and 0.90, respectively. Since RF values

Figure 1

Densitograms of AVO, OS, and PBS standards after development withmobile phases A and B ( = 300 nm).


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346 Journal of Planar Chromatography 25 (2012) 4

obtained on RP-18 stationary phase for oil-soluble filters underinvestigation (AVO and OS) were too close for their effectivequantification [11], this idea was discontinued. Attention wasthen turned towards NP chromatography on silica gel 60 withtwo alternative mobile phases ethyl acetateethanol water 70:35:30 ( v/v) or acetonitrilewater 9:1 ( v/v). Althoughthese mobile phases were unsuitable for AVO and OS( RF 0.95), retention behavior of PBS was in these conditionsfar more promising ( RF 0.80 and 0.82, respectively). At thisstage it was decided that chromatographic plates should be

developed twice at first with the non-aqueous mobile phase Asuitable for oil-soluble filters and then with the aqueous mobilephase B (ethyl acetateethanolwater 70:35:30 ( v/v) or acetoni-trilewater 9:1 ( v/v)) to elute PBS. Finally, after due considera-

tion of method specificity and toxicology aspects, the followingmobile phases were selected for further investigations: mobilephase A (cyclohexanediethyl ether 5:1 ( v/v)) and mobile phaseB (ethyl acetateethanolwater 70:35:30 ( v/v)).

Analytical wavelengths suitable for UV filters investigated inthe course of this study were selected on the basis of multiwave-

length scanning of chromatograms from 200 to 420 nm at 20 nmintervals: 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 noingredients absorbing between 300 and 400 nm other than OS,AVO, and PBS. It was nevertheless important to check if thechromatographic method proposed in this paper is suitable alsofor formulations containing, e.g., parabens. It was establishedthat mobile phases A and B separate parabens from the UV fil-ters under investigation:

Mobile phase A RF (OS) 0.85, RF (AVO) 0.50, RF (propyl-paraben) 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 analysisof the cream sample was confirmed by UV/vis spectra of sun-screens acquired directly from chromatographic plates inreflectance mode. Spectra collected at two different points of particular peaks obtained for the sample solution were comparedwith spectra acquired for the standards ( Figure 2 ).

3.2.2 Calibration

Calibration plots were obtained by plotting peak areas againstamount of substances in the range 0.061 g spot 1 . In all cases,non-linearity of calibrating plots was clearly visible. Calibrationplots were finally generated for all sunscreens in the form of sec-ond degree polynomials ( Table 1 ), and their quality wasassessed 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] byreplicating the entire method on the same day, using the samecosmetic preparation, batches of solvents, and chromatographic

plates, by the same analyst (Day 1, Analysis A and B). Interme-diate precision was verified according to Ref. [27] by repeatingthe procedure on the same cosmetic preparation but on a differ-ent 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 AVO PBS

Equation 522.9 x2 + 1499.7 x + 11.3 2745.4 x2 + 6980.4 x 175.5 1419.6 x2 + 3246.4 x + 145.5

R 0.9973 R = 0.9996 R = 0.9988

LOD (g spot 1 ) 0.02 0.03 0.02

LOQ (g spot 1 ) 0.06 0.08 0.06

Figure 2

UV spectra of OS, AVO, and PBS acquired from chromatograms of thecream sample (2, 3) and from the standards (1).


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and chromatographic plates (Day 2). The results of these exper-iments ( Table 2 ) prove that the method precision is sufficient forroutine product analysis.

3.2.4 Limits of Detection and Quantification

The limits of detection and quantification for AVO, OS, and PBSdetermined experimentally by calculating the signal-to-noiseratio according to [28] are given in Table 1.

3.2.5 Robustness

After due consideration of factors that can influence the analysisresults, it was concluded that the critical points are the quality of

chromatographic plates (HPTLC vs. TLC) and the method of spotting. The same cosmetic preparation was analyzed onHPTLC silica gel 60 chromatographic plates with automaticspotting and on standard TLC silica gel 60 plates with manualspotting with a Hamilton 10-L TLC microsyringe. The resultsof these analyses (Table 2) are similar with coefficients of vari-ations being slightly worse for manual spotting.

3.2.6 Accuracy

Blank cosmetic cream was spiked with AVO, PBS, and OS atthree concentrations 1%, 2%, and 3% ( v/v) of each sunscreencorresponding to 0.30, 0.60, and 0.90 g spot 1 (3 L spot 1 of

Journal of Planar Chromatography 25 (2012) 4 347

Table 2

Results of repeatability, intermediate precision, and robustness tests.

HP TLC plates, automatic spotting ( n = 3) TLC plates, manual

Day 1, Analyst 1 Day 2, Analyst 2 spotting (n

= 3)Analysis A Analysis B

OS g spot 1 0.50 0.50 0.49 0.48

% in formulation 5.00 5.00 4.90 4.8

CV% 2.15 1.70 2.42 6.57

AVO g spot 1 0.40 0.41 0.40 0.38

% in formulation 0.40 0.41 0.40 0.38

CV% 2.46 2.63 1.02 4.55

PBS g spot 1 0.40 0.41 0.41 0.43

% in formulation 4.0 4.1 4.1 4.3

CV% 1.30 2.24 1.43 5.80

Figure 3

Residues for AVO, OS, and PBS calibration plots.


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348 Journal of Planar Chromatography 25 (2012) 4

the cream solution prepared according to Section 2.2). The ana-lytical 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

Mixed standard solutions of AVO, PBS, and OS, as well as solu-tions of individual sunscreens were refrigerated between theexperiments and not exposed to light except for time needed forplates spotting. The stability of all solutions was in these condi-tions excellent as tested by UV/vis spectroscopy over the periodof 2 weeks.

4 Concluding Remarks

Oil- and water-soluble UV filters may be effectively separatedfrom each other and from other components of cosmetic prepa-rations by normal-phase, multiple-development HPTLC on sili-ca gel 60. Chromatographic plates were first developed with thenon-aqueous mobile phase that was suitable for analysis of oil-soluble UV filters (e.g. AVO and OS) and, after densitometricscanning, developed again with the more polar mobile phasecontaining water (in these conditions water-soluble UV filterssuch as PBS elute). The proposed strategy for simultaneousquantification of oil- and water-soluble sunscreens is simple,quick and relatively inexpensive, requires no toxic solvents, andmay be therefore recommended for routine analysis of cosmeticproducts. This method can be applied with acceptable results

with both automatic and manual spotting, on HPTLC and stan-dard TLC plates.

Acknowledgments

This work was supported by an internal grant from the MedicalUniversity of Lodz, Poland (no. 503/3-016-03)503-01. Thanksare due to Merck for free samples of sunscreens used throughthis investigation.

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Ms received: September 27, 2011Accepted: January 3, 2012

Table 3

Recovery tests ( n = 3).

OS AVO PBS

0.30 g spot 1 Received 0.30 0.29 0.31

% recovery 100.0 96.6 103.3

CV% 3.35 2.80 2.05

0.60 g spot 1 Received 0.63 0.59 0.58

% recovery 105.0 98.3 96.7

CV% 2.56 2.85 3.83

0.90 g spot 1 Received 0.91 0.94 0.88

% recovery 101.1 104.4 97.8

CV% 3.48 2.13 2.74

pbs avo os publikacja jpc2012

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