154-159

7/30/2019 154-159

1/6

154

Summary

TLC separation of the UV filters avobenzone (AVO) and octocry-

lene (OCR) on RP-18 and silica gel 60 as stationary phases, with

numerous mobile phases, revealed that these compounds have simi-

lar retention properties under almost all the chromatographic con-

ditions 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 suc-

cessful for OCR in the presence of AVO. This involved chromatog-

raphy 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.

1 Introduction

Butyl methoxydibenzoylmethane (avobenzone, AVO; Figure 1)

is the most popular UVAfilter, authorized in Europe, USA, Aus-tralia, and Japan [1]. This compound affords high UVA protec-

tion 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 fil-

ter 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 par-

ticular cosmetic preparation.

Keeping in mind the significance of the avobenzoneoctocry-lene 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 octocry-

lene have been simultaneously quantified by column chro-

matography or related techniques, for example RP HPLC

[613], GC [1416], and microemulsion electrokinetic chro-

matography (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 RPHPTLCspec-

trodensitometric 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 theliterature, not an easy task, and avobenzone is chromatographi-

cally, 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 sali-

cylate [6, 10, 19], homosalate [6, 7], octyldimethyl PABA [10],

or octyl methoxycinnamate [8, 10, 20]. Separation of these com-

pounds requires gradient elution [6, 10, 12, 21, 22] or modifica-

tion 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 sim-

ilarly migrating UV filters listed above, complicating their quan-

tification 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.

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

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

Anna W. Sobanska* and Elzbieta Brzezinska

Key Words

Avobenzone

Octocrylene

TLC

Spectrodensitometry

Figure 1

The structural formulas of avobenzone and octocrylene.

Journal of Planar Chromatography 24 (2011) 2, 154159 DOI: 10.1556/JPC.24.2011.2.140933-4173/$ 20.00 Akadmiai Kiad, Budapest

7/30/2019 154-159

2/6

TLC Analysis of Avobenzone and Octocrylene

The objective of this research was to develop a simple and cost-

effective method of densitometric analysis of avobenzone and

octocrylene combined in a sunscreen preparation.

2 Experimental

2.1 Chemicals, Materials, and Solutions

Eusolex 9020 (avobenzone) and Eusolex OCR (octocrylene)

were kindly donated by Merck. Uvinul T 150 (ethylhexyl tria-

zone, ET) was a free sample from BASF. Ethylparaben was pur-

chased from SigmaAldrich. Methanol, cyclohexane, diethyl

ether, isopropanol, toluene, ethyl acetate, acetic acid, tetra-

hydrofuran, 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 Cosmet-

ics, Poland.

Eusolex 9020 (avobenzone), 500 mg, and Eusolex OCR

(octocrylene), 500 mg, were weighed accurately into 100-mL

volumetric flasks. Both UV filters were dissolved in adequate

amounts of methanol and diluted to volume with the same sol-

vent 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 as500 g mL1 solutions in acetone and methanol, respectively. All

flasks were wrapped with aluminum foil and kept refrigerated.

2.2 Sample Preparation

Suncare lotion (1000 mg) was weighed 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 alu-

minum foil and left to stand for 60 min.

2.3 Thin-Layer Chromatography

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 solu-

tions of AVO and OCR) and developed with either cyclohexa-

nediethyl ether 1:1 (v/v), Method A, or cyclohexanepiperidine

15:1 (v/v), Method B. Plates were developed in a vertical chro-

matographic chamber lined with filter paper and previously sat-

urated 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, plateswere 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 investiga-

tion, obtained as described above, are shown in Figures 2 and 3.

2.4 Analysis of Avobenzone and Octocrylene in the

Sunscreen Lotion

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 was4 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 com-

pounds 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

Journal of Planar Chromatography 24 (2011) 2 155

Figure 2

Densitograms obtained from AVO standard and the product at

380 nm (Method A).

Figure 3

Densitograms obtained from OCR standard and the product at

300 nm (Method B).

7/30/2019 154-159

3/6


156 Journal of Planar Chromatography 24 (2011) 2

above two stationary phases (RP-18 and silica gel 60) and sev-

eral mobile phases were tested. The results of these tests are

summarized in Tables 1 and 2.

On the basis of comparison ofRF

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 advanta-

geous when avobenzone is present in the formulation. Other

Table 1

RF

of AVO, OCR, ET, and ethylparaben on RP-18.

Mobile phase AVO OCR ET Ethylparaben

components Ratio (v/v)

MeOHH2O 7:3 0.02 0.02 0 0.38

MeOHTHFpH 13 buffer 50:35:15 0.40 0.45 0.06 0.72

MeOHTHFH2O 50:35:15 0.37 0.42 0.06 0.72

MeCNH2O 9:1 0.32 0.40 0 0.70

MeOHH2O 9:1 0.25 0.35 0 0.70

MeOHpH 13 buffer 9:1 0.21 0.32 0 0.68

MeOHpH 3 buffer 9:1 0.20 0.30 0 0.63

MeOHEDTA 0.002 mol L1 9:1 0.23 0.35 0 0.68

MeOHNH3 2.0 mol L1 9:1 0.22 0.34 0 0.66

EtOHH2OAcOH 70:29.5:0.5 0.08 0.12 0 0.52

MeOHH2O 4:1 0.10 0.12 0 0.54

MeOHMeCN 9:1 0.50 0.61 0.18 0.74

i-PrOHAcOEtH2O 6:1:2 0.44 0.54 0.24 0.79

Table 2

RF

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

Mobile phase AVO OCR ET Ethylparaben

components Ratio (v/v)

Cyclohexaneetheri-PrOH 15:1:1 0.55 0.55 0.23 0.22

CyclohexaneetherAcOEt 15:1:1 0.33 0.36 0.02 0.07

Cyclohexaneether 1:1 0.55 0.60 0.23 0.30

Cyclohexaneether 5:1 0.37 0.40 0 0.08

Cyclohexaneether 10:1 0.30 0.34 0 0.02

Cyclohexaneether 15:1 0.20 0.15 0 0

Cyclohexaneetheracetone 15:1:1 0.34 0.34 0.02 0.08

Cyclohexaneetheracetone 15:1:2 0.42 0.44 0.10 0.15

Cyclohexaneetheracetone 15:1:3 0.42 0.43 0.21 0.19Cyclohexanei-PrOH 15:1 0.51 0.51 0.10 0.14

CyclohexaneAcOEt 15:1 0.31 0.34 0 0.05

CyclohexaneCHCl3 1:1 0.38 0.38 0 0

Toluene 0.33 0.27 0 0

TolueneAcOEt 15:1 0.50 0.68 0.08 0.12

TolueneAcOEtAcOH 15:1:0.2 0.63 0.67 0.12 0.15

TolueneAcOEtAcOH 15:1:1 0.67 0.72 0.36 0.31

Cyclohexaneetheri-PrOHAcOEt 15:1:1:1 0.62 0.67 0.29 0.28

Cyclohexaneetheri-PrOH 15:1:0.2 0.37 0.41 0 0.05

Cyclohexanepiperidine 15:1 0.03 0.29 0 0

Cyclohexanepiperidine 5:1 0.18 0.56 0.30 0.03

7/30/2019 154-159

4/6


mobile phases typically used in RP TLC also resulted in moder-

ate separation. Attempts to change the migration properties of

AVO by varying mobile phase pH (Table 1) were also unsuc-

cessful. 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 reactiv-ity of octocrylene compared with avobenzone, formally contain-

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 select-

ed for further tests because of its suitable physicochemical prop-

erties (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, subse-

quently denoted Method A (for avobenzone) and Method B (for

octocrylene).

3.2 Method A

Method A involved application of the standard NP mobile

phase cyclohexaneether 1:1 (v/v). Under these conditions sepa-

ration of common preservatives (parabens) and UV filters such

as ethylhexyl triazone from co-eluting AVO and OCR was easi-

ly 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, howev-

er, 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 per-formed at 380 nm; statistical data for the calibration plot

obtained are given in Table 3. Limits of detection and quantifi-

cation 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 present-

ed in Table 5. The specificity of analysis of AVO at the proposed

wavelength (380 nm) was confirmed by comparing multiwave-

length scans acquired for AVO and OCR (Figure 5).

3.3 Method B

The change in the retention behavior of avobenzone compared

with octocrylene in Method B made it possible to determine

octocrylene densitometrically without interference from avo-

benzone. 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 sun-

screen 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 recoverytests are summarized in Table 5. The purity of the OCR peaks


Table 3

Linear regression data for AVO and OCR, on the basis of peak areas

(n= 10).

AVO (Method A) OCR (Method B)

Slope (b Sb) 1507.96 63.01 1956.56 94.26

Y-intercept (a Sa) 466.25 78.20 304.34 106.08

LOD (calculated) [ng/spot] 170 180

LOQ (calculated) [ng/spot] 510 537

Sxy (Residual SD) 114.47 146.02

Correlation coefficient,R 0.9931 0.9920

Linearity range [ng per spot] 2001800 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 1490

CAVO [% w/w] 3.87 3.72

RSD [%] 3.20 2.16

OCR (Method B) CAVO [ng per spot] 1010 980

CAVO [%] 10.1 9.8

RSD [%] 4.97 6.42

Figure 4

UV spectra obtained from AVO and OCR adsorbed on silica gel 60, by

use of the Desaga CD 60.

7/30/2019 154-159

5/6


158 Journal of Planar Chromatography 24 (2011) 2

was confirmed by comparing UV spectra acquired at three dif-

ferent points on the OCR peak obtained from the sunscreen

product and the spectrum acquired for the OCR standard

(Figure 6).

4 Conclusions

UV filters avobenzone and octocrylene may be effectively sepa-

rated 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 quanti-

fied, in the presence of octocrylene, by densitometry at 380 nm,

where octocrylene does not absorb. There is no analytical wave-

length 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 avoben-

zone substantially by using a different mobile phase, cyclohexa-

nepiperidine 15:1 (v/v) and to quantify octocrylene densitomet-

rically 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 recom-mended for routine analysis of cosmetic products.

Acknowledgments

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 sun-

screens used throughout this research.

References

[1] A. Salvador, A. Chisvert,Anal. Chim. Acta537 (2005) 114.

[2] C. Bonda, Cosmet. Toilet. 123 (2008) 4960.

[3] C. Mendrok-Edinger, K. Smith, A. Janssen, J. Vollhardt, Cosmet.

Toilet. 124 (2009) 4754.

[4] L.R. Gasper, P.M.B.G. Maia Campos, Int. J. Pharm. 307 (2006)

123128.

[5] S. Scalia, M. Mezzena, AAPS PharmSciTech 10 (2009) 384390.

[6] D.J. Schakel, D. Kalsbeek, K. Boer, J. Chromatogr. A 1049 (2004)

127130.

[7] A. Salvador, A. Chisvert, Anal. Chim. Acta537 (2005) 1524.

[8] S. Simeoni, R. Tursilli, A. Bianchi, S. Scalia, J. Pharm. Biomed.

Anal. 38 (2005) 250255.

[9] D. De Orsi, G. Giannini, L. Gagliardi, R. Porra, S. Berri, A. Bolas-

co, I. Carpani, D. Tonelli, Chromatographia64 (2006) 509515.

[10] S.C. Rastogi, G.H. Jensen, J. Chromatogr. A 828 (1998) 311316.

[11] R. Rodil, S. Schrader, M. Moeder, J. Chromatogr. A 1216 (2009)48874894.

Table 5

Recovery of AVO and OCR, on the basis of peak areas (n= 3).

AVO (Method A) OCR (Method B)

Spiked amount Found Recovery RSD Spiked amount Found Recovery RSD

[ng per spot] [ng per spot] [%] [%] [ng per spot] [ng per spot] [%] [%]

375 380 101.3 5.51 375 385 102.7 3.751000 1050 105.0 1.20 800 790 98.8 0.84

1600 1603 100.0 2.36 1600 1620 101.2 5.95

Figure 5

Multiwavelength scans of AVO and OCR, obtained by use of the

Desaga CD 60.

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.

7/30/2019 154-159

6/6


[12] R. Danovaro, L. Bongiorni, C. Corinaldesi, D. Giovanelli,

E. Damiani, P. Astolfi, L. Greci, A. Pusceddu, Environ. Health Per-

spect. 116 (2008) 441447.

[13] R. Rodil, J.B. Quintana, P. Lopez-Mahia, S. Muniategui-Lorenzo,

D. Prada-Rodriguez, Anal. Chem. 80 (2008) 13071315.

[14] T. Poiger, H.-R. Buser, M.E. Balmer, P.-A. Bergqvist, M.D. Mller,

Chemosphere 55 (2004) 951963.[15] P. Cuderman, E. Heath, Anal. Bioanal. Chem. 387 (2007)

13431350.

[16] R. Rodil, M. Moeder, J. Chromatogr. A 1179 (2008) 8188.

[17] C.W. Klampfl, T. Leitner, E.F. Hilder, Electrophoresis 23 (2002)

24242429.

[18] J. Fisher, J. Sherma, J. Planar Chromatogr. 13 (2000) 388

390.

[19] A. Chisvert, M.C. Pascual-Marti, A. Salvador, J. Chromatogr. A

921 (2001) 207215.

[20] R. Jiang, C.G.J. Hayden, R.J. Prankerd, M.S. Roberts, H.A.E. Ben-

son, J. Chromatogr. B 682 (1996) 137145.

[21] J. Awatramani, J.E. Nucci, Cosmet. Toilet. 120 (2005) 6974.

[22] E.R.M. Kedor-Hackmann, M.L. De Lourdes Perez Gonzalez,

A.K. Singh, M.I.R.M. Santoro, Int. J. Cosmet. Sci. 28 (2006)

219224.

[23] E. Westgate, J. Sherma, J. Liq. Chrom. Relat. Technol. 23 (2000)609615.

[24] B. Musial, J. Sherma, J. Planar Chromatogr. 10 (1997) 368371.

[25] B. Musial, J. Sherma, Acta Chromatogr. 8 (1998) 512.

[26] E. Westgate, J. Sherma, American Lab. (2000) 1318.

[27] P. Konieczko, J. Namiesnik, Ocena i kontrola jakoci wynikw

pomiarw analitycznych, Wydawnictwo Naukowo-Techniczne,

Warszawa, 2007.

Ms received: January 20, 2010

Accepted: June 14, 2010


154-159

Documents