Liquid Chromatographic Determination of UV Absorbers in Sunscreens Mary R. Davis Van Dyk, Main and William Streets, Belleville, NJ 07109 Michael N. C4uigley1 Richard King Mellon Hall of Science, Duquesne University, Pittsburgh, PA 15282 Commercial sunscreens contain various compounds that absorb or physically block UV radiation-properties that can he exploited when determining the concentrations of these compounds. Although UV absorbance spectroscopy has been used to evaluate sunscreens ( I , 21, separation of components is preferred. Thin-layer chromatography (3) and gas-liquid chromatography (3, 4 ) have been investigated, but liquid chromatography with UV absorbance detection is better (3,5-9). E a r l y s u n s c r e e n s contained p-aminobenzoic acid (PABA), and the UV absorbing qualities of this compound are well-documented (10, 11). The popularity of PABA in the manufacture of cosmetics during the 1960's and 70's led to widespread use by people intent on acquiring a suntan without risking serious sunburn or worse. The American Cancer ~ocietykstimatesthat more than half a million new cases of skin cancer are diagnosed every year and that about 90% are caused by exposure to the sun. In a recently published article in this Journal, Taylor noted that 1in 10 Americans will contract skin cancer during their lifetime (12). Uufortunatelv, it later became apparent that PABA can .. induct! :illw&!c n!;iclit~risin sensitive mdividuals 13-22,. This led cosrnetolorrists to investiwte thc efT,ctivc.ncssof various alternatives (231, particularly octyl salicylate (CAS no. 118-60-5; h max 310 nm), benzophenone-3 (CAS no. 131-57-7; h max 325 nm), octyl dimethyl PABA (CAS no. 21245-02-3; h max 310 nm), and octyl p-methoxycinnamate (CAS no. 5466-77-3; h rnax 311 nm). Most modern sunscreens on the North American market contain a t least one of these compounds. Each compound is classified as a W - A or UV-B absorber, depending upon the region of the spectrum in which i t is effective. UV-A radiation (wavelengths 3 2 0 4 0 0 nm) represents about 5.5% of the total solar energy reaching the earth, but causes less erythema (redness of the skin) than UV-B radiation (wavelengths 290-320 nm), which accounts for only about 0.5% of the same total energy. W - C radiation (wavelengths 200-290 nm) is absorbed by atmospheric ozone but has become more significant due to ozone depletion by chlorofluorocarbons. See the table for UV-Aor W - B designations and Figure 1for molecular structures. Although pretan accelerators and self-tanning creams are becorkng more popular, sunscreens still holdihr lirgrsr shrirr ofthr rnxkct (34 . Many manufacturers provide a range of sunscreens with varying concentrations of absorbing agent(s). This has resulted in consumer familiarity with sun protection factors (SPF's) (25-27). In order to define SPF, the minimal erythema dose (MED) must first be considered. MED is the minimum amount of energy re'Author to whom correspondence should be addressed. Current address: Bradfield and Emerson Halls, Cornell University, Ithaca, NY
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14853.
2Mainand William Streets, Belleville, NJ 07109,
quired to produce the first sign of redness of the skin. SPF is then calculated a s a ratio. SPF value = MEDprotectedskin
MEDunprotectedkin
Hence, a sunscreen with a n SPF value of 2 allows a person to be exposed to the sun two times longer than would be possible without protection. An SPF of 3 allows three times the length of exposure, and so on. Experimental procedures for isocratic (3,9) and gradient elution (5-7) LC determinations of UV absorbers in sunscreens have been previously reported and led us to investigate the use of our own isocratic, reverse-phase procedure in the undergraduate laboratory. Even a basic HPLC UV detector, and system (one pump, injector, C I column, ~ strip-chart recorder) can provide good results. Experimental All reagents must be of analytical grade, Mobile Phase Prepare a 1-L solution of 90:lO (vlv) acetonitrilelwater. Standard Solutions Obtain octyl salicylate (Escalol 587, Van DykZ),benzophenone-3 (Escalol 5671, octyl dimethyl-PABA (Escalol 5071, and octyl p-methoxycinnamate (Escalol 557). Prepare dilutions of each one from 1to 10% (wlv) in methanol.
Figure 1. Compounds commonly used as sun-blocking agents: (a) Octyl salicylate (C,,HZ2O3);(b) BenzophenoneJ (C,4H,,0,); (c)Octyl dimethyl PABA (C,,H2,O2N); ( d ) Octyl pmethoxyclnnarnate (ci8H2603).
Volume 72 Number 3 March 1995
279
Commercial Sunscreens and Their Active ingredients
Sunscreena
Type of ~bsorbe?
Active IngredientC
Escalol Llesignationd
% Concn
Label Panama Jack (SPF 4)
uv-B
Octyidimethyi PABA
Coppertone (SPF 6)
uv-B UV-B
Homosalate (Homomenthylsalicyiate) Octyldimethyl PABA
uv-B
Avobenzone Padimate 0 (Octyldimethyl PABA)
UV-B
Butylmetnoxyo oenzoyl metnane Pao mate 0 (Octyldlmelny PABA)
Kids (SPF 30)
uv-BIA uv-B
Oxybenzone (Benzophenoned) Ethylhexyl pmethoxycinnamate (Octvl o-methoxvcinnamate)
Sundown (SPF 30)
UV-BIA
uv-B PB
Oxybenzone (Benzophenone-3) Octy melhoxyc nnamale Octylsalicylate Titanium dioxide
UV-BIA uv-B uv-B PB
Benzophenone-3 Octyl pmethoxycinnamate Octyl salicyiate Titanium dioxide
Filteray (SPF 15) Photoflex (SPF 15+)
Piz Buin (SPF 30+)
uv-B
UV-BIA Oxybenzone (BenzophenoneB) Octyldimethyl PABA uv-B 'Sunscreens have been placed in order of increasing SPF value (see definition in the text) "See re*! for descr pton ol *V-A ano LV-B oes qnatnons Des gnatran as a - V - 8 A absorber ndmles lnal oanmphenono-3nas oroaa specIra ln s aosorbance exltnas nlo h e JV.C region. Scc ref 18. PB refors to a pnysca b o c ng ~ aqenl concerwal ons grealcr lnan A a ue nqreo e m s are as lhsleo oy !ne s~rscreenrnan.lact,rer Slanoaru re0 names are glven n parenlneses %ur commonly used UV radiation absorbers are available from Van Dyk under the Escalol trade name.
Found
PreSun (SPF 39)
aowroance At
Apparatus The exact experimental setup will depend on the instrumentation availahle. At Duquesne, mobile phase is fed to a high Dresswe reci~rocatineDumD (Waters 6000A Solvent ~ & & ySystem) ihrough 56-in.-i.d. TFE tubing (Waters). Stainless steel tubing (1116-in. 0.d. and 0.03-in. i d . ) is used to connect the pump to the injector (Waters U6K). Zero dead volume fittings and 1116-in.-o.d., 0.007-in.4.d. stainless steel tubing (Alltech) are used to connect sequentially, an in-line filter (Rheodyne) and a guard column (Waters, C18Guard Pak) to the analytical column (Waters Cl8 ~ B o n dapak). Absorbance measurements are obtained a t 254 n m using a n UV absorbance detector (Waters 440). Data is recorded with a n electronic integrator (Shimadzu CR501 Chromatopac). Solutions are filtered using 0.5ym Millex-SRsyringe filters (Millipore), before injection into the chromatograph using a 10-pL syringe (Hamilton). Asyringe cleaner is used between injections (28).
0
2
6
TIME (MINUTES) Figure 2. Chromatogram of a sunscreen extract solution. The sunscreen is Filteray. The peak marked a is due to the presence of octyidimethyi PABA(Ci7Hz02N).
Procedure Accurately weigh 1g of the sunscreen into a centrifuge tube. Add 500 pL of 2 M sulfuric acid solution and 10 mL of methanol. Ultrasonicate for 30 min, and then centrifuge a t 1000 G for 10 min. If such equipment is unavailable, use a small conical flask to contain the sunscreen and reagents. Mechanically shake this for several hours and finally allow to stand overnight. Dilute 5.0 mL of the supernatant to 25 mL with methanol. Filter through a 0.45-urn membrane filter. This solution is ready for *LC analisis. Continuously degas the mobile phase by sparging with helium. Equilibrate the C18 column for several hours by
280
Journal of Chemical Education
passing mobile phase through the column a t a flow rate of 0.1 mumin. For analysis, use a flow rate of 2.0 mumin. Inject the standard solutions into the chromatograph, and obtain peak height or peak area measurements. Use these data to construct a calibration curve of response versus concentration. Inject the test solution(s) into the chromatograph, obtain the peak height(s1 or peak area(s), and determine the concentration of active ingredient by interDolation to the calihration curve. Because manv manufacturers do not quote the concentrations of active ingredients on their products, making results difficult to verify, the
procedure is best viewed a s a comparative exercise. Retention times provide the identity of a given species, and comparison of peak heights or areas for the same species indicates the level of W protection. As may be seen from Figure 2, the procedure is satisfactory for most investigations, and gradient elution is required only for those sunscreens that contain poorly resolved components (5-7). Results for those sunscreens tested by the authors are given i n the table. Literature Cited 1. Yogelman. J. H.: Nieuer. E.: Btind, J. L.: Nash. R. A ; Orentreich, N. J. Appl CosmeDl. 1989.3. 1. 2. Evans, G. O.,II J. C h r m Educ 1978,53,315. 3. Shaath. N. A ; GtiEn. E M.; Andernicael, G. I. In Sunscmns: Deuelopmanf, Eiolud i m and Repulalory Aspects: Lowe, N. J.: Shaath, N. A , Eds.: Marcel Dekker, 1990: 00 . . 505436. 4. Cumpclt. H. M. Cosmel. Toildries 1982.97, 67. 5. Gsgliardi, L.; Arnato. A ; Tuchetfo, L.; Cavazzutti. OAnol. Lotierr 1890.23.2123. 6. Gagliardi. L.; Arnato, A,; Basili. A ; Cauazzutti, G.; Tonelli, D. J. Chiomlogr. 1987, 408, 409.
7. Dagliardi, L.: Amato. A ; Basili. A : Cauazzutti, 0.: Dattaveeehia. E.: Tonelli, D. J. Chromatom. 1988.362.450. 1985, 17.67. 6. Hillier.E.:CottprR.:Andrews.M.Am.Loh. 9. Bruse, M.; Fregert. S.; Gnvberger. B. Phofaiwmntologv 1984, 1,277. 10. Vogeimsn, J. H.;Niever.E.;Bn"a,J. L.;Nash,R. A.:Orentreich.N. J A p p I . cormeto,
15. 16. 17. 16. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
I**'
7 1
Stenberg,C.: Mellstrand. T.; Larko, 0 . Pholodermoioiog~1987.4.201. Gasparno, F E Photodirmotology 1986,3,61. Gaqarra, E E Pholodermnlology 1985,2, 151. Roelsndfs, R.:Vsnhee,J.:Bonsrnie. A,: Kerkhofs, L.: Degreef H.Ini.J. Drrmntoloxy 1983.22,247. Marrnelzst. J.;Rapsport, M. J. Contocl Derrnoiifis 1980.6.230, Horio, T.; Hipchi, T. Dermnlolngicn 1978, 156,124. Mathia8.C. G. T.;Miabach. H. I.: Epetein. J.Arch. Dermoinl. 1918. 114. 1665. Thompson, G.: Maibech. H.; Epstein, J. Arch D~rmalol.1971, 113, 1252. Willis. I.; Kligma, A. M. Arch Dermnlol. 1910,lOZ. 405. Davts, M. R. Sunscreens: Van Dyk: Bellevilie, NJ. Brown, S.; DiFfey, B. L. Photochem. Phohhiol. 1986.44,509. Fair. P M.; Diffeey. H. L. Br J. Dermofol. 1985.112.113. Sayre. R. M.; Poh A@. P. Soc Cosmet Cbrm. 1984,35,439. Quiglel M . N. J. Chem Educ 1881,68, 520.
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