The Analysis of Sunscreen

Home , Avobenzone, Homosalate, Oxybenzone, Sunscreen

APPLICATION NOTE

Liquid Chromatography

Authors: Catharine Layton Wilhad M. Reuter PerkinElmer, Inc. USA

The Analysis of Sunscreen- Active Ingredients and Parabens Introduction Individuals typically use 5-20 cosmetics in Lotions and Lip Balms by per day, many of which contain sunscreen to prevent skin damage from the sun’s UHPLC with PDA Detection radiation, and antimicrobial preservatives called parabens. Although sunscreen-active ingredients are designed to block UV radiation, some cell damage may be caused when these ingredients are illuminated by sunlight after absorption into the skin.1,2 For example, oxybenzone, an ingredient considered safe by the FDA (Food and Drug Administration), is believed to contribute to the recent rise in melanoma cases by increasing the production of DNA-attacking free radicals upon UV exposure. 3 Additionally, studies have shown oxybenzone to behave similarly to the hormone estrogen, suggesting that it may also contribute to the development of breast cancer.4 Parabens are absorbed through the skin via cosmetic applications and can be found in nearly all adult urine samples, with the highest concentrations observed in adult females and adolescents.5 Furthermore, parabens are thought to have estrogenic activity, which affects the expression of genes regulated by the natural form of estrogen, leading to early puberty in girls and an increased risk for the development of breast cancer. While some parabens are banned in European countries, in the U.S., the FDA maintains that they are safe in cosmetic products at levels up to 25%.6 Since the usage of cosmetics formulated with parabens and Solvents, Standards and Samples sunscreen-active ingredients comes with the above mentioned All solvents and diluents used were HPLC grade. health concerns, it is important for the skincare industry to All stock paraben and sunscreen ingredient standards were develop robust analytical methods to maintain compliance purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). regarding safety regulations. With this in mind, this application Parabens included methylparaben, ethylparaben and provides the UHPLC method conditions and performance data propylparaben. Sunscreen ingredients included octocrylene, for the analysis of parabens and sunscreen-active ingredients homosalate, octinoxate, avobenzone, octisalate, and oxybenzone. found in variations of three cosmetic formulations. Performance data includes repeatability, linearity and LOD/LOQ determination. The following cosmetic samples were obtained from a local drug store: 1) a sunscreen lotion; 2) a skin moisturizer formulated with Experimental sunscreen; 3) a lip balm formulated with sunscreen; 4) a skin Hardware/Software moisturizer without sunscreen; and 5) a lip balm without sunscreen. A PerkinElmer UHPLC system with a PDA (photodiode array) All standard and sample dilutions were prepared in methanol as detector (PerkinElmer, Shelton, CT, USA) was used for this study. a diluent, which was also used for all blank injections. Paraben A PerkinElmer Brownlee™ SPP C18 2.7-μm 50 x 2.1-mm column and sunscreen standard solutions were prepared at 1 mg/mL was used for all determinations (PerkinElmer, Shelton, CT, USA). (1000 ppm), followed by a dilution to 1 µg/mL and 0.1 mg/mL, Method Parameters respectively. The latter were used as working stock standard The HPLC method parameters are shown in Table 1. solutions. For sample solutions, 200 mg of each sample was first added to a tared 50-mL volumetric flask. The flask was then Table 1. HPLC Method Parameters. filled to mark with diluent and sonicated for 15 minutes. HPLC Conditions Prior to injection, all standards and samples were filtered through PerkinElmer Brownlee™ SPP C18 2.7 µm, a 0.22-µm Nylon filter to remove any residual particles. Column: 50 x 2.1 mm column (Part # N9308402)

Solvent A: 0.2% acetic acid in water Results and Discussion Solvent B: 50/50 methanol/ethanol Figure 1 shows the peak profile of three paraben and six Time Flow Rate %A %B %C %D Curve (min) (mL/min) sunscreen standards at 254 nm. All paraben and sunscreen-active Mobile Phase 1 Initial 0.500 70.0 30.0 0.0 0.0 Initial ingredients are well separated, with the exception of octinoxate Gradient: 2 2.00 0.500 20.0 80.0 0.0 0.0 6 and avobenzone. However, it is unlikely that these two 3 6.00 0.500 5.0 95.0 0.0 0.0 6 4 8.00 0.500 5.0 95.0 0.0 0.0 6 components are present in the same sunscreen product, as 5 8.10 0.500 70.0 30.0 0.0 0.0 6 octinoxate is highly susceptible to photolysis when formulated 7 Injection delay time between injections: 10 min. with avobenzone. Detector: PDA; wavelength: 254 nm Though not further characterized, two isomers, labeled Isomer 1 Oven Temp.: 30 ºC and Isomer 2, eluted at 2.8 min and 4.5 min. These were 8,9 Injection Volume: 1 µL associated with avobenzone and homosalate, respectively. Sampling (Data) Rate: 10 pts/sec As shown in Figure 2, chromatographic repeatability was Pressure (max.): ~4500 psi confirmed via ten injections of the sunscreen lotion. For the paraben and sunscreen-active ingredients present, retention time repeatability ranged between 0.13 and 0.33 % RSD.

2 Overlay Report

0.65

0.60

0.55 Oxybenzone

0.50

0.45

0.40

0.35 Octocyclene Avobenzone Homosalate AU 0.30 Ethylparaben Isomer 1 0.25 Octisalate Propylparaben Methylparaben Isomer 2 0.20

0.15 Octinoxate

0.10

0.05

0.00

1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes octinoxate; Sample Set Id 3858; Injection Id 3870; Injection Volume 1.00 Figure 1. UHPLC chromatogram of a standard mix of three oxybenzone; parabens Sample and six sunscreen-activeSet Id 3858; Injection ingredients. Id 3906; IsomerInjection 1 Volumeand Isomer 1.00 2 are associated with avobenzone and homosalate, respectively. methyl std; Sample Set Id 5342; Injection Id 5406; Injection Volume 1.00Overlay Report one plot ethyl std; Sample Set Id 5342; Injection Id 5411; Injection Volume 1.00 propyl std; Sample Set Id 5342; Injection Id 5423; Injection Volume 1.00 octocrylene; Sample Set Id 3858; Injection Id 3885; Injection Volume 1.00 0.40 Octisalate3ul; Sample Set Id 6246; Injection Id 6248; Injection Volume 3.00 Homosalate high; Sample Set Id 4374; Injection Id 4469; Injection Volume 3.00 0.38 Avobenzone5ul; Sample Set Id 6203; Injection Id 6226; Injection Volume 5.00 0.36

0.34

0.32

0.30 Reported by User: System Project Name: Sunscreen 0.28Report Method: Overlay Report Date Printed: Report Method ID: 7112 7112 4/1/2015 0.26Page: 1 of 1 2:24:07 PM US/Eastern

0.24

0.22

0.20 AU

0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes Figure 2. Chromatographic repeatability of sunscreen selectivity; lotion; Channel wavelength: Description 254 nm. PDA Ch1 [email protected]; Injection Id 3972; Sample Set Id 3961 selectivity; Channel Description PDA Ch1 [email protected]; Injection Id 3986; Sample Set Id 3961 selectivity; Channel Description PDA Ch1 [email protected]; Injection Id 4028; Sample Set Id 3961 selectivity; Channel Description PDA Ch1 [email protected]; Injection Id 4014; Sample Set Id 3961 selectivity; Channel Description PDA Ch1 [email protected]; Injection Id 4000; Sample Set Id 3961

Reported by User: System Project Name: Sunscreen Report Method: Overlay Report one plot Date Printed: Report Method ID: 5107 5107 3/9/2015 Page: 1 of 1 1:09:14 PM US/Eastern

3 CLCal Report IndivPUBLICATION

Processing Method: Suns_Ethyllin_PM Project Name: Sunscreen Processing Method ID: 4716 System: NewAcqui t y Channel: PDA Ch1 [email protected] Calibration ID: 4768 Proc. Chnl. Descr.: PDA Ch1 [email protected] Date Calibrated: 3/24/2015 10:18:07 AM EDT

Cal Curve Id 4769

Using ethylparaben and oxybenzone as representative examples, Figures 3 and 4 show the respective linearity 160000.0 Ethylparaben 140000.0 results. The concentration ranges were 0.5-5 µg/mL and 254 nm 0.055-0.55 mg/mL, respectively; the concentration range 120000.0 reflecting the levels most commonly found in U.S.-marketed 100000.0 products for each component.10,11 80000.0 Area 60000.0 Table 2 lists the LOQ and LOD levels that were obtained for four typical paraben and sunscreen components. These levels were 40000.0 20000.0 based upon the indicated minimum signal-to-noise (s/n) limits. CLCal Report IndivPUBLICATION 0.0 R² = 0.99994 Table 2. Calculated LOQ and LOD levels for ethylparaben, propylparaben, oxybenzone -20000.0 and octinoxate at 254 nm. Processing Method: Sun_oxybenzone_PM Project Name: Sunscreen Processing Method0.00 ID: 42071.00 2.00 3.00 System: 4.00 NewAcqui t y5.00 LOQ (µg/mL (ppm)) LOD (µg/mL (ppm)) Channel: PDA Ch1 [email protected] ug/ml Calibration ID: 4264 Component Proc. Chnl. Descr.: **** Name Ethylparaben; ; R^2 0.999943 (s/n ≥ 10/1) (s/n ≥ 3/1) FigureDate Calibrated:3. Linearity plot of3/4/2015 ethylparaben; 11:14:52 AM EST concentration range: 0.5-5 µg/mL; Injection Id 4403 wavelength:Cal Curve Id 4265 254 nm. Ethylparaben 0.009 0.003 Sample Set Id 4374 Propylparaben 0.012 0.004 600000 Oxybenzone 0.402 0.121 Oxybenzone Octinoxate 0.303 0.091 500000 254 nm

400000 A comparison of lip balms formulated with and without sunscreen is shown in Figure 5. Methylparaben and propylparaben were 300000 Area present in both formulations, while, as expected, oxybenzone 200000 and octinoxate were only present in the lip balm formulated with sunscreen. 100000

0 R² = 0.99891

-100000 0.00 0.10 0.20 0.30 0.40 0.50 mg / ml Name Oxybenzone; ug; R^2 0.998914 Figure 4. Linearity plot of oxybenzone; range: 0.055-0.55 mg/mL; wavelength: 254 nm. Injection Id **** Overlay Report

Sample Set Id ****

0.38

0.36

0.34 Oxybenzone 0.32

0.30

0.28

0.26

0.24

0.22

0.20

0.18 AU

0.16

0.14 Octinoxate

0.12

0.10

0.08 Methylparaben

0.06 Propylparaben

0.04

0.02

0.00

-0.02

1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes Figure 5. Overlay of a lip balm formulated blank; with Sample sunscreen Set (red), Id 3915; lip Resultbalm formulated Id 5042; Injection without Id sunscreen 3937; Channel (blue), Description and subsequent PDA Ch1 blank [email protected] injection (black). chap; Sample Set Id 3915; Result Id 5057; Injection Id 3952; Channel Description PDA Ch1 [email protected] chap+sun; Sample Set Id 3915; Result Id 5043; Injection Id 3942; Channel Description PDA Ch1 [email protected]

Reported by User: System Project Name: Sunscreen Report Method: Overlay Report Date Printed: Report Method ID: 6079 6079 3/19/2015 Page: 1 of 1 11:10:57 AM US/Eastern Figure 6 shows an overlay of moisturizing lotion formulated with and without sunscreen. Methylparaben, ethylparaben, and propylparaben were observed in both lotions, while, as expected, oxybenzone and octinoxate were present only in the skin moisturizer formulated with sunscreen. Overlay Report

0.80

0.75

0.70

0.65 Oxybenzone

0.60

0.55

0.50

0.45

0.40

AU 0.35 Octinoxate

0.30 Methylparaben

0.25

0.20 Ethylparaben Propylparaben

0.15

0.10

0.05

0.00

-0.05

-0.10 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes blank; Sample Set Id 3915; Injection Id 3937; Channel Description PDA Ch1 [email protected]; Result Id 5042 Figure 6. Overlay of skin moisturizer formulated lotion; Sample with sunscreenSet Id 3915; (red), Injection skin Idmoisturizer 3932; Channel formulated Description without PDA sunscreen Ch1 [email protected]; (blue) and Result subsequent Id 5101 blank injection (black). lotion+sun; Sample Set Id 3915; Injection Id 3922; Channel Description PDA Ch1 [email protected]; Result Id 5100 As shown in Figure 7, the sunscreen lotion was found to contain three parabens and five sunscreen-active ingredients. As shown overlaid in Figures 5-7, a subsequent water blank injection after each sample injection confirmed that there was no noticeable sample carryover in each case. Overlay Report Reported by User: System Project Name: Sunscreen Report Method: Overlay Report Date Printed: Report1.90 Method ID: 5102 5102 3/6/2015 Page: 1 of 1 3:42:57 PM US/Eastern 1.80

1.70 Oxybenzone 1.60

1.50

1.40

1.30

1.20

1.10

1.00

AU 0.90

0.80

0.70

0.60

0.50 Methylparaben 0.40 Octinoxate

0.30 Propylparaben Ethylparaben Octocyclene Homosalate

0.20 Isomer 1 Octisalate Unknown 1 0.10 Isomer 2

0.00

-0.10 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes Figure 7. Chromatographic overlay of a sunscreenblank; Sample lotion Set (blue) Id 3915; and Result the subsequent Id 5579; Injection blank injectionId 3937; Channel (black). Description PDA Ch1 [email protected] sunscreen; Sample Set Id 3858; Result Id 6298; Injection Id 3896; Channel Description PDA Ch1 [email protected] 5

Reported by User: System Project Name: Sunscreen Report Method: Overlay Report Date Printed: Report Method ID: 6315 6315 3/23/2015 Page: 1 of 1 4:57:44 PM US/Eastern In Table 3, the calculated amount for each analyzed component Conclusion and the recovery of sunscreen ingredients in each of the three This work demonstrated the fast, effective chromatographic formulations are listed. It should be noted that octinoxate, separation of three parabens and six sunscreen ingredients using a oxybenzone, avobenzone and homosalate are unstable and PerkinElmer UHPLC system with PDA detector. The results exhibited decompose over time. Therefore, depending upon the shelf-life exceptional linearity for the representative paraben and sunscreen of the product, recoveries of these ingredients may not always active ingredients over the tested concentration ranges. The results be as per the label claim. Also, although the recoveries for also demonstrated more than ample sensitivity for verifying the label octocrylene and oxybenzone were higher than label claim, these claim of all the analyzed components having published claim values. two components are primarily added to help preserve the photo and chemical integrity of the more unstable active ingredients.12,13 References

Table 3. Calculated amounts and recoveries of parabens and sunscreen-active ingredients 1. Hayden CG, et al. “Systemic absorption of sunscreen after in moisturizer, lip balm and sunscreen lotion, compared to label claim. topical application”. Lancet, 350 (9081):863-4, 1997. Note: for components having a label claim, the amount (%) was entered so as to match the significant figures provided in the label claim. 2. Hanson, KM, et al. “Sunscreen enhancement of UV-induced reactive oxygen species in the skin”. Free Radical Biology & Moisturizer + Label Claim (%) Amount (%); n=2 Recovery (%) Sunscreen Medicine. 1205–1212, 2006. Methylparaben Not Provided 0.17 - 3. Knowland, John, et al. “Sunlight-induced mutagenicity of Ethylparaben Not Provided 0.09 - a common sunscreen ingredient”, FEBS Letters, 324(3): 309-313, 1993. Propylparaben Not Provided 0.10 - Oxybenzone 2 2 100 4. Krause M, et al. “Sunscreens: are they beneficial for health? An overview of endocrine disrupting properties of UV-filters”. Octinoxate 6 6 100 2012 International Journal of Andrology. 35(3):424-36, 2012. Lip Balm + Label Claim (%) Amount (%); n=2 Recovery (%) 5. Byford JR, et al. “Oestrogenic activity of parabens in MCF7 Sunscreen human breast cancer cells”. Journal of Steroid Biochemistry & Methylparaben Not Provided 0.07 - Molecular Biology, 80:49-60, 2002. Propylparaben Not Provided 0.06 - 6. US Food and Drug Administration, Cosmetic Products and Oxybenzone 3.5 3.1 86 Ingredients, “Parabens”, 2015. Octinoxate 7.5 6.1 81 7. Sayre RM, et al. “Unexpected photolysis of the sunscreen octinoxate in the presence of the sunscreen avobenzone”. Sunscreen Label Claim (%) Amount (%); n=2 Recovery (%) Lotion Journal of Photochemical Photobiology, 81(2):452-6, 2005. Methylparaben Not Provided 0.32 - 8. Jan Zawadiak, et al., “UV absorption and keto–enol tautomerism Ethylparaben Not Provided 0.19 - equilibrium of methoxy and dimethoxy 1,3-diphenylpropane-1, 3-diones”, Spectrochimica Acta Part A, 75 925–929, 2010. Propylparaben Not Provided 0.26 - Oxybenzone 6 8 133 9. Chisvert, A et al. “Determination of the UV filters worldwide authorized in sunscreens by high-performance liquid Octocrylene 2.8 5.0 179 chromatography - Use of cyclodextrins as mobile phase modifier”, Avobenzone 3 3 100 Journal of Chromatography A; 921(2):207-15, 2001. Octisalate 5 5 100 10. US Food and Drug Administration, “ Cosmetics: Parabens”. 2015. Homosalate 10 10 100 11. US Food and Drug Administration, CFR - Code of Federal Regulations Title 21, Part 352.10. 2015. 12. The Dermatology Review, “Octocrylene”, 2014. 13. NIH US National Library of Medicine, Toxicology Data Network, “2-Hydroxy-4-Methoxybenzophenone”, CASRN: 131-57-7, 2015

PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com

For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs

137331 PKI