Simultaneous determination of two UV filters (octylmethoxycinnamate, octylsalicylate) in sunscreen creams by high performance thin layer chromatography (HPTLC)

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Octylmethoxycinnamate (OMC), octylsalicylate (OS) are UV filters commonly used in sunscreen creams for skin protection against UV radiations. In this study, an HPTLC method was developed and validated for simultaneous determination of OMC and OS in sunscreen creams.

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Nội dung Text: Simultaneous determination of two UV filters (octylmethoxycinnamate, octylsalicylate) in sunscreen creams by high performance thin layer chromatography (HPTLC)

Journal of Pharmaceutical Research and Drug Information 2025; 00(00); 000–000 Journal homepage: jprdi.vn/JP Journal of Pharmaceutical Research and Drug Information An official journal of Hanoi University of Pharmacy Research article Simultaneous determination of two UV filters (octylmethoxycinnamate, octylsalicylate) in sunscreen creams by high performance thin layer chromatography (HPTLC) Minh Thuy Ngoa, Thi Bich Dao Danga, Dinh Chi Leb, Thi Thanh Vuong Tonga,* a Department of Analytical Chemistry and Drug Quality Control, Hanoi University of Pharmacy, 13-15 Le Thanh Tong Street, Hoan Kiem District, Hanoi, Vietnam b National Institute of Pharmaceutical Technology, Hanoi University of Pharmacy, 13-15 Le Thanh Tong Street, Hoan Kiem District, Hanoi, Vietnam. * Corresponding author: Thi Thanh Vuong Tong - vuongttt@hup.edu.vn Article history: Received 05 December 2024 Resived 10 January 2025 Accepted 17 January 2025 ABSTRACT Octylmethoxycinnamate (OMC), octylsalicylate (OS) are UV filters commonly used in sunscreen creams for skin protection against UV radiations. In this study, an HPTLC method was developed and validated for simultaneous determination of OMC and OS in sunscreen creams. The method was carried out using an silica gel 60 F254 thin layer plate (20 cm × 10 cm) with a mixture of cyclohexane - aceton (10:1, v/v) as mobile phase. Samples were applied on thin layer plate as 4-mm bands with applied volume at 7.0 µL per band and the distance between two consecutive bands was 9 mm. The chromatograms were developed over a distance of 80mm, then scanned and recorded at 307 nm for peak detection and calculation of peak area. The method was fully validated according to requirements on analytical method performance of AOAC International. The linearity ranges of the method were from 17.9 to 32.2 µg/mL for OMC and from 16.3 to 29.4 µg/mL for OS, respectively, and its accuracy (recovery rate from 97.4% to 102.2% for _________________________________________ *Correspondence: vuongttt@hup.edu.vn; 1 http://doi.org/10.59882/1859-364X/264
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000 OMC and from 98.0% to 102.4% for OS) and its precision (RSD ≤ 2.6% for OMC and ≤ 2.4 % for OS) were sufficient for simultaneous determination of these two UV filters. Therefore, validation results confirmed its reliability and suitability for intended application. The method was employed to determine the content of OMC and OS in commercial sunscreen creams. Keywords: HPTLC, UV filter, sunscreen cream, octyl methoxycinnamate, octyl salicylate INTRODUCTION Exposure to ultraviolet (UV) radiation is harmful for human health. To minimize direct exposure of human skin to UV radiation, many sunscreen products in different form (creams, emulsions, etc.) are available on the market. The protective effects against UV radiation of these products comes from the UV filters that they contain. UV filters are chemical compounds capable of neutralizing harmful assaults of UV radiations on skin surface by scattering them (physical mechanism) or by absorbing them (chemical mechanism) [1]. Octylmethoxycinnamate (OMC), also known as octinoxate or ethylhexylmethoxycinnamate, and octylsalicylate (OS), also known as octisalate or ethylhexyl salicylate, are chemical UV filters with proven capacity in absorbing UV radiation [2]. According to Annex VII of ASEAN Cosmetic Directive, both OMC and OS are UV filters allowed to be use in cosmetic such as sunscreen creams with highest accepted limits at 10% (w/w) and 5% (w/w), respectively [3,4]. These limits are also currently imposed by Vietnamese authorities. To control the quality of these UV filters in pure material, OMC and OS were quantified by gas chromatography using dimethylpolysiloxane column and flame ionization detector [5, 6]. OMC was determined in sunscreen products with UV-Vis absorption spectroscopy [7] and with reversed-phase HPLC on C18 column [8, 9]. OS and OMC were quantified simultaneously in sunscreen emulsion using HPLC [10]. LC-MS/MS was employed to determine OS in human placeta tissue [11]. High performance thin layer chromatography (HPTLC) was applied to analyze OMC [12] and to quantify OS in sunscreen products [13]. Up to now, some authors have expressed concerns or reported cases on harmful effects of OMC [14] and OS [15]. Therefore, to assure the protective effects of sunscreen products, they must contain the right kind and amount of UV filters and the content of UV filters in sunscreen products must not exceed authorized limit to avoid harmful effects that may occur due to abusive use of UV filters. These practical requirements demand reliable and available analytical tool for monitoring UV filters’ content in commercial cosmetic products on market. In this study, an HPTLC method was developed for simultaneous determination of OMC and OS in sunscreen creams. The method was fully validated according to current guidance of AOAC International [16] to assure its suitability and reliability for its intended purpose. MATERIALS AND METHODS Reference standards, reagents and samples Reference standards of OMC (purity: 97.9% w/w, batch No.: 20672909TO) and OS (purity: 93.9% w/w, batch No: OS#1070220) were kindly provided by National Institute of
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000 Drug Quality Control (Hanoi, Vietnam). Cyclohexane, aceton, diethyl ether, methanol PA grade were purchased from Merck Vietnam (Ho Chi Minh City, Vietnam). Representative placebo matrix was a commercial cream which did not contain OMC and OS and was used as such after preliminary analysis confirmed that it did not contain OMC and OS. The composition of the representative placebo consisted of water, zinc oxide, caprylic/capric triglyceride, propylene glycol, cetyl alcohol, titanium dioxide, dimethicone, ethylhexylglycerin, magnesium aluminum silicate, xanthan gum, disodium EDTA. Samples were four commercial sunscreen creams containing at least one of the analytes (according to label claim) purchased on market. Their label provided only the name of components without any quantitative information about the content of each component in product. Standard solutions Stock standard solutions of OMC and OS were prepared separately using methanol as solvent at concentration of 1000 µg/mL. The mixed standard solutions for routine analysis and method validation were prepared by diluting stock standard solutions with the same solvent to targeted concentrations. Sample preparation About 0.25 g of sample was accurately weighed by using a weighing boat and transferred into a 100-mL stoppered flask and dispersed with 30 mL of methanol by sonicating in 30 minutes at 55-60oC to dissolve analytes. The stoppered flask was then cooled down to room temperature and the content was transferred in to a 50-mL volumetric flask and made to volume with the same solvent and mixed. The obtained liquid was left 15 minutes at 4oC, the liquid was then transferred into a centrifuging tube and centrifuged at 10000 rpm in 10 minutes at same temperature. The supernatant was applied on thin layer plate. Apparatus and Chromatographic conditions Chromatographic separation was done on silica gel 60 F254 thin layer plates purchased from Merck Vietnam (Ho Chi Minh City, Vietnam) and activated at 115oC in 15 minutes before use. HPTLC system Camag CAT No. 027.6200 of Camag (Muttenz, Switzerland) consisted of semi-automatic sample application device Linomat 5, ADC2 developing chamber, TLC Scanner 4 device for thin layer plate scanning, and UV cabinet with UV sources at 254 nm and 366 nm for observing thin layer plate. The system was controlled by WinCAT software of Camag (Muttenz, Switzerland) for chromatographic process and data collection. The composition of mobile phase, the wavelength for scanning and recording chromatograms were investigated during method development step to obtain suitable conditions (the results were provided in Method development section). For final method, the mobile phase was a mixture of cyclohexane and aceton (10:1, v/v). The volume of mobile phase put into developing chamber was 10 mL. The relative humidity inside developing chamber was maintained at 46-48% at room temperature during developing procedure. The developing chamber was saturated with vapor of mobile phase for 20 minutes before placing thin layer plate into developing chamber. Standard and sample solutions were applied on plate in 4 mm-long bands. The distance between two adjacent bands was 9 mm. The volume of sample applied on each band was 7.0 µL. Developing distance of mobile phase was 80 mm. When the developing procedure finished, the developed plate was taken out of developing chamber and left inside Hood cabinet for
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000 drying. The chromatogram of each band was scanned and recorded at 307 nm. Method validation For linearity validation, mixed standard solutions of OMC and OS were prepared at 5 different concentration levels for each analyte (from 17.9 to 32.2 µg/mL for OMC; from 16.3 to 29.4 µg/mL for OS). For accuracy validation in spiked samples, exact quantities of OMC and OS were added into representative placebo. The spiked samples were prepared in triplicate at each spiking level and at 3 different spiking levels for each analyte (final concentrations for applying on plate were about 20 µg/mL, 25 µg/mL, and 30 µg/mL for OMC; 18 µg/mL, 23 µg/mL and 28 µg/mL for OS) and processed as with sample preparations. The accuracy of method was assessed by recovery rates between spiked quantities and detected ones of analytes in spiked samples. The recovery rates and the RSD (%) of quantitative results at each spiking level for each analyte must lie within ones recommended by AOAC International [16]. For precision validation on real samples, 6 sample preparations of same commercial product were analyzed in the same day and by same analyst for repeatability assessment, and 12 sample preparations of same commercial product were analyzed by two analysts and in two different days for intermediate precision assessment. The repeatability and intermediate precision were assessed by relative standard deviation (RSD) calculated from quantitative results of each analytes in sample preparations. The values of RSD for each analyte must not exceeded those recommended by AOAC International [16]. In order to estimate LOQ and LOD of the method, spiked samples were prepared by adding OMC and OS at diferent concentrations into representative placebo and analyzed to calculate peak height of analytes. The signal-to-noise ratios at LOQ and LOD levels were calculated by comparing peak height obtained with each analyte on chromatograms of spiked samples at corresponding concentrations and the peak height obtained on chromatogram of representative placebo at the same Rf value of each analyte. The limit of quantitation (LOQ) of each analyte was the lowest concentration at which the linearity of the method was maintained and yielding the signal to noise ratio on chromatogram not less than 10. The limit of detection (LOD) of each analyte was the lowest concentration giving the signal to noise ratio not less than 3. RESULTS AND DISCUSSIONS Method development To obtain good separation of OMC and OS on thin layer plate, several mobile phase mixtures have been tried to test their suitability during preliminary studies, including: Cyclohexane-aceton (5:1, v/v), cyclohexane-diethylether (5:1, v/v), cyclohexane-aceton (7:1, v/v), cyclohexane-aceton (10:1, v/v), and cyclohexane-diethylether-aceton (15:1:2, v/v/v). The Rf values of OMC and OS after chromatographic separation using these mobile phases were summerized in Figure 1. Representative chromatograms obtained with several mobile phases were provided in Figure 2 (A-C).
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000 1.00 0.90 0.80 0.70 0.60 Rf 0.50 0.40 0.30 0.20 0.10 0.00 OMC OS Mobile phase Figure 1. Rf values of OMC and OS with different mobile phases. (A) (B) (C) Figure 2. Chromatograms obtained in preliminary studies with different mobile phases: (A) Cyclohexane-diethyl ether-aceton (15:1:2); (B) Cyclohexane-aceton (10:1); (C) Cyclohexane-diethyl ether (5:1).
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000 Among these mobile phases, only the mixture cyclohexane-aceton (10:1, v/v) giving suitable Rf values for both OMC (about 0.43) and OS (about 0.64) to assure good chromatographic separation between these analytes and the position of their peaks on chromatogram sufficiently distanced from the frontline of mobile phase to yield good peak shape. In contrast, Rf values of OS were over 0.8, even close to 1.0 (for instance, with the mixture of cyclohexane and diethylether (5:1, v/v), the spot of OS was on the frontline of the mobile phase, as shown in Figure 2, C) with other mobile phases, unsuitable for quantitative application. Therefore, the mixture of cyclohexane and aceton (10:1, v/v) was selected as mobile phase for final method. Figure 3. UV-Vis absorption spectra of OMC and OS. To choose appropriate wavelength for scanning and recording chromatograms, the UV absorption spectra of analytes were analyzed directly on their spots on plate after chromatographic separation (Figure 3). Both analytes had absorption maximum at 307 nm, so this wavelength was selected for scanning chromatograms and calculating peak area of OMC and OS for quantitative purpose. Method validation In terms of specificity, chromatograms of mixed standard solution (Figure 4, A) and of placebo spiked with standards of OMC and OS (Figure 4, C) showed peak of OMC and that of OS at the same value of Rf and these two peaks were completely separated from each other. In contrast, on chromatograms of placebo (Figure 4, B) and methanol (diluent for standard and sample solutions, Figure 4, F) no peak appeared at Rf values corresponding to those of OMC and OS on chromatogram of standard solutions. The UV-Vis spectra scanned from spots corresponding to OMC and OS on chromatograms of mixed standard solution and sample solution had similarity close to 1.000 for all analytes (Figure 5). These results proved that the method was sufficiently specific for simultaneous determination of OMC and OS in sunscreen creams.
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000 OMC OS Figure 4. Typical chromatograms at 307 nm: (A) Mixed standard solution: (B) Representative placebo; (C) Representative placebo spiked with standards of OMC and OS; (D) Standard solution of OS; (E) Standard solution of OMC; (F) Methanol. Figure 5. Comparison of UV-Vis spectra extracted from spots of OMC (A, similarity: 0.99988) and OS (B, similarity: 0.99956) obtained with standard solution and sample Asolution. B The validation results for system suitability, linearity, precision, accuracy of method as well as the determination of LOQ and LOD were summarized in Table 1. In terms of system suitability, the RSD (%) of Rf values and peak areas for both analytes were below 2.0% after six independent analysis on thin layer plate (Table 1), so the chromatographic conditions of final method were suitable for quantitative analysis of OMC
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000 and OS. Table 1. Summary of method validation results. Criteria Parameter OMC OS RSD (%) of Rf values (n = 6) 1.3 0.8 System suitability RSD (%) of peak areas (n = 6) 1.7 1.7 Range of concentrations (µg/mL) 17.9 - 32.2 16.3 - 29.5 Linearity Coefficient of determination (R2) 0.990 0.999 Recovery (%) at low level (n = 3) 97.4 - 102.2 101.5 - 102.4 RSD (%) at low level (n = 3) 2.6 0.4 Recovery (%) at medium level (n = 3) 97.4 - 99.1 98.8 - 101.7 Accuracy RSD (%) at medium level (n = 3) 0.9 1.5 Recovery (%) at high level (n = 3) 97.6 - 101.4 98.0 - 102.0 RSD (%) at high level (n = 3) 2.1 2.0 Requirements for recovery rates (%) according to AOAC 97.0 - 103.0 97.0 - 103.0 International [16] at the concentrations of recovery studies RSD (%) for repeatability (n = 6, same 2.5 2.2 day, same analyst) Precision RSD (%) for intermediate precision (n = 2.6 2.4 12, 2 days, 2 analysts) Requirements of RSD (%) according to AOAC International 2.7 2.7 [16] at concentration of precision studies Concentration (µg/mL) 17.9 16.3 LOQ Content (%, w/w) in cream 0.36 0.32 Concentration (µg/mL) 2.7 2.3 LOD Content (%, w/w) in cream 0.05 0.04 In linearity studies, the relation between the peak area and the concentration of analyte was investigated in concentration range from 17.9 to 32.2 µg/mL with OMC and from 16.3 to 29.4 µg/mL with OS. And validation results proved that the linearity of the method was good for all analytes within the assessed ranges. In precision studies, the RSD values for assessing repeatability and intermediate precision were all lower than limits recommended by AOAC International. In accuracy studies, the recovery rates for all analytes at all spiked levels also lied within the ranges recommended by AOAC International. The LOQs were estimated at 17.9 µg/mL (equivalent to 0.36% (w/w) in cream samples) for OMC and 16.3 µg/mL (equivalent to 0.32% (w/w) in cream samples) for OS, because at this concentration the linearity between applied amount of analyte on plate and peak area was assured and the signal to noise ratio for peak of respective analyte in spiked samples was higher than 10. The LODs were estimated at 2.7 µg/mL (equivalent to 0.05% (w/w) in cream samples) for OMC and 2.3 µg/mL (equivalent to 0.04% (w/w) in cream samples) for OS. These results proved that the method was precise, accurate for simultaneous quantitation of OMC and OS in sunscreen creams. Application on commercial samples The method was applied to determine OMC and OS in 04 different commercial sunscreen creams claiming least one of them on their labels. Among the tested products, only
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000 one claimed both OMC and OS on its label, the other threes claimed only OMC. In all samples, the detected contents of analytes conformed to the acceptable limits imposed by Vietnamese authorities as shown in in Table 2. And the UV filter(s) actually found in all products were in accordance with their respective label claims. Representative chromatograms of sample solutions were provided in Figure 6. Table 2. Summary of results obtained with commercial sunscreen creams. Content of OMC Content of OS Regulatory limits 𝑆 𝑆 Samples (%, average  𝑡(95%,2) × 𝑛, (%, average  𝑡(95%,2) × 𝑛, (%, w/w) √ √ n = 3) n = 3) M1 2.7  0.4 not detected M2 3.6  0.2 3.1  0.2 OMC: 10% M3 6.6  0.8 not detected OS: 5% M4 6.8  0.8 not detected A OMC B OMC OS Figure 6. Chromatograms of M1 product (A) and M2 product (B). CONCLUSION
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000 A simple HPTLC method was developed for simultaneous quantitation of two UV filters (OMC and OS) in sunscreen creams. The method has been fully validated according to requirements of AOAC International [15] and was proved as reliable for intended application. The method has been employed to analyze 04 samples purchased on market. The detected UV filters were in accordance with label claims, and their amounts in all products conformed with regulatory limits imposed by Vietnamese authorities and ASEAN Cosmetic Directive. ACKNOWLEDGMENTS This study was done using reference standards of OMC and OS kindly provided by National Institute of Drug Quality Control. CONFLICTS OF INTEREST None. REFERENCES 1.Woźnica P, Starosta R. Physicochemical properties of selected compounds used as ultraviolet filters in cosmetics. Aesth Cosmetol Med. 2022, 11: 137-145. https://doi.org./10.52336/acm.2022.02. 2. Nitulescu G, Lupuliasa D, Adam-Dima I, Nitulescu G. Ultraviolet Filters for Cosmetic Applications. Cosmetics. 2023, 10: 101. https://doi.org/10.3390/cosmetics10040101. 3.ASEAN Cosmetic Directive, Annex VII – Part 1: List of permitted UV filters which cosmetic products may contain, entry No.12, 2021. 4.ASEAN Cosmetic Directive, Annex VII – Part 1: List of permitted UV filters which cosmetic products may contain, entry No.20, 2021. 5.United Pharmacopoeia, Octinoxate monograph (electronic edition), USP-NF 2024, 2024. 6.United Pharmacopoeia, Octisalate monograph (electronic edition), USP-NF 2024, 2024. 7.Kanlayavattanakul M, Kasikawatana N, Lourith N. Analysis of octyl methoxychinnamate in sunscreen products by a validated UV-spectrophotometric method. J Cosmet Sci. 2016, 67(3): 167-173. 8.Azizoğlu GA, Azizoğlu E, Tanriverdi ST, Özer Ö. A validated HPLC method for simultaneous estimation of Melatonin and Octyl Methoxycinnamate in combined pharmaceutical applications. Marmara Pharm J. 2017, 21 (4): 921 - 930. 9. Martins T, De Oliveira Pinto C, De Oliveira A, Lima F, Velasco M, Rodrigues L, Baby. RP-HPLC simultaneous quantification of rutin, avobenzone, and octyl methoxycinnamate in the presence of hydroxypropyl β-cyclodextrin (HPβCD) and sulfobutyl ether β- cyclodextrin (SBEβCD). Braz J Pharm Sci. 2022, 58: e20284. Doi: https://doi.org/10.1590/s2175-97902022e20284. 10. Dutra E, Kedor-Hackmann E, Santoro M. Validation of a high performance liquid chromatography method for sunscreen determination in cosmetics. Int J Cosmet Sci. 2002, 24: 97-102. 11. Jiménez-Díaz I, Molina-Molina J, Zafra-Gómez A, Ballesteros O, Navalóna A, Real M et al. Simultaneous determination of the UV-filters benzyl salicylate, phenyl salicylate, octyl salicylate, homosalate, 3-(4-methylbenzylidene) camphor and 3-benzylidene camphor in human placental tissue by LC–MS/MS. Assessment of their in vitro endocrine activity. J Chromatogr B. 2013, 936: 80-87. 12. Sobanska A, Brzezinska E, Simultaneous NP. TLC Analysis of the Sunscreens
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000 Diethylamino Hydroxybenzoyl Hexyl Benzoate and Octyl Methoxycinnamate. J Planar Chromatogr. 2011, 24 (3): 227-231. 13.Sobanska A, Pyzowsk J. Simultaneous Multiple-Development HPTLC Quantification of Water- and Oil-Soluble Sunscreens. J Planar Chromatogr. 2012, 25 (4): 344-348. 14. Klammer H, Schlecht C, Wuttke W, Jarry H. Multi-organic risk assessment of estrogenic properties of octyl-methoxycinnamate in vivo A 5-day sub-acute pharmacodynamic study with ovariectomized rats. Toxicology. 2005, 215: 90-96. 15. Singh M, Beck M. Octyl salicylate: a new contact sensitivity. Contact Dermatitis. 2007, 56: 48. 16. AOAC International. Appendix F: Guidelines for standard method performance requirements. AOAC International Official Methods of Analysis. 2016.