Sunscreen preparations are use for protection of epidermis from ultraviolet rays of sunlight, which cause skin allergy, skin aging, skin burn and skin cancer. Bemotrizinol is added to the different sunscreen preparations for absorbing the harmful sun rays like UVB (290 nm-320nm) as well as UVA (320nm-400nm) rays. The stability of Bemotrizinol is very important in different stress condition to obtain the maximum skin protecting effect from sun rays. The stress condition like photolytic dry and wet exposure, thermal exposure, oxidation, acid hydrolysis and base hydrolysis of Bemotrizinol was study with photo diode array detector and a cost effective high performance liquid chromatographic method. The chromatographic condition like mobile phase methanol 100 percent, flow rate 2.5 ml per minutes, column octadecylsilane C-18 (30 x 0.39 cm, 10 micron) and detection was done at 254 nm. The photolytic and thermal stability of Bemotrizinol ware conformed by the dry, wet and thermal chromatogram compare with standard chromatogram, where no such degradation peak was found. Oxidative, acid and base decomposition ware found by gating the degradation peak in the chromatogram compare with standard chromatogram. The developed analytical method of Bemotrizinol obeys linearity 70 – 130 microgram per ml with correlation coefficient 0.996 and statistically validated for accuracy. The excellent recovery was 100.01% to 105.04% and RSD of precision was 0.9576%. The simple economic specific linear method is use to determine the Bemotrizinol as well as identify the degradation of the sunscreen preparations.
Research Article
Determination of Bemotrizinol degradation in different stress condition by high performance liquid chromatography in sunscreen preparations
https://doi.org/10.21203/rs.3.rs-2629843/v1
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Photolytic stability
Bemotrizinol
Forced degradation
HPLC
Sunscreen
The fat soluble Bemotrizinol has broad spectrum affect which increase the efficiency of sunscreen to protect against harmful UV sun rays. The chemical name of Bemotrizinol is 2,2′-[6-(4-Methoxyphenyl)-1,3,5-triazine-2,4-diyl]bis{5-[(2-ethylhexyl)oxy]phenol}(Fig. 1) and absorb at 310 and 340 nm [1–2]. Bemotrizinol is yellow crystals and having melting point 83–85°.
In the United States Bemotrizinol is not permitted in sunscreen preparation but Australia, European union and some other world ware permitted to use in topical preparation [3–8]. The degradation of Bemotrizinol during stability study is very important to obtain the maximum skin protection from sun rays. It is investigated by applying the different stress condition like photolytic dry and wet exposure, thermal exposure, oxidation, acid hydrolysis and base hydrolysis of Bemotrizinol. However, no studies were found for determination of its degradation.
In this experiment we developed a simple, economic, linear method for determination of Bemotrizinol and successfully identify the degradation in sunscreen preparations.
Instrument
HPLC, Shimadzu model no.SPD-M10AVP, LC solution software, binary pump (model no.LC-10ATVP and LC20AD), column cabinet (model no.CTO-10-AS-VP), autosampler (model no.SIL-20-A-HT) and PDA detector.
Chromatographic condition
The isocratic elution of Bemotrizinol was done with mobile phase methanol 100 percent, flow rate 2.5 ml per minutes, column octadecylsilane C-18 (30 x 0.39 cm, 10 micron), at 27°C, application volume 20 µ1 and measurement was done at 254 nanometer. Methanol, chloroform ware used in standard and sample preparation. The chromatograms are shown in Fig. 2 and Fig. 3.
Procedure
Standard preparation: Standard Bemotrizinol 130 microgram / ml was prepared in 100 ml volumetric flask containing 10 ml chloroform. Sonicated for 20 minutes and made up to volume with methanol.
Sample preparation: Accurately weight sample equivalent to 25 mg of Bemotrizinol in 50 ml volumetric flask containing 10 ml chloroform. Sonicated for 20 minutes and volume make up with methanol. Pipette 5 ml and dilute to 20 ml with methanol.
The brand was assay by differentiate between the response of sample and standard. The result was found 99.23% (Photoderm MAX SPF50+) Table 1. The assay result indicates that the method is very selective to determine the Bemotrizinol from sunscreen formulations without any interference of pharmaceutical base.
Table 1: Assay of Bemotrizinol in sunscreen preparations
|
Brand |
Assay % (w/w) |
|
Photoderm MAX SPF50+ (Bioderma) |
99.23 |
Method validation
Chromatographic suitability
This parameter was used to verify the chromatographic method and performance of column and which ware acceptable for deliberate application. The %RSD area was less than 2.0 % in the developed analytical method of Bemotrizinol. The retention time, theoretical plates and tailing factor ware presented in Table 2.
Table 2: Chromatographic Parameters
|
Parameters |
Bemotrizinol |
|
Linear dynamic range |
70-130 microgram /ml |
|
R2 value |
0.996 |
|
Retention time |
17.599 |
|
Theoretical plates |
4680.915 |
|
Tailing Factor |
0.958 |
|
LOD |
0.12(µg/mL) |
|
LOQ |
0.38(µg/mL) |
|
Inter day |
100.125% |
|
Intra day |
100.248% |
|
Specificity |
Specific |
|
Recovery |
100.01% to 105.04% |
|
RSD% |
0.33 |
Linearity
The linearity concentration of Bemotrizinol was look over the range 70 – 130 microgram /ml. In the calibration, graph was established by comparison of concentration versus peak response and least squares linear regression equation. In the calibration curve, y-intercept and slope of was recorded. The regression equation for the calibration curve was graphically formatted in a mathematical equitation Y= 7715X-15320. R2=0.996. The correlation coefficient (R2) value was graphically shown in Fig. 4.
LOD and LOQ
The LOD and LOQ were computed by of the response (σ) standard deviation and slope of the linear regression graph of Bemotrizinol (S) as express in ICH guideline Q2(R1). Based on the response (σ) standard deviation and slope, LOD was determining by using equation 3.3(σ)/S and LOQ 10(σ)/S. The values of LOQ, LOD ware 0.38 microgram /ml and 0.12 microgram /ml respectively Table 3.
Table 3 Limit of detection (LOD) and limit of quantification (LOQ)
Accuracy
The recovery was work out with this accuracy study of the Bemotrizinol in sunscreen matrix. In this study, known content of analyte was spiking to the pre analyze sample and determination carried out with the newly developed assay method at 80%, 100% and 120% concentration stage Table 4.
Table 4 Recovery study
|
% Target |
% Recovery |
|
80% |
100.52 |
|
100% |
105.04 |
|
120% |
100.01 |
Selectivity / specificity
In the analytical method validation, specificity of the newly originated assay method was checked by blank interference. The diluents, standard and sample were arranging as in the standard operative protocol and injected. Figure 5. The chromatogram of standard and sample were indicating in Fig. 2 and Fig. 3.
Forced degradation
The forced degradation of Bemotrizinol was illustrated through examination of the sample on oxidative, acid, alkaline, and photolytic decomposition. The sample was treated with this environment and the principal peak was studied, thus specify that this procedure successfully differentiate the peaks of degraded substance from the principal ingredient Fig. 6 and it was also confirmed by the assay results Table 5.
Table 5 Assay results in different stress conditions
|
Test |
Assay % (w/w) |
|
Acid Degradation |
41.31 |
|
Alkaline Degradation |
40.44 |
|
Oxidative degradation Photolysis in dry stage Photolysis in wet stage Thermal degradation |
40.38 98.66 98.26 98.60 |
Acid Degradation
About 25 mg of Bemotrizinol was accurately weighted and reflex with 1 ml 5N HCl at 70° for five hours. Then transferred to 50ml volumetric flask containing 10 ml chloroform and volume make up with methanol. Pipette 5 ml and dilute to 20 ml with methanol. Inject the solution into the HPLC system to get the chromatograms Fig. 7.
Alkaline Degradation
About 25 mg of Bemotrizinol was accurately weighted and reflex with 1 ml 5N NaOH at 70° for five hours. Then transferred to 50ml volumetric flask containing 10 ml chloroform and volume make up with methanol. Pipette 5 ml and dilute to 20 ml with methanol. Inject the solution into the HPLC system to get the chromatograms Fig. 8.
Oxidative degradation
About 25 mg of Bemotrizinol was accurately weighted and reflex with 1 ml 30% H2O2 at 70° for two hours. Then transferred to 50ml volumetric flask containing 10 ml chloroform and volume make up with methanol. Pipette 5 ml and dilute to 20 ml with methanol. Inject the solution into the HPLC system to get the chromatograms Fig. 9.
Photolysis
About 25 mg of Bemotrizinol was accurately weighted and kept in a UV cabinet in dry and wet stage at 254nm for twenty hours separately. Then transferred to 50ml volumetric flask containing 10 ml chloroform and volume make up with methanol. Pipette 5 ml and dilute to 20 ml with methanol. Inject the solution into the HPLC system to get the chromatograms Fig. 10 and Fig. 11.
Thermal degradation
About 25 mg of Bemotrizinol was accurately weighted and kept in an oven 105° for six hours. Then transferred to 50ml volumetric flask containing 10 ml chloroform and volume make up with methanol. Pipette 5 ml and dilute to 20 ml with methanol. Inject the solution into the HPLC system to get the chromatograms Fig. 12.
A reversed-phase analytical technique was succeeding to estimate Bemotrizinol at 254nm with an octadecylsilane C-18 (30 x 0.39 cm, 10 micron) column. Methanol (100%) was using as mobile phase for symmetrical peak in this HPLC (high performance liquid chromatographic) with retention time 17.599. The flow was extending from 2.0 ml/minute to2.5 ml/ minute. Total run time was increased at flow rate 2.0 ml/min. This problem was solved at flow 2.5 ml/minute. It was our target to use methanol as mobile phase to increase the life time of both the column and the chromatographic pump and it also reduced the total cost of the analysis. In this chromatographic condition retention time was 30 minutes and this recognizes that multiple analyses to be analyzed with this reduced time. The trade formulation is inspected and the recovery percentage values of Bemotrizinol from the prior examine production batch was in between 100.01–105.04% (Table 2). It was observed that the linearity of Bemotrizinol obeys concentration range within 70–130 µg/ml and a linear line was going through the origin (Fig. 4). The LOQ was obtained 0.38µg/ml and the LOD was obtained 0.12µg/ml. In the robustness, the developed assay method was confirmed by introducing little changes in the chromatographic conditions which cover column temperature (25 and 29°), wavelength (252 and 256 nm) and flow (2.25 and 2.75 ml). The RSD% of assay for the corresponding sample under primary and robustness procedure was not more than 2.0%, which indicate that the analytical procedure was robust, cost effective, precise, simple and accurate for Bemotrizinol quantification in pure and its sunscreen products and also report that the frequently used additives and excipients in the cosmeceuticals formulation was not hamper in this recommended method.
Degradation of Bemotrizinol was not notice during stress study like thermal and photolytic except in base hydrolysis, acid hydrolysis and oxidation (Table 5). Bemotrizinol was decomposed in acidic hydrolysis and forms degradation peaks at 2.773min and 14.907min (Fig. 7). During basic hydrolysis we found degradation peaks at 14.720min (Fig. 8) and oxidation the degradation peak was at 14.793min (Fig. 9). The assay of Bemotrizinol is unaffected in sunscreen preparation authenticate its stability indicative power of the method.
This RP-HPLC method was perfect for verification of Bemotrizinol in pharmaceutical cosmetic formulations. The method is very sensitive, simple, precise, specific, accurate and stability indicating. Hence, this method can be use for routine assay and stability analysis of Bemotrizinol in sunscreen formulations.
ACKNOWLEDGMENTS
The authors are grateful to Palsons Derma Pvt. Ltd. kolkata for supplying gift sample of pure Bemotrizinol. All above authors are also thankful to School of Pharmacy, Techno India University, West Bengal for providing research support to carry out this experiment successfully and also grateful to scholars and my friends for their continues support from time to time at each and every step of this method development work.
The authors have no relevant financial or non-financial interests to disclose.
Competing Interests
The authors have no competing interests to declare that are relevant to the content of this article.
- Vielhaber G, Grether-Beck S, Koch O, Johncock W, krutmann J, (2006), Photo. Photobio. Sci. 5, 275-282. https://link.springer.com/article/10.1039/b400806e
- Chisvert A, Pascaul-Marti M. C, and Salvador A, (2001), Jour. of Chrom. A. 921, 207–215. https://doi.org/10.1016/S0021-9673(01)00866-4
- He,Q.S.Xu, Liao N, (2011), Chin.J.Chromatogr.29,762-767. https://www.chrom- china.com/EN/10.3724/SP.J.1123.2011.00762
- FDA, Department of Health and Human Services, 21CFR Parts 310, 352, 700 and 740, RIN 0910-AA01, (1999), Sunscreen Drug Products for over-the-counter Human Use, Final Monograph, Federal Register, Rules and Regulations, 64, 27666.
- Hygienic Standard for Cosmetics. Ministry of Health, People’s Republic of China. Beijing, 2007. http://www. moh.gov.cn/open/web_edit_file/20070124145740.pdf ,accessed January 5, 2015.
- Couteau C, Faure A, Fortin J, Paparis E, and Coiffard L. J. M, Jour. of Pharma. and Bio.(2007), Anal. 44, 270–273. https://doi.org/10.1016/j.jpba.2007.01.052
- Ikeda K, Suzuki S, and Watanabe Y, (1990), Jour. of Chromato. A. 513, 321–326. https://doi.org/10.1016/S0021-9673(01)89449-8
- Simeoni S, Tursilli R, Bianchi A, and Scalia S, (2005) Jour. of Pharma. and Biom. Anal. 38,250–255. https://doi.org/10.1016/j.jpba.2004.12.021
No competing interests reported.
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