RESEARCH ARTICLE Challenges of HPLC Method Development and Validation for the Assay of Bemotrizinol from Complex Matrix in Cosmeceutical Preparation Kallol Jana*, Beduin Mahanti School of Pharmacy, Techno India University, West Bengal, EM 4, Sector-V, Salt Lake, Kolkata-700091, West Bengal, India Received: 27th June, 2020; Revised: 14th July, 2020; Accepted: 06th August, 2020; Available Online: 25th September, 2020 ABSTRACT A simple high performance liquid chromatography (HPLC) method was developed for the assay of bemotrizinol (Tinosorb-S) from the complex pharmaceutical cosmetics matrix. Unlike the existing methods, the proposed mobile phase used in this method is very simple and excluding buffer. The use of buffer reducing column longevity and also a time-consuming process which increases the cost of analysis. To overcome all the referred problems, the present article was developed and validated as per International Council for Harmonization (ICH) guidelines. The reverse-phase chromatography was performed on Shimadzu model no. SPD-M10A VP with LC solution software, µBondapack (3.9 × 300 mm, 10-micron particle size) column with methanol (100%) as mobile phase at a flow rate 2.5 mL per minutes and UV detection at 254 nm. The retention time of bemotrizinol was found in 17.599 minutes, and the linear regression analysis data for the calibration plots showed a good linear relationship in the concentration range 70 to 130 µg/mL. The value of the correlation coefficient, slope, and intercept were 0.996, 7,715, and 15,320, respectively. The limit of quantification (LoQ) and limit of detection (LoD) were found to be 1.32 and 0.44, respectively. The relative standard deviation (RSD) for intra-day sample A 1.0858, sample B 0.8859, and inter-day sample A 0.9921, sample B 0.967 which were found to be lesser than 2%. The developed method was validated with regard to linearity, accuracy, precision, selectivity, and robustness, and the method was found to be simple, cost-effective, precise, accurate, linear, and specific for the successful identification and determination of bemotrizinol in pharmaceutical cosmetic preparation. Keywords: Bemotrizinol (Tinosorb-S), HPLC, Validation. International Journal of Pharmaceutical Quality Assurance (2020); DOI: 10.25258/ijpqa.11.3.2 How to cite this article: Jana K, Mahanti B. Challenges of HPLC method development and validation for the assay of bemotrizinol from complex matrix in cosmeceutical preparation. International Journal of Pharmaceutical Quality Assurance. 2020;11(3):310-316. Source of support: Nil. Conflict of interest: None INTRODUCTION bisoctrizole, ethylhexyl triazone, or iscotrizinol. However, The use of sunscreen products is widely accepted for excessive addition of sunscreen agents for the long term may the prevention of skin cancer due to the ultraviolet (UV) increase the risk of developing a skin allergy to light, as well 3 sunlight and it is also used for primary prevention of as, birth defects. So the types and amounts of sunscreen long-term exposure to strong sunlight which may result agents in cosmetics are strictly regulated. The permitted active in skin burns (i.e., “sunburn”). Bemotrizinol is (Figure 1) ingredients of sunscreen agents in sunscreen cosmetics in the chemically known as bis-ethylhexyloxyphenol methoxyphenyl triazine, molecular formula C38H49N3O5, molecular weight 627.81 g/mol. Bemotrizinol is an oil-soluble broad-spectrum organic compound that is added to sunscreen to absorb UV rays, UVB (290–320), as well as, UVA (320–400) rays. It is highly efficient at low concentration and perfect for long- lasting sun care and face care products that offer a distinctive sensory experience. It has two absorption peaks, 310 and 340 nm.1 It is highly photostable. Even after 50 minimal erythemal doses (MEDs), 98.4% remains intact. It helps prevent the photodegradation of other sunscreen actives, like avobenzone.2 Bemotrizinol has strong synergistic effects on the sun protection factor (SPF) when formulated with Figure 1: Bemotrizinol *Author for Correspondence: [email protected] HPLC method Development and Validation of Bemotrizinol in Cosmeceutical Preparation European Union (EU), the United States (USA), China (CHN), of bemotrizinol cream as an alternative method. This method and Japan (JP) are listed in Table 1.4-7 can be used for the analysis of a large number of samples. Some sunscreen cosmetics are still adding phenyl salicylate The HPLC method was validated in terms of linearity, which may cause potential human health problems that are not precision, accuracy, robustness, LoD, and LoQ according to listed in these regulations.3 Therefore, it is necessary to detect ICH guideline.13-16 multiple sunscreen agents in different sunscreen formulations. AIMS AND OBJECTIVES The application of HPLC has been applied extensively for quantitative determination.8-12 Very few methods are reported To develop a simple, cost-effective, time-effective, and efficient in the literature and for the quantitative determination of HPLC method for the determination of bemotrizinol, a bemotrizinol; buffer is used for mobile phase preparation. sunscreen agent from the complex pharmaceutical cosmetics The reported methods in the literature suffer from one or the matrix in different sunscreen products due to the potential other disadvantage, such as, long run time, poor sensitivity, developing a skin allergy to light and birth defects in humans. column longevity, very narrow linearity range, etc. Since MATERIALS AND METHODS pharmacopeias do not describe a suitable method for the determination of bemotrizinol in pharmaceutical cosmetic The sunscreen cream Sunmate SPF 30+ gel cream from Palsons formulations, in the present work we developed a simple, Derma Pvt. Ltd. and Photoderm MAX SPF50+ from Bioderma, cost-effective, precise, accurate, linear, and specific reverse respectively, used for analysis. Pure drug sample bemotrizinol phase liquid chromatographic method for the determination was kindly supplied as a gift sample by Palsons Derma Table 1: Active ingredients of sunscreen agents in sunscreen cosmetic and their permitted concentrations in different parts of the world Permitted concentration Active ingredients USA USA EU P CHN H JP N JP p-aminobenzoic acid 15% Forbidden 5% Forbidden 2-hydroxy-4-methoxybenzophenone 6% 10% 10% 5% 3,3,5-trimethylcyclohexyl salicylate 15% 10% 10% 10% 2-ethylhexyl-4-methoxycinnamate 7.5% 10% 10% 20% 2-ethylhexyl salicylate 5% 5% 5% 10% 2-hydroxy-4-methoxy-benzophenone-5-sulfonic acid 10% 5% 5% 10% Avobenzone butylmethoxydibenzoylmethane 3% 5% 5% 10% 3-(4-methylbenzylidene) camphor Forbidden 4% 4% Forbidden Octocrylene 10% 10% 10% 10% Menthyl anthranilate 5% Forbidden Forbidden Forbidden Trolamine salicylate 12% Forbidden Forbidden Forbidden Ecamsule 3% 10% 10% Forbidden Titanium dioxide 25% 25% 25% No limit Zinc oxide 25% 25% 25% No limit Tinosorb M Forbidden 10% 10% 10% Tinosorb S Forbidden 10% 10% 3% Neo heliopan AP Forbidden 10% Forbidden Forbidden Mexoryl XL Forbidden 15% 15% Forbidden Benzophenone-9 Forbidden Forbidden Forbidden 10% Uvinul T 150 Forbidden 5% Forbidden 3% Uvinul A plus Forbidden 10% Forbidden 10% Uvasorb HEB Forbidden Forbidden 10% 5% Parsol SLX Forbidden 10% 10% 10% Isopentenyl-4-methoxycinnamate 10% 10% 10% Forbidden Padimate-O 8% Forbidden 8% 10% Phenylbenzimidazole sulfonic acid 4% 8% 8% 3% Cinoxate 3% Forbidden Forbidden Forbidden Dioxybenzone 3% Forbidden Forbidden Forbidden Note: USA (United States of America); EU (European Union); CHN (China); JP (Japan) IJPQA, Volume 11 Issue 3 July 2020 – September 2020 Page 311 HPLC method Development and Validation of Bemotrizinol in Cosmeceutical Preparation Pvt. Ltd. The sample is used without further purification. respectively. The results were shown in Table 2. The assay HPLC grade methanol, chloroform was procured from Merck, result indicates that the method is very selective to determine Qualigen, and Spectrochem, Rankem, respectively. 0.2 µm the bemotrizinol from different sunscreen formulations without PTFE syringe filter, and also 0.2 µm nylon syringe filter were any interference of the pharmaceutical base. procured from Millipore. HPLC Method Validation and Optimization Instrumentation and Chromatographic Condition Column and mobile phase selection were done simultaneously. The HPLC system Shimadzu model no. SPD-M10A VP with The development was started with BDS Hypersil (4.6 × LC solution software consisted of a binary pump (model 250 mm, 5-micron particle size) column with acetonitrile 100% no. LC-10AT VP and LC20AD), autosampler (model no. as mobile phase at a flow rate of 2.5 mL per minute. Under SIL-20AHT), column oven (model no. CTO-10AS VP), and this condition, resolution, and symmetry of the peak was not PDA detector. Isocratic elution was achieved on µBond pack satisfactory, so the mobile phase was changed to methanol column, (3.9 × 300 mm, 10 microns) and column temperature 100%. Peak broadening with less plate count of the peak was maintained at 27ºC by using a column oven. Methanol 100% observed. Further trial was carried out with different columns, was used as a mobile phase. Chloroform and methanol such as, HyperClone (4.6 × 250 mm, 5-micron particle were used as diluents. Other parameters, such as, run time size) keeping the mobile phase and rest of chromatographic 30 minutes, 2.5 mL/minutes as flow rate, and the injection conditions was the same, and peak broadening with less volume was 20 µL. Detection was done by spectrophotometer plate count of the peak was observed. The next trial column set at 254 nm. The chromatogram of standard and sample was µBondapack (3.9 × 300 mm, 10-micron particle size) solution is shown in Figures 2 and 3. and the symmetrical peak was well resolved with good Procedure peak area by using 100% methanol, which provides the best chromatographic response for further studies. Standard Solution Preparation Method Validation Weigh accurately about 0.013-gram of bemotrizinol working standard in a 100 mL volumetric flask; add about 10 mL of System Suitability chloroform, sonicated for five minutes in an ultrasonic bath to To verify the system, method, and column performance the dissolve and volume makeup with the methanol.