DEVELOPMENT AND VALIDATION OF AN HPLC METHOD FOR THE QUANTIFICATION OF BENZOCAINE AND FUSIDIC ACID SIMULTANEOUSLY IN MICROEMULSION FORMULATION

Öz

Objective: The purpose of this study was to develop and validate an analytical technique for quantification in the microemulsion formulation containing fusidic acid and benzocaine developed for the treatment of mild and moderate wounds. Material and Method: Acetonitrile and acetic acid (0.1%) were used as a mobile phase in the HPLC method. The flow rate and injection volume were determined. The ICH Q2 guideline was followed in the validation of the developed method. Benzocaine (2%) and fusidic acid (2%) were added to the blank microemulsion prepared using ethyl oleate, propylene glycol, ethanol, and Cremophor EL, and the selectivity of the HPLC method for both these substances was investigated. Result and Discussion: The ratio of acetonitrile to acetic acid (0.1%) was determined to be 70:30 (v/v). The injection volume of 20 µl and the flow rate of 1 ml·min-1 were set. The developed method, which demonstrated a high correlation coefficient (0.9999) and a low variation coefficient (<2%), was simple, inexpensive, convenient, selective, and suitable for the analysis of both substances at the same wavelength (210 nm).

Anahtar Kelimeler

• Analytical method
• benzocaine
• fusidic acid
• HPLC
• microemulsion

MİKROEMULSİYON FORMÜLASYONUNDA BENZOKAİN VE FUSİDİK ASİT MİKTAR TAYİNİ İÇİN HPLC METODU GELİŞTİRİLMESİ VE VALİDASYONU

Öz

Amaç: Bu çalışmanın amacı, hafif ve orta dereceli yaraların tedavisi için geliştirilen benzokain ve fusidik asit içeren mikroemülsiyon formülasyonunda miktar tayini için analitik yöntem geliştirilmesi ve valide edilmesidir. Gereç ve Yöntem: HPLC metodunda mobil faz olarak asetonitril ve asetik asit (0.1%) kullanıldı. Akış hızı ve enjeksiyon hacmi belirlendi. Geliştirilen metodun doğrulamasında ICH Q2 kılavuzuna uyulmuştur. Etil oleat, propilen glikol, etanol ve Cremophor EL kullanılarak hazırlanan boş mikroemülsiyona benzokain (%2) ve fusidik asit (%2) ilave edildi ve HPLC metodunun bu iki madde için seçiciliği araştırıldı. Sonuç ve Tartışma: Asetonitrilin asetik aside (%0.1) oranı 70:30 (h/h) olarak belirlendi. Enjeksiyon hacmi 20 μl ve akış hızı 1 ml·dk-1 olarak ayarlandı. Yüksek korelasyon katsayısı (0.9999) ve düşük varyasyon katsayısı (<%2) gösteren geliştirilen yöntem basit, ucuz, seçici, kullanışlı ve her iki maddenin aynı dalga boyunda (210 nm) analizi için uygundu.

Anahtar Kelimeler

• Analitik metot
• benzokain
• fusidik asit
• HPLC
• mikroemülsiyon

Kaynakça

1. 1. Ahmad, N. (2023). In vitro and in vivo characterization methods for evaluation of modern wound dressings. Pharmaceutics, 15(1). [CrossRef]
2. 2. Kaya, S., Derman, S. (2023). Properties of ideal wound dressing. Ankara Üniversitesi Eczacılık Fakültesi Dergisi, 47(3), 1119-1131. [CrossRef]
3. 3. Aksu, N.B., Yozgatlı, V., Okur, M.E., Ayla, Ş., Yoltaş, A., Üstündağ Okur, N. (2019). Preparation and evaluation of QbD based fusidic acid loaded in situ gel formulations for burn wound treatment. Journal of Drug Delivery Science and Technology, 52, 110-121. [CrossRef]
4. 4. Paudel, K.S., Milewski, M., Swadley, C.L., Brogden, N.K., Ghosh, P., Stinchcomb, A.L. (2010). Challenges and opportunities in dermal/transdermal delivery. Therapeutic Delivery, 1(1), 109-131. [CrossRef]
5. 5. Arpa, M.D., Arslan, T., Eraslan, H., Üstündağ Okur, N. (2024). Sprayable microemulsion of diphenhydramine hydrochloride for dermal delivery. Acta Pharmaceutica Sciencia, 62(2), 462-479. [CrossRef]
6. 6. Arpa, M.D., Çağlar, E.Ş., Güreşçi, D., Sipahi, H., Üstündağ Okur, N. (2024). Novel microemulsion containing benzocaine and fusidic acid simultaneously: Formulation, characterization, and in vitro evaluation for wound healing. AAPS PharmSciTech, 25(53). [CrossRef]
7. 7. El-Kadem, A.H., Negm, W.A., Elekhnawy, E., Binsuwaidan, R., Attallah, N.G.M., Moglad, E., Sultan, A.A. (2024). Neohesperidin-loaded microemulsion for improved healing of Acinetobacter baumannii-infected wound. Journal of Drug Delivery Science and Technology, 98, 105905. [CrossRef]
8. 8. Okur, M.E., Ayla, Ş., Yozgatlı, V., Aksu, N.B., Yoltaş, A., Orak, D., Sipahi, H., Üstündağ Okur, N. (2020). Evaluation of burn wound healing activity of novel fusidic acid loaded microemulsion based gel in male Wistar albino rats. Saudi Pharmaceutical Journal, 28(3), 338-348. [CrossRef]

9. 9. de Assis, K.M.A., da Silva Leite, J.M., de Melo, D.F., Borges, J.C., Santana, L.M.B., dos Reis, M.M.L., Moreira, V.M., da Rocha, W.R.V., Ramalho Catão, R.M., dos Santos, S.G., da Silva Portela, A., de Sousa Silva, S.M., de Oliveira, T.K.B., da Silveira, J.W.S., Pires, E.G., Weege Nonaka, C.F., Castriani Sanches, F.A., de Lima Damasceno, B.P.G. (2020). Bicontinuous microemulsions containing Melaleuca alternifolia essential oil as a therapeutic agent for cutaneous wound healing. Drug Delivery and Translational Research, 10(6), 1748-1763. [CrossRef]
10. 10. Zhao, C.Y., Sui, H., Xue, E., Li, L., Zhang, J., Xu, T., Liang, X, Yang, Y. (2022). Determination of benzocaine in pharmaceutical formulations by indirect SERRS assay combined with azo coupling. Molecules, 27(14). [CrossRef]
11. 11. Elmotasem, H., El-Marasy, S.A., Mohamed, A.L. (2023). Benzocaine mesoporous silica nanoparticles/bio polysaccharides-based hydrogels loaded cotton bandage as a platform for topical anesthesia. Results in Chemistry, 5, 100830. [CrossRef]
12. 12. De Araujo, D.R., Padula, C., Cereda, C.M.S., Tófoli, G.R., Brito, R.B., De Paula, E., Nicoli, S., Santi, P. (2010). Bioadhesive films containing benzocaine: Correlation between in vitro permeation and in vivo local anesthetic effect. Pharmaceutical Research, 27(8), 1677-1686. [CrossRef]
13. 13. Paczkowska, M., Wiergowska, G., Miklaszewski, A., Krause, A., Mroczkowka, M., Zalewski, P., Cielecka-Piontek, J. (2018). The analysis of the physicochemical properties of benzocaine polymorphs. Molecules, 23(7). [CrossRef]
14. 14. Waqas, M.K., Sadia, H., Khan, M.I., Omer, M.O., Siddique, M.I., Qamar, S., Zaman, M., Butt, M.H., Mustafa, M.W., Rasool, N. (2022). Development and characterization of niosomal gel of fusidic acid: In-vitro and ex-vivo approaches. Designed Monomers and Polymers, 25(1), 165-174. [CrossRef]
15. 15. Rasool, N., Kanwal, Q., Waseem, M., Mehrunnisa, Khan, M.I. (2021). Analytical method development and determination of hydrocortisone acetate and fusidic acid simultaneously in cream formulation, by reversed-phase HPLC. Biomedical Chromatography, 35(3), 0-2. [CrossRef]
16. 16. Üstündağ Okur, N., Çağlar, E.Ş., Arpa, M.D., Karasulu, H.Y. (2017). Preparation and evaluation of novel microemulsion-based hydrogels for dermal delivery of benzocaine. Pharmaceutical Development and Technology, 22(4), 500-510. [CrossRef]
17. 17. Erkmen, C., Uslu, B. (2023). Green procedure index assessment of the novel stability-indicating RP-HPLC method for the determination of captopril from pharmaceutical dosage form. Ankara Üniversitesi Eczacılık Fakültesi Dergisi, 47(3), 894-906. [CrossRef]
18. 18. Rao, W., Li, L., Zhang, C., Zheng, J., Fan, X., Luan, B., Sun, J., Qiu, M., Wu, S., Li, Y., Wang, Z. (2023). Development and validation of a stability-indicating RP-HPLC method for the determination of fifteen impurities in rivaroxaban. Journal of Pharmaceutical and Biomedical Analysis, 228, 115325. [CrossRef]
19. 19. Curbete, M.M., Salgado, H.R.N. (2016). Stability-indicating RP-LC method for quantification of fusidic acid in cream. Brazilian Journal of Pharmaceutical Sciences, 52(3), 447-457. [CrossRef]
20. 20. Hirpara, M., Patel, P., Patel, N., Kulkarni, G., Patel, B.P. (2015). Development and validation of analytical method for simultaneous estimation of diclofenac sodium and benzocaine in gel dosage form. World Journal of Pharmaceutical Sciences, 3(6), 1095-1103.
21. 21. International Conference for Harmonisation (ICH) (2005). Validation of analytical procedures: Text and methodology Q2(R1). Geneva, 1-15.
22. 22. Farn, S.S., Yeh, Y.H., Li, C.C., Yang, C.S., Chen, J.T., Yu, C.S., Lin, W.J. (2017). Development and validation of a reversed-phase HPLC method for analysis of radiochemical purity in [123I]IBZM. Applied Radiation and Isotopes, 127, 61-67. [CrossRef]
23. 23. Angelo, T., Pires, F.Q., Gelfuso, G.M., da Silva, J.K.R., Gratieri, T., Cunha-Filho, M.S.S. (2016). Development and validation of a selective HPLC-UV method for thymol determination in skin permeation experiments. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 1022, 81-86. [CrossRef]
24. 24. Rao, G., Goyal, A. (2016). An overview on analytical method development and validation by using HPLC. The Pharmaceutical and Chemical Journal, 3(2), 280-289.
25. 25. Santhosh, G., Nagasowjanya, G., Ajitha, A., Uma, Y., Rao, M. (2014). HPLC method development and validation: An overview. International Journal of Pharmaceutical Sciences and Research, 4(4), 274-280.
26. 26. Hoa, L.T.H., Hung, V.T.N., Trang, D.T., Thu, T.N.H., Le, D.C. (2019). Development and validation of an HPLC method for simultaneous assay of MCI and MI in shampoos containing plant extracts. International Journal of Analytical Chemistry, 2019. [CrossRef]
27. 27. Singh, R. (2013). HPLC method development and validation-An overview. Indian Journal of Pharmaceutical Education and Research, 4(1), 26-33.
28. 28. Papadoyannis, I.N., Samanidou, V.F. (2004). Validation of HPLC instrumentation. Journal of Liquid Chromatography & Related Technologies, 27(5), 753-783. [CrossRef]
29. 29. Wadhwa, S., Singh, B., Sharma, G., Raza, K., Katare, O.P. (2016). Liposomal fusidic acid as a potential delivery system: A new paradigm in the treatment of chronic plaque psoriasis. Drug Delivery, 23(4), 1204-1213. [CrossRef]
30. 30. Grillo, R., De Melo, N.F.S., De Araújo, D.R., De Paula, E., Dias Filho, N.L., Rosa, A.H., Fraceto, L.F. (2009). Validation of an HPLC method for quantitative determination of benzocaine in PHBV-microparticles and PLA-nanoparticles. Latin American Journal of Pharmacy, 28(3), 393-399.
31. 31. Manikandan, K., Lakshmi, K.S., Pai, A. (2019). QbD approach in RP-HPLC method development for the assay of benzocaine and diclofenac in dosage forms. AIP Conference Proceedings, 020083, 1-19. [CrossRef]
32. 32. Spagnol, C.M., Isaac, V.L.B., Corrêa, M.A., Salgado, H.R.N. (2016). Validation of HPLC-UV assay of caffeic acid in emulsions. Journal of Chromatographic Science, 54(3), 305-311. [CrossRef]
33. 33. Yaseen, A., Ghareeb, M.M., Alsaffar, D.F., Ming, T.S., Gazzali, A.M. (2023). Development and validation of HPLC method for the detection of fusidic acid loaded in non-ionic and cationic nanoemulsion-based gels. Biomedical & Pharmacology Journal, 16(1), 573-580. [CrossRef]