FENİTOİNİN YEŞİL HPLC TAYİNİ VE METOT VALİDASYONU

Year 2024, Volume: 48 Issue: 3, 1021 - 1032, 10.09.2024

Ertuğrul Faruk Ökmen , Ebru Çubuk Demiralay , İlkay Konçe , Yaşar Doğan Daldal

https://doi.org/10.33483/jfpau.1454525

Abstract

Amaç: Bu çalışmada antiepileptik ilaç fenitoinin kromatografik davranışı yeşil HPLC yöntemi ile belirlenmiştir. Geliştirilen yöntemin optimizasyonu, değişen su-etanol ikili karışımlarındaki fenitoinin kapasite faktörü değerlerine ve bileşiğin analiz edildiği mobil fazdaki etanol derişimine dayandırılmıştır.
Gereç ve Yöntem: RPLC yöntemiyle geliştirilen metot optimizasyonunda %35, %40 ve %45 (h/h) etanol içeren etanol-su ikili karışımları kullanılmıştır. Bileşiğin alıkonma süreleri Zorbax SB-CN (150x4.6 mm, 3.5 µm ID) kolonu ile belirlenmiştir. Analizler sabit akış hızı (0.3 ml/dakika) ve kolon sıcaklığında (37°C) gerçekleştirilmiştir. Kantitatif analiz için optimum koşul, pH'ı 6.5 olan %40 (h/h) etanol içeren bir etanol-su ikili karışımı olarak belirlenmiştir.
Sonuç ve Tartışma: Bu çalışmada logk-φ ilişkisini kullanılarak fenitoinin hidrofobisitesi hesaplanmıştır. Elde edilen kromatografik veriler kullanılarak optimum koşul belirlenmiş, ticari tablet formülasyonunda fenitoinin kantitatif tayini dahili standart yöntemiyle yapılmıştır. Bu koşullar altında 0.8-2.8 μg/ml derişim aralığında mükemmel doğrusallık (r>0.99) elde edilmiştir. Geliştirilen yöntemin tespit limiti 0.021 μg/ml; miktar belirleme sınırı 0.064 μg/ml olarak hesaplanmıştır. Yöntemin geri kazanım değeri %99.61 olarak belirlenmiştir. Kesinlik, doğruluk ve metot sağlamlığı parametrelerinin validasyon prosedürleri için uygun olduğu sonucuna varılmıştır.

Keywords

Antiepileptik ilaçlar, ikili karışım, RPLC, yeşil kimya, yöntem optimizasyonu

GREEN HPLC DETERMINATION OF PHENYTOIN AND METHOD VALIDATION

Abstract

Objective: In this study, the chromatographic behavior of the antiepileptic drug phenytoin was determined by the green HPLC method. The optimization of the developed method was based on the capacity factor values of phenytoin in varying water-ethanol binary mixtures and the ethanol concentration in the mobile phase where the compound was analyzed.
Material and Method: Ethanol-water binary mixtures containing 35%, 40%, and 45% (v/v) ethanol were used in the optimization for the determination performed by the RPLC method. Retention times of the compound were determined with the Zorbax SB-CN (150x4.6 mm, 3.5 µm ID) column. Analyzes were performed at a constant flow rate (0.3 ml/min) and column temperature (37°C). The optimum condition for quantitative analysis was determined as an ethanol-water binary mixture containing 40% (v/v) ethanol with a pH of 6.5.
Result and Discussion: In this study, the hydrophobicity of phenytoin was calculated using the logk-φ relationship. The optimum condition was determined using the obtained chromatographic data, and the quantitative determination of phenytoin in the commercial tablet formulation was made by the internal standard method. Under these conditions, excellent linearity (r>0.99) was obtained in the concentration range of 0.8-2.8 μg/ml. The detection limit of the developed method is 0.021 μg/ml; the limit of quantitation was calculated as 0.064 μg/ml. The recovery value of the method was determined as 99.61%. It was concluded that the parameters of precision, accuracy, and method robustness were appropriate for the validation procedures.

Keywords

Antiepileptic drugs, binary mixture, green chemistry, method optimization, RPLC

Ethical Statement

None

References

• 1. Jiang, Y., Lu, Y., Yang, L. (2022). An epileptic seizure prediction model based on a time-wise attention simulation module and a pretrained ResNet. Methods, 202, 117-126. [CrossRef]
• 2. Brunton, L.L., Lazo, J.S., Parker, K.L. (2008). Goodman & Gilman's: The Pharmacology Basis of Therapeutics, McGraw-Hill, ABD, p.350.
• 3. Keppel Hesselink, J.M., Kopsky, D.J. (2017). Phenytoin: 80 years young, from epilepsy to breast cancer, a remarkable molecule with multiple modes of action. Journal of Neurology, 264(8), 1617-1621. [CrossRef]
• 4. Jones, G.L., Wimbish, G.H., McIntosh, W.E. (1983). Phenytoin: Basic and clinical pharmacology. Medicinal Research Reviews, 3(4), 383-434. [CrossRef]
• 5. Sakaguchi, Y., Arima, R., Maeda, R., Obayashi, T., Masuda, A., Funakoshi, M. (2023). Development of a useful single reference HPLC method for therapeutic drug monitoring of phenytoin and carbamazepine in human plasma. Analytical Science, 39, 447-454. [CrossRef]
• 6. Flores, J., Alexander, S., Babayeva, M.A. (2018). Novel HPLC method for determination of phenytoin in human plasma. Journal of Pharmaceutical Research International, 22(6), 1-7. [CrossRef]
• 7. Dural, E., Bolayır, A., Çiğdem, B. (2021) Determination of phenytoin in human plasma by a validated hplc method: Application to therapeutic drug monitoring study. ACTA Pharmaceutica Sciencia, 59(1), 149-170. [CrossRef]
• 8. Sungthong, B., Rattarom, R., Sato, V.H., Sato, H. (2019). Development and validation of a new RP-HPLC-UV Method for the simultaneous determination of phenytoin ımpurities, benzophenone and benzil. Acta Chromatographica, 31(4), 241-245. [CrossRef]
• 9. Kazakevich, Y., Lobrutto, Y. (2007). HPLC for Pharmaceutical Scientists, Wiley-Interscience, ABD, p.141.
• 10. Meyer, V.R. (2010). Practical High-Performance Liquid Chromatography, John Wiley and Sons, ABD, p.174.
• 11. Yabré, M., Ferey, L., Somé, I.T.,Gaudin, K. (2018). Greening reversed-phase liquid chromatography methods using alternative solvents for pharmaceutical analysis. Molecules, 23, 1065. [CrossRef]
• 12. Ibrahim, F.A., Elmansi, H., Fathy, M.E. (2019). Green RP-HPLC method for simultaneous determination of moxifloxacin combinations: Investigation of the greenness for the proposed method. Microchemical Journal, 148, 151-161. [CrossRef]
• 13. Roses, M., Bosch, E. (2002). Influence of mobile phase acid-base equilibria on the chromatographic behaviour of protolytic compounds. Journal of Chromatography A, 982, 1-30. [CrossRef]
• 14. Demiralay, E.C., Cubuk, B., Alsancak, G, Ozkan, S.A. (2010). Combined effect of polarity and pH on the chromatographic behaviour of some angiotensin II receptor antagonists and optimization of their determination in pharmaceutical dosage forms. Journal of Pharmaceutical and Biomedical Analysis, 53(3), 475-482. [CrossRef]
• 15. Erdemgil, F.Z., Sanli, S., Sanli, N., Ozkan, G., Barbosa, J. (2007). Determination of pKa values of some hydroxylated benzoic acids in methanol-water binary mixtures by LC methodology and potentiometry. Talanta, 72, 489-496. [CrossRef]
• 16. Horváth, C., Melander, W., Molnár, I. (1977). Liquid chromatography of ionogenic substances with nonpolar stationary phases. Analytical Chemistry, 49(1), 142-154. [CrossRef]
• 17. Subirats, X., Bosch, E., Rosés, M. (2006). Retention of ionisable compounds on high-performance liquid chromatography XV. Estimation of the pH variation of aqueous buffers with the change of the acetonitrile fraction of the mobile phase. Journal of Chromatography A, 1121, 170-177. [CrossRef]
• 18. Secilmis, H.C., Demiralay, E.C., Alsancak, G, Ozkan, S.A. (2012). The combined effect of the organic modifier content and pH of the mobile phase on the chromatographic behavior of some arylpropionic and arylacetic acids to optimize their liquid chromatographic determinations. Chromatographia, 75, 711-720. [CrossRef]
• 19. Poole, C.F., Poole, S.K. (1991). Chromatography Today, Elsevier Science, Holland, p.311.
• 20. Snyder, L.R., Dolan, J.W.,Gant, J.R. (1979). Gradient elution in high-performance liquid chromatography: I. Theoretical basis for reversed-phase systems. Journal of Chromatography A, 165(1), 3-30. [CrossRef]
• 21. Snyder, L.R., Dolan, J.W. (1998). Advances in Chromatography, CRC Press, ABD, p.24.
• 22. Baczek,T., Markuszewski, M., Kaliszan, R., van Straten, M.A., Claessens, H.A. (2000). Linear and quadratic relationships between retention and organic modifier content in eluent in reversed phase high-performance liquid chromatography: A systematic comparative statistical study. Journal of High Resolution Chromatography, 23(12), 667-676. [CrossRef]
• 23. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. Validation of analytical procedure: Text and Methodology Q2 (R1), 2005.
• 24. Helrich, K. (1990). Official Methods of Analysis, Analytical Chemists. Inc., USA, p.548.
• 25. Rondinini, S., Mussini, P.R., Mussini, T., Vertova, A. (1998). pH measurements in non-aqueous and mixed solvents: Predicting pH (PS) of potassium mixtures hydrogen phthalate for alcohol-water. Pure and Applied Chemistry, 70, 1419-1422. [CrossRef]
• 26. Baucke, F.G.K., Neumann, R., Alexander-Weber, C. (1993). Multiple-point calibration with linear regression as a proposed standardization procedure for high-precision pH measurements. Analytical Chemistry, 65(22), 3244-3251. [CrossRef]
• 27. Swiss ADME program. http://www.swissadme.ch/index.php/. Accessed date: 2023-07-26.
• 28. Chemicalize program. https://chemicalize.com/#/calculation/. Accessed date: 2016-03.10.
• 29. McNally, R. (2000). The United States Pharmacopoeia. 24th revision. Easton.
• 30. Horwitz, W., Albert, R. (2006). The Horwitz Ratio (HorRat): A useful index of method performance with respect to precision. Journal of AOAC International, 89(4), 1095-1109. [CrossRef]
• 31. Kumar, V.S., Vinayan, K.P. (2022). Anticonvulsant and acute toxicity evaluation of phenytoin sodium-loaded polymeric nanomicelle in MES rat model. Journal of Nanoparticle Research, 24(8), 6-16. [CrossRef]
• 32. Nair, S.C., Vinayan, K.P., Mangalathillam, S. (2021). Nose to brain delivery of phenytoin sodium loaded nano lipid carriers: Formulation, drug release, permeation and in vivo pharmacokinetic studies. Pharmaceutics, 13 (10), 2-23. [CrossRef]
• 33. Sungthong, B., Rattarom, R., Sato, V.H., Sato, H. (2019). Development and validation of a new RP-HPLC-UV method for the simultaneous determination of phenytoin impurities, benzophenone, and benzil. Acta Chromatogr, 31(4), 241-245. [CrossRef]
• 34. Rahman, Z., Dharani, S., Barakh Ali, S.F., Nutan, M.T., Khan, M.A. (2020). Effects of diluents on physical and chemical stability of phenytoin and phenytoin sodium. An Official Journal of the American Association of Pharmaceutical Scientists, 21, 1-14. [CrossRef]
• 35. Kole, P., Parameswaran, S. (2023). Simultaneous estimation and validation of four antiepileptic drugs from bulk and formulations using reverse phase HPLC. Brazilian Journal of Pharmaceutical Sciences, 58, 1-9. [CrossRef]
• 36. Ayman, A., EL-Shabrawy, Y., Zeid, A.M., Wahba, M.E.K. (2021). A diagnostic intoxication tool for clozapine and phenytoin using hybrid micelle liquid chromatography. Journal of Taibah Universityf or Science, 15(1), 87-96. [CrossRef]