Development and Validation of the RP-HPLC Method for Dexamethasone Sodium Phosphate Determination in Nasal Chitosan Microsphere Preparations
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Keywords:
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Method Development and Validation, Dexamethasone Sodium Phosphate, Microspheres, Entrapment Efficiency, Chitosan
Abstract
The purpose of this work was to provide a robust, sensitive, accurate, and straightforward analytical method for measuring dexamethasone sodium phosphate (DSP) in chitosan microspheres preparedn using the spray drying method. DSP was quantitatively analyzed using RP-HPLC with an ultraviolet detector at 254 nm, a mobile phase that contained a mixture of acetonitrile and 0.1% sodium dihydrogen phosphate monohydrate (50:50) operating isocratically at a flow rate of 1.0 mL/min, and a stationary phase that was a C18 PrincetonSPHER-100 C18-QB 100A HPLC Column (250 × 4.6 mm, 5 um). The ICH recommendations were followed in the validation of the analytical method. DSP had a retention duration of 2.899 minutes and a tailing factor of 0.827. The RP-HPLC method was linear (R = 0.9992) in the 15-60 ug/mL concentration range. The limits of quantitation (LOQ) and detection (LOD) were 4.425 ug/mL and 1.327 ug/mL, respectively. The relative standard deviations for the intra-day and inter-day precisions were 0.057-0.876% and 0.780-0.949%, respectively. The recovery percentages at 50, 100, and 200% concentration levels were within the 99.269-100.980% range. The validated method has been successfully applied to determine DSP entrapment efficiency in chitosan microspheres. A linear, sensitive, accurate, precise, and robust technique of determining DSP in chitosan microsphere preparations is offered by the established RP-HPLC method.
References
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Boros, B., Jakabová, S., Madarász, T., Molnár, R., Galambosi, B., Kilár, F., Felinger, A., & Farkas, A. (2014). Validated HPLC Method for Simultaneous Quantitation of Bergenin, Arbutin, and Gallic Acid in Leaves of Different Bergenia Species. Chromatographia, 77(17–18), 1129–1135.
Bratskaya, S., Privar, Y., Nesterov, D., Modin, E., Kodess, M., Slobodyuk, A., Marinin, D., & Pestov, A. (2019). Chitosan Gels and Cryogels Cross-Linked with Diglycidyl Ethers of Ethylene Glycol and Polyethylene Glycol in Acidic Media. Biomacromolecules, 20(4), 1635–1643.
Brugnera, M., Vicario De la Torre, M., Andrés Guerrero, V., Bravo Osuna, I., Molina Martínez, I. T., & Herrero Vánrell, R. (2022). Validation of a Rapid and Easy-to-Apply Method to Simultaneously Quantify Co-Loaded Dexamethasone and Melatonin PLGA Microspheres by HPLC-UV: Encapsulation Efficiency and In Vitro Release. Pharmaceutics, 14(2), 288.
Cárdenas, G., Chávez-Canales, M., Espinosa, A. M., Jordán-Ríos, A., Malagon, D. A., Murillo, M. F. M., Araujo, L. V. T., Campos, R. L. B., Wong-Chew, R. M., González, L. E. R., Cresencio, K. I., Velázquez, E. G., de la Cerda, M. R., Leyva, Y., Hernández-Ruiz, J., Hernández-Medel, M. L., León-Hernández, M., Quero, K. M., Monciváis, A. S., & Sciutto, E. (2024). Intranasal Versus Intravenous Dexamethasone to Treat Hospitalized COVID-19 Patients: A Randomized Multicenter Clinical Trial. Archives of Medical Research, 55(2), 102960.
Chhajed, S., Sangale, S., & Barhate, S. D. (2011). Advantageous Nasal Drug Delivery System: A Review. IJPSR, 2(6), 1322–1336.
Czechowska-Biskup, R., Wojtasz Pająk, A., Sikorski, J., Henke, A., Ulanski, P., & Rosiak, J. M. (2007). Aqueous Solutions of Hydrochloric Acid as Simple Solvents of Chitosan for Viscosity-and Light-Scattering-Based Molecular Weight Determination. Polish Chitin Society, Monograph XII, 87–94.
Desnita, R., Noviana, E., Zai, K., & Sulaiman, T. N. S. (2023). An Up-To-Date Review: Microspheres as a New Drug Delivery System. International Journal of Applied Pharmaceutics, 15(6), 82–89.
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González, A. G., & Herrador, M. A. (2007). A Practical Guide to Analytical Method Validation, Including Measurement Uncertainty and Accuracy Profiles. TrAC - Trends in Analytical Chemistry, 26(3), 227–238.
Gupta, R. K. (2020). Advantages of Analytical Validation Method. JournalNX, 6(8), 200–204.
Hagesaether, E. (2011). Permeation Modulating Properties of Natural Polymers - Effect of Molecular Weight and Mucus. International Journal of Pharmaceutics, 409(1–2), 150–155.
Joshi, R. R., & Maresh, A. (2018). Iatrogenic Cushing’s Syndrome and Adrenal Insufficiency in Infants on Intranasal Dexamethasone Drops for Nasal Obstruction – Case Series and Literature Review. International Journal of Pediatric Otorhinolaryngology, 105, 123–126.
Kowalska, M., Woźniak, M., Kijek, M., Mitrosz, P., Szakiel, J., & Turek, P. (2022). Management of Validation of HPLC Method for Determination of Acetylsalicylic Acid Impurities in a New Pharmaceutical Product. Scientific Reports, 12(1), 1.
Le, T. H. H., Phung, T. H., & Le, D. C. (2019). Development and Validation of an HPLC Method for Simultaneous Assay of Potassium Guaiacolsulfonate and Sodium Benzoate in Pediatric Oral Powder. Journal of Analytical Methods in Chemistry, 2019(1), 6143061.
McMillan, J. (2016). Principles of Analytical Validation. In Proteomic Profiling and Analytical Chemistry: The Crossroads: Second Edition. Elsevier, 2nd edition.
Meneses, G., Gevorkian, G., Florentino, A., Bautista, M. A., Espinosa, A., Acero, G., Díaz, G., Fleury, A., Pérez Osorio, I. N., & del Rey, A. (2017). Intranasal Delivery of Dexamethasone Efficiently Controls LPS-Induced Murine Neuroinflammation. Clinical and Experimental Immunology, 190(3), 304–314.
Merkus, F. W., Verhoef, J. C., Schipper, N. G., & Marttin, E. (1998). Nasal Mucociliary Clearance as a Factor in Nasal Drug Delivery. Advanced Drug Delivery Reviews, 29(1–2), 13–38.
Mouez, M. A., Zaki, N. M., Mansour, S., & Geneidi, A. S. (2014). Bioavailability Enhancement of Verapamil HCl via Intranasal Chitosan Microspheres. European Journal of Pharmaceutical Sciences, 51(1), 59–66.
Naseef, H., Moqadi, R., & Qurt, M. (2018). Development and Validation of an HPLC Method for Determination of Antidiabetic Drug Alogliptin Benzoate in Bulk and Tablets. Journal of Analytical Methods in Chemistry, 2018(1), 1902510.
Norman, P. S. X., Winkenwerder, W. L., George, W., Parsons, J. W., & Murgatroyd, G. W. (1966). Evidence for the Local Action of Intranasal Dexamethasone Aerosols in the Suppression of Hay Fever Symptoms. Journal of Allergy, 38(2), 93–99.
Pandey, J., & Tripathi, P. K. (2016). Formulation and Evaluation of Levocetirizine Loaded Mucoadhesive Microspheres for Nasal Delivery. International Journal of Pharmaceutical Sciences and Research, 7(5), 2242–2251.
Patil, S. D., & Chalikwar, S. S. (2024). A Brief Review on Application of Design of Experiment for The Analysis of Pharmaceuticals Using HPLC. Annales Pharmaceutiques Françaises, 82(2), 203–228.
Pereswetoff-Morath, L. (1998). Microspheres as Nasal Drug Delivery Systems. Advanced Drug Delivery Reviews, 29(1–2), 185–194.
Prasada Rao, P. T. S. R. K. (2022). HPLC Method Development and Validation of Lercanidipine HCl and Atenolol, Characterization of Its Degradants by LC-MS/MS. International Journal of Applied Pharmaceutics, 14(2), 125–134.
Puti, K. M. A. (2023). Validation of Analytical Method for The Dissolution Test of Vitamin D3 5000 IU Chewable Tablets with HPLC and Stability Testing. Journal of Science and Technology Research for Pharmacy.
Sapra, A., Hm, H., Amin, S. M., Syahrani, F. A., Kelsi, Nur, S., & Permana, A. D. (2024). Development of Mucoadhesive Microspheres for Intranasal Delivery of Fluconazole as an Alternative Treatment of Cryptococcal Meningitis Infection in Patients with Acquired Immunodeficiency. Annales Pharmaceutiques Françaises, 82(5), 813–821.
Sellappan, M., & Devakumar, D. (2021). Development and Validation of RP-HPLC Method for The Estimation of Escitalopram Oxalate and Flupentixol Dihydrochloride in Combined Dosage Form and Plasma. International Journal of Pharmacy and Pharmaceutical Sciences, 13, 61–66.
Sonar, P., Shaikh, K., & Harer, S. (2022). Development and Validation of Novel RP-HPLC-DAD Method for Quantification of Lapatinib Ditosylate in Newer Nano-Liposome Formulation: A Quality by Design (QbD) Approach. International Journal of Applied Pharmaceutics, 14(6), 239–250.
Suresh, S., & Bhaskaran, S. (2005). Nasal Drug Delivery: An Overview. Indian Journal of Pharmaceutical Sciences, 67(1), 19–25.
Toffoletto, N., Salema-Oom, M., Nicoli, S., Pescina, S., González-Fernández, F. M., Pinto, C. A., Saraiva, J. A., Alves de Matos, A. P., Vivero-Lopez, M., Huete-Toral, F., Carracedo, G., Saramago, B., & Serro, A. P. (2024). Dexamethasone Phosphate and Penetration Co-Eluting Contact Lenses: A Strategy to Enhance Ocular Drug Permeability. International Journal of Pharmaceutics, 650, 123685.
Wang, S., Sun, Y., Zhang, J., Cui, X., Xu, Z., Ding, D., Zhao, L., Li, W., & Zhang, W. (2020). Astragalus Polysaccharides/Chitosan Microspheres for Nasal Delivery: Preparation, Optimization, Characterization, and Pharmacodynamics. Frontiers in Pharmacology, 11, 1–9.
Wu, J., Shu, Q., Niu, Y., Jiao, Y., & Chen, Q. (2018). Preparation, Characterization, and Antibacterial Effects of Chitosan Nanoparticles Embedded with Essential Oils Synthesized in an Ionic Liquid Containing System. Journal of Agricultural and Food Chemistry, 66(27), 7006–7014.
AlAani, H., & Alnukkary, Y. (2016). Stability-Indicating HPLC Method for Simultaneous Determination of Chloramphenicol, Dexamethasone Sodium Phosphate, and Tetrahydrozoline Hydrochloride in Ophthalmic Solution. Advanced Pharmaceutical Bulletin, 6(1), 137–141.
Amjad, M., & Hussain, S. (2020). Development and Validation of HPLC Method for the Simultaneous Determination of Dexamethasone Sodium Phosphate and Prednisolone Acetate in Injectable Veterinary. The Pharmaceutical and Chemical Journal, 7(5), 48–65.
Apridamayanti, P., Sari, R., & Pratiwi, L. (2023). Development Validation of Quercetin Compounds Using RP-HPLC and In Vitro Activity Studies on Melastoma malabathricum Leaf Nanocream Foundation Preparations. International Journal of Applied Pharmaceutics, 15(5), 317–324.
Boros, B., Jakabová, S., Madarász, T., Molnár, R., Galambosi, B., Kilár, F., Felinger, A., & Farkas, A. (2014). Validated HPLC Method for Simultaneous Quantitation of Bergenin, Arbutin, and Gallic Acid in Leaves of Different Bergenia Species. Chromatographia, 77(17–18), 1129–1135.
Bratskaya, S., Privar, Y., Nesterov, D., Modin, E., Kodess, M., Slobodyuk, A., Marinin, D., & Pestov, A. (2019). Chitosan Gels and Cryogels Cross-Linked with Diglycidyl Ethers of Ethylene Glycol and Polyethylene Glycol in Acidic Media. Biomacromolecules, 20(4), 1635–1643.
Brugnera, M., Vicario De la Torre, M., Andrés Guerrero, V., Bravo Osuna, I., Molina Martínez, I. T., & Herrero Vánrell, R. (2022). Validation of a Rapid and Easy-to-Apply Method to Simultaneously Quantify Co-Loaded Dexamethasone and Melatonin PLGA Microspheres by HPLC-UV: Encapsulation Efficiency and In Vitro Release. Pharmaceutics, 14(2), 288.
Cárdenas, G., Chávez-Canales, M., Espinosa, A. M., Jordán-Ríos, A., Malagon, D. A., Murillo, M. F. M., Araujo, L. V. T., Campos, R. L. B., Wong-Chew, R. M., González, L. E. R., Cresencio, K. I., Velázquez, E. G., de la Cerda, M. R., Leyva, Y., Hernández-Ruiz, J., Hernández-Medel, M. L., León-Hernández, M., Quero, K. M., Monciváis, A. S., & Sciutto, E. (2024). Intranasal Versus Intravenous Dexamethasone to Treat Hospitalized COVID-19 Patients: A Randomized Multicenter Clinical Trial. Archives of Medical Research, 55(2), 102960.
Chhajed, S., Sangale, S., & Barhate, S. D. (2011). Advantageous Nasal Drug Delivery System: A Review. IJPSR, 2(6), 1322–1336.
Czechowska-Biskup, R., Wojtasz Pająk, A., Sikorski, J., Henke, A., Ulanski, P., & Rosiak, J. M. (2007). Aqueous Solutions of Hydrochloric Acid as Simple Solvents of Chitosan for Viscosity-and Light-Scattering-Based Molecular Weight Determination. Polish Chitin Society, Monograph XII, 87–94.
Desnita, R., Noviana, E., Zai, K., & Sulaiman, T. N. S. (2023). An Up-To-Date Review: Microspheres as a New Drug Delivery System. International Journal of Applied Pharmaceutics, 15(6), 82–89.
Fuchs, M., Wetzig, H., Kertscher, F., Täschner, R., & Keller, E. (1999). Iatrogenic Cushing Syndrome and Mutatio Tarda Caused by Dexamethasone Containing Nose Drops. HNO, 47(7), 647–650.
González, A. G., & Herrador, M. A. (2007). A Practical Guide to Analytical Method Validation, Including Measurement Uncertainty and Accuracy Profiles. TrAC - Trends in Analytical Chemistry, 26(3), 227–238.
Gupta, R. K. (2020). Advantages of Analytical Validation Method. JournalNX, 6(8), 200–204.
Hagesaether, E. (2011). Permeation Modulating Properties of Natural Polymers - Effect of Molecular Weight and Mucus. International Journal of Pharmaceutics, 409(1–2), 150–155.
Joshi, R. R., & Maresh, A. (2018). Iatrogenic Cushing’s Syndrome and Adrenal Insufficiency in Infants on Intranasal Dexamethasone Drops for Nasal Obstruction – Case Series and Literature Review. International Journal of Pediatric Otorhinolaryngology, 105, 123–126.
Kowalska, M., Woźniak, M., Kijek, M., Mitrosz, P., Szakiel, J., & Turek, P. (2022). Management of Validation of HPLC Method for Determination of Acetylsalicylic Acid Impurities in a New Pharmaceutical Product. Scientific Reports, 12(1), 1.
Le, T. H. H., Phung, T. H., & Le, D. C. (2019). Development and Validation of an HPLC Method for Simultaneous Assay of Potassium Guaiacolsulfonate and Sodium Benzoate in Pediatric Oral Powder. Journal of Analytical Methods in Chemistry, 2019(1), 6143061.
McMillan, J. (2016). Principles of Analytical Validation. In Proteomic Profiling and Analytical Chemistry: The Crossroads: Second Edition. Elsevier, 2nd edition.
Meneses, G., Gevorkian, G., Florentino, A., Bautista, M. A., Espinosa, A., Acero, G., Díaz, G., Fleury, A., Pérez Osorio, I. N., & del Rey, A. (2017). Intranasal Delivery of Dexamethasone Efficiently Controls LPS-Induced Murine Neuroinflammation. Clinical and Experimental Immunology, 190(3), 304–314.
Merkus, F. W., Verhoef, J. C., Schipper, N. G., & Marttin, E. (1998). Nasal Mucociliary Clearance as a Factor in Nasal Drug Delivery. Advanced Drug Delivery Reviews, 29(1–2), 13–38.
Mouez, M. A., Zaki, N. M., Mansour, S., & Geneidi, A. S. (2014). Bioavailability Enhancement of Verapamil HCl via Intranasal Chitosan Microspheres. European Journal of Pharmaceutical Sciences, 51(1), 59–66.
Naseef, H., Moqadi, R., & Qurt, M. (2018). Development and Validation of an HPLC Method for Determination of Antidiabetic Drug Alogliptin Benzoate in Bulk and Tablets. Journal of Analytical Methods in Chemistry, 2018(1), 1902510.
Norman, P. S. X., Winkenwerder, W. L., George, W., Parsons, J. W., & Murgatroyd, G. W. (1966). Evidence for the Local Action of Intranasal Dexamethasone Aerosols in the Suppression of Hay Fever Symptoms. Journal of Allergy, 38(2), 93–99.
Pandey, J., & Tripathi, P. K. (2016). Formulation and Evaluation of Levocetirizine Loaded Mucoadhesive Microspheres for Nasal Delivery. International Journal of Pharmaceutical Sciences and Research, 7(5), 2242–2251.
Patil, S. D., & Chalikwar, S. S. (2024). A Brief Review on Application of Design of Experiment for The Analysis of Pharmaceuticals Using HPLC. Annales Pharmaceutiques Françaises, 82(2), 203–228.
Pereswetoff-Morath, L. (1998). Microspheres as Nasal Drug Delivery Systems. Advanced Drug Delivery Reviews, 29(1–2), 185–194.
Prasada Rao, P. T. S. R. K. (2022). HPLC Method Development and Validation of Lercanidipine HCl and Atenolol, Characterization of Its Degradants by LC-MS/MS. International Journal of Applied Pharmaceutics, 14(2), 125–134.
Puti, K. M. A. (2023). Validation of Analytical Method for The Dissolution Test of Vitamin D3 5000 IU Chewable Tablets with HPLC and Stability Testing. Journal of Science and Technology Research for Pharmacy.
Sapra, A., Hm, H., Amin, S. M., Syahrani, F. A., Kelsi, Nur, S., & Permana, A. D. (2024). Development of Mucoadhesive Microspheres for Intranasal Delivery of Fluconazole as an Alternative Treatment of Cryptococcal Meningitis Infection in Patients with Acquired Immunodeficiency. Annales Pharmaceutiques Françaises, 82(5), 813–821.
Sellappan, M., & Devakumar, D. (2021). Development and Validation of RP-HPLC Method for The Estimation of Escitalopram Oxalate and Flupentixol Dihydrochloride in Combined Dosage Form and Plasma. International Journal of Pharmacy and Pharmaceutical Sciences, 13, 61–66.
Sonar, P., Shaikh, K., & Harer, S. (2022). Development and Validation of Novel RP-HPLC-DAD Method for Quantification of Lapatinib Ditosylate in Newer Nano-Liposome Formulation: A Quality by Design (QbD) Approach. International Journal of Applied Pharmaceutics, 14(6), 239–250.
Suresh, S., & Bhaskaran, S. (2005). Nasal Drug Delivery: An Overview. Indian Journal of Pharmaceutical Sciences, 67(1), 19–25.
Toffoletto, N., Salema-Oom, M., Nicoli, S., Pescina, S., González-Fernández, F. M., Pinto, C. A., Saraiva, J. A., Alves de Matos, A. P., Vivero-Lopez, M., Huete-Toral, F., Carracedo, G., Saramago, B., & Serro, A. P. (2024). Dexamethasone Phosphate and Penetration Co-Eluting Contact Lenses: A Strategy to Enhance Ocular Drug Permeability. International Journal of Pharmaceutics, 650, 123685.
Wang, S., Sun, Y., Zhang, J., Cui, X., Xu, Z., Ding, D., Zhao, L., Li, W., & Zhang, W. (2020). Astragalus Polysaccharides/Chitosan Microspheres for Nasal Delivery: Preparation, Optimization, Characterization, and Pharmacodynamics. Frontiers in Pharmacology, 11, 1–9.
Wu, J., Shu, Q., Niu, Y., Jiao, Y., & Chen, Q. (2018). Preparation, Characterization, and Antibacterial Effects of Chitosan Nanoparticles Embedded with Essential Oils Synthesized in an Ionic Liquid Containing System. Journal of Agricultural and Food Chemistry, 66(27), 7006–7014.
Authors
Desnita, R., Noviana, E., Zai, K., & Sulaiman, T. N. S. (2025). Development and Validation of the RP-HPLC Method for Dexamethasone Sodium Phosphate Determination in Nasal Chitosan Microsphere Preparations. Science and Technology Indonesia, 10(1), 165–172. https://doi.org/10.26554/sti.2025.10.1.165-172
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