Development and Validation of High Performance Liquid Chromatography Method for Quantification of Hesperetin in a Topical Formulation
Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, 1Department of Dentistry, Melaka Manipal Medical College, Manipal Academy of Higher Education, Udupi, Karnataka 576 104, 2Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Vaishali, Bihar 844 102, India
L. Kumar, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Vaishali, Bihar 844 102, India, E-mail: email@example.com
Hesperetin is a flavanone glycoside abundantly available in citrus fruits. Hesperetin is known to have various pharmacological activities, including anti-oxidant, anti-inflammatory, anti-allergic etc. In this current study, a simple, isocratic, fast, accurate and robust high-performance liquid chromatographic analytical method was developed for the estimation of hesperetin in a topical gel. A good chromatographic separation was achieved with an inertsil ODS 3V column with acetonitrile and acidified water as mobile phase. The method was validated over a linearity range of 25 to 2500 ng/ml following regulatory guidelines for various parameters, including specificity, system suitability, linearity, accuracy, precision and robustness. A topical gel containing hesperetin was prepared using Carbopol as a gel-forming agent. The total drug content of this formulation was determined using the validated high-performance liquid chromatographic method. The validated method was found to be linear over the selected range. Limit of detection and limit of quantitation was calculated as 1.61 and 4.89 ng/ml respectively. The method was found to be precise with % relative standard deviation of less than 1 % for intraday precision and less than 2 % for inter-day precision experiments. The method was also found to be robust, with small and deliberate changes in various method parameters. The drug content of topical gel formulation was found to be more than 98 % when analyzed using the validated high-performance liquid chromatographic method. The developed method will be useful for the routine quantitative analysis of hesperetin in topical gel formulations.