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Development and Validation of Stability-Indicating RP-HPLC Method for Estimation of Fisetin in Novel Cubosomal Nanoformulation: Application to the Marketed Formulation and Selected Plant Extracts



Fisetin (FIS) is a bioactive flavonoid found in various plants, reported for many pharmacological activities, and presently marketed as a nutraceutical. To overcome less water solubility and bioavailability issues, FIS cubosomal nanoformulation has been prepared and characterized.



To estimate FIS in prepared novel cubosomes, an RP-HPLC analytical method development with the most sensitivity, economical, robust, and wide applicability in marketed FIS formulations and plant extracts also.



An RP-HPLC method was developed and validated as per ICH Q2R1 guidelines by using C-18 Phenomenex Luna 5µ, 100A0 column, LC-20 AD pump, and Shimadzu LC solution 1.25 software. The combination of acetonitrile and formic acid (0.1%v/v) in the ratio of 25:75 v/v was used as a mobile phase for chromatographic separation using a PDA detector at 360 nm and a flow rate of 1 ml/min.



In the range of 0.1 to 16 µg/ml, the developed method was remarkably linear (R2 ˃ 0.999). This method was found to be accurate (recovery 98.24 to 100.65 %), precise, robust (% RSD ˂ 2), and more sensitive than the earlier reported method with LOD and LOQ values of 17.26 and 52.31 ng/ml respectively. The FIS estimation was also performed using the developed method in the marketed FIS formulation Doctor’s Best ® Fisetin, and different plants extracts such as strawberry, grapes, black tea, and green tea. The forced degradation study suggests FIS was unstable in alkaline and oxidative stress conditions.



For FIS estimation in cubosomal nanoformulation, a widely applicable, novel, robust, most sensitive, and economical RP-HPLC method was developed and validated, and also applied to marketed formulation and plant extracts.
