Development and Validation of an RP-HPLC Method for the Quantification of Selected Anti-Cancer Standards (Rutin, Chrysin, Piperine and Berberine) in Botswana Medicinal Plants

Method validated using (RP-HPLC) reversed-phase high-performance liquid chromatographic method of this research was four selected Anti-Cancer standards. The newly developed method is novel, simple, precise, accurate, reproducible and less time consuming and it can be used for raw material of quantitative analysis and quality control. The separation of Rutin, Chrysin, Piperin and Berberine was conducted in less than ۳ minutes and chromatographic separation was achieved using a stationary phase C۱۸ J’Sphere column (internal diameter۱۵۰ mm x ۴.۶ mm and particle size ۴µ) and mobile phase contained acetonitrile: ۰.۱% formic acid (pH ۲.۷ ) in isocratic elution of (۸۵:۱۵ v/v) ratio, with a flow rate of ۱mL/min and column temperature was maintained at ۳۰ ̊ C with injection volume ۲۰ µl, UV detection wavelength of at ۳۳۶ nm were used. Rutin, Chrysin, Piperine and Berberine had retention times of ۱.۳, ۱.۸, ۲.۱ and ۲.۶ minutes, respectively. Calibration curves were accepted and reported good linear correlation coefficients (r۲ ˃ ۰.۹۹۹) within the range of ۱-۳.۵µg/mL. Mean percent recoveries for the standards were found to be within the acceptance limits (۸۰-۱۱۵%). Relative standard deviation (according to FDA and ICH guidelines) which indicates the method is precise. The developed method was applied in various medicinal plants Solanum aculeastrum dunal, Elephantorrhiza elephantina, Cadaba aphylla (Thunb) and Adenia glauca used to manage by traditional doctors in Botswana. All three plants were reported Rutin and Chrysin compounds except S. aculeastrum, it was reported only Rutin present whereas, Piperine and Berberine was not detected in all four plants.