Analytical Quality by Design Approach for Simultaneous Determination of Phytomarkers Orientin, Quercetin and Piperine in Nilaveembu Kudineer by RP-HPLC Method

Orientin, quercetin and piperine are significant phytoconstituents present in Nilaveembu Kudineer (NK), NK is a polyherbal formulation consisting of 9 medicinal plants used in the management of dengue fever, inflammation, pyrexia, digestive and liver disorders etc. The present objective is to develop a Quality by Design (QbD)-based RP-HPLC method for standardization of orientin, quercetin and piperine phytomarkers present in NK by using Box Behnken Design. The Independent variables selected for QbD method are flow rate, acetic acid concentration, injection volume and the dependent variables selected are retention time, theoretical plate and tailing factor. Effective chromatographic separation of orientin, quercetin and piperine was established on C18 (150 mm x 4.6 mm x 5µm) column using moving fluid comprised of a combination of ACN and H2O (pH adjusted with 0.5 % v/v GAA) in gradient elution, with 1 mL/min flow rate, the temperature was set at 40°C and the injection volume was 20 μL. The Rt for orientin was found to be 4.108 min, quercetin 7.450 min and piperine 12.92 min respectively. Lambda max (λmax) was 340 nm. The method was found to be specific for the concurrent analysis of orientin, quercetin and piperine in NK, with (>90%) of accuracy and less then (%RSD < 2%) of precision. The novel RP-HPLC approach was effectively applied for the quantification of orientin, quercetin and piperine in NK. The developed RP-HPLC method could be further used for the simultaneous quantification of these phytoconstituents present in other herbal preparations.