Development of a high performance liquid chromatography method for quantification of isomers β-caryophyllene and α-humulene in copaiba oleoresin using the Box-Behnken design.

The sesquiterpene isomers, β-Cariofileno (CAR) and α-Humuleno (HUM) are the primary constituents of the copaiba oleoresin species. These natural products are primarily used by the Amazonian population and marketed as phytotherapies and cosmetics. The aim of this study was to develop and validate a method that simultaneously assays the isomers present in copaiba oleoresins by high performance liquid chromatography using the Box-Behnken design. After preliminary studies, the reverse phase chromatographic system was selected using a cyano column and a mobile phase consisting of acetonitrile and phosphate buffer. The Box-Behnken design was applied at three levels and with four independent variables: flow rate (X1), gradient slope time (X2), proportion of organic compounds at the end of the gradient (X3) and at the beginning of the gradient (X4). Also, the responses of the dependent variables: CAR retention time (Y1) and the resolution between the CAR and HUM peaks (Y2) was assessed. The mathematical model obtained from the regression results was satisfactory (R(2)>0.98, n=27) and showed a quadratic relationship where the effects of interactions between the variables, was observed by response surface graphs. The simultaneous optimization method was used to establish the best compromise of the resolution between the CAR and HUM isomers while adjusting the retention time of CAR. This method was successfully optimized by BBD obtaining chromatographic peaks with good symmetry, resolution and separation efficiency. The validation of the developed method confirmed its specificity, precision, accuracy and linearity in the range of 5.0-11.0 and 0.4-1.0μg/mL for CAR and HUM, respectively, and is considered suitable for routine applications which assure quality control.