Development and Validation of an Analytical Method to Quantify Bioactive Neolignans of Piper rivinoides Kunth Extracts

Piper rivinoides Kunth (Piperaceae) is a native species from Brazil and is registered from North to South. Investigations about the chemistry of P. rivinoides indicate the presence of bioactive benzofuran neolignans eupomatenoid-5 (EUP-5), eupomatenoid-6 (EUP-6) and conocarpan (CNC) in its extract. Therefore, the extract of P. rivinoides Kunth, rich in neolignans, has the potential to become a phytotherapeutic product. A newly High-performance Liquid Chromatography coupled to Diode-Array Detector (HPLC-DAD) method for simultaneous determination and quantification of EUP-5, EUP-6 and CNC was developed and validated in this work. A modified-silica based column C-18 (250 mm x 4.6 mm, 5 mu m) with mobile phase composed by acetonitrile and ultrapure water (80:20 v/v) was used, along with temperature of 50 degrees C, flow rate at 1 mL/min, injection volume of 20 mu L and total analysis time of 8 min. The method was validated, showing to be selective and linear for the concentration ranges 3.125 - 100 mu g/mL; 3.125 - 75 mu g/mL and 1 - 20 mu g/mL for EUP-5, EUP-6 and CNC, respectively; precise (CV <2.0%); exact (CV <5.0%); robust (CV <3.0%), with recovery values close to 100% and limits of quantification and detection that vary in the range of 50 - 100 ng/mL and 25 - 37.5 ng/mL. The method developed and validated is unprecedented and will contribute to adequate phytochemical analysis, as well as to the establishment of quality control parameters for extracts of P. rivinoides.