PROPOSAL OF A HPLC-PAD METHOD FOR THE DETERMINATION OF GRANDIFLORENIC ACID AND KAURENIC ACID FROM COESPELETIA TIMOTENSIS
A simple, rapid, and selective high-performance liquid chromatography (HPLC) method with photo diode array detection (PAD) was developed for the determination of grandiflorenic acid (G -ac), kaurenic acid (k-ac) and iso-kaurenic acid (iK-ac) in Coespeletia timotensis. Effective chromatographic separation was achieved using a Waters-XBridge C18 (3 mm x 50 mm; 3.5 μm particle size) column, maintained at 50 ºC in an oven, with a mobile phase composed of 0.1% phosphoric acid solution, acetonitrile, and methanol in the ratio of 30:49:21 (v/v). The mobile phase was pumped isocratically at a flow rate of 0.6 mL min -1 , and quantification of each ent-kaurenoic acid was based on measuring its peak area at 220 nm. The retention time for G-ac was about 3.0 min with acceptable resolution from K-ac (4.0 min) and iK-ac (4.4 min) in a total run of just 10 min. The reliability and analytical performance of the proposed HPLC procedure were statistically validated. Peak identity was confirmed using pure standards of ent-kaurenoic acids. Calibration curves were linear in the range of 10 – 50 ng μL -1 with an acceptable correlation coefficient. The validated HPLC method was successfully applied to the determination of G -ac in resin´s acidic fraction of aerial parts of C. timotensis.