Manscript Submission

PROPOSAL OF A HPLC-PAD METHOD FOR THE DETERMINATION OF GRANDIFLORENIC ACID AND KAURENIC ACID FROM COESPELETIA TIMOTENSIS

Alida Perez
Instituto de Investigaciones,Facultad de Farmaciay Bioanálisis, Universidad de Los Andes, Mérida, Venezuela.
José F Ovalles
Departamento de Análisis y Control, Facultad de Farmaciay Bioanálisis, Universidad de Los Andes, Mérida, Venezuela
Ana Medina
Departamento de Ciencias de los Alimentos, Facultad de Farmaciay Bioanálisis, Universidad de Los Andes, Mérida, Venezuela
Rosa Aparicio
Instituto de Investigaciones,Facultad de Farmaciay Bioanálisis, Universidad de Los Andes, Mérida, Venezuela.
Alfredo Usubillaga
Instituto de Investigaciones,Facultad de Farmaciay Bioanálisis, Universidad de Los Andes, Mérida, Venezuela.

Reversed phase liquid chromatography ,HPLC-PAD ,Grandiflorenic acid ,Kaurenic acid ,Kaurenoic acid ,Andean giant rosette ,Frailejón ,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.

4 , 2 , 2014