Desenvolvimento de método cromatográfico (HPLC-UV) para a determinação de ácidos fenólicos extraídos por ultra-som de forrageiras tropicais

Abstract

Livestock performance can be improved by increasing the digestibility of forage. The p-coumaric acid and ferulic acid are associated with the components of the cell wall by linking ester or ether, which can negatively influence the digestibility of forage. This paper proposes a method for ultrasound based extraction and separation by HPLC for ferulic acid, p-coumaric acid, m-coumaric acid, ocoumaric acid, caffeic acid and shikimic acid, and quantification of ferulic acid and p-coumaric acid ester linked to cell wall in samples of tropical forages. The method of analysis was optimized in a high performance liquid chromatography with UV-VIS detector MWD and a reverse-phase column C-18. Composition and pH of the mobile phase and wavelengths of detection were parameters studied in the optimization of the method. In the study of the composition of mobile phase were tested various organic solvents and aqueous solutions at different pH values, where the best condition was obtained with isocratic elution using as mobile phase acetonitrile / methanol /H3PO4 pH 2.05 (13:12 , 5:74,5). In order to reduce the time in the processing of samples, the extraction was studied using the ultrasound, where it proved to be a more rapid and reproducible compared to the bath with programmed temperature. Was the assessment of method applying external calibration and internal calibration. As the internal calibration using the o-coumaric acid as internal standard and the m-coumaric acid as surrogate standard showed good results, including detection limits of 0.09 and 0.04 mg / L for p-coumaric acid and ferulic acid, respectively, and the recovery values ranging from 83 to 99%, we chose to quantify the samples using this method. Thus, the optimized methodology was applied to 43 samples of forage (supplied by Embrapa Gado de Leite) of four species: Cynodon nlemfuensis cv. Florona, Cynodon dactylon cv. Florakirk, Panicum maximum cv. Mombaça and Brachiaria brizantha cv. Marandu. We analyzed stem and leaves of these samples, the range of values found was from 3.63 to 9.04 mg / g dry weight for the p-coumaric acid and 3.35 to 7.69 mg / g dry weight to ferulic acid.