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Determination of Amino Acids in Tunisian Animal Feedstuffs by HPLC-FLD


Intissar Limam, Raouf Medimagh, Fayçal Hellal

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Keywords

Amino acids, animal feedstuffs, HPLC-FLD, doehlert factorial design

Abstract

The amino acids supplemented to animal feedstuffs cannot be determined directly. Thus, we need to optimize the sample preparation step as well as the analytical method. In this study, two hydrolysis mode were performed, one using 6 mol.L-1 hydrochloric acid at 105°C for 24 h and the other based on microwave radiation-induced hydrolysis. Moreover, a chromatographic method was developed for simultaneous determination of methionine and lysine in feedstuffs, using conventional High Performance Liquid Chromatography equipment equipped with an automated precolumn derivatization system using ortho-phthalaldehyde (OPA) / mercapto-2-ethanol (ME) reagents and the fluorescence detector. Reversed-phase chromatography appears to be the preferred method, with a C18 stationary phase and a gradient elution. Doehlert matrix was used to get the optimal separation for 18 amino acids. System Suitability Test (SST), Linearity, Limit of Detection (LOD), Limit of Quantification (LOQ), Repeatability and Specificity of the method were studied. The LOD calculated were 93 and 9.4 pmol.μL-1 and the LOQ were 173.5 and 36.2 pmol.μL-1 for methionine and lysine, respectively.