Sequential determination of metabolites involved in the biosynthesis of aromatic amino acids after ultrasound-assisted extraction from plants and reverse LC separation

A dual method is proposed for the determination of metabolites involved in the shikimate pathway which are biomarkers of the effects of glyphosate action on plants exposed to this herbicide. Extraction of the target metabolites (phenylalanine, tryptophan, tyrosine and shikimic acid) from a wheat model plant was accelerated by ultrasound energy. After centrifugation and micro-filtration, 1 μL of extract was injected into the chromatograph in an isocratic regime for 4 min to determine shikimate by absorption at 254 nm. In the mean time, a 130 μL aliquot of extract was subjected to derivatization with o-phthaldialdehyde and 2-mercaptoethanol for 1 min, the reaction stopped and 1 μL of the solution chromatographied in a gradient regime prior to laser-induced fluorescence detection of the derivatized amino acids. The characterization of the dual method provided limits of detection around 0.03 μg mL−1 for the aromatic amino acids and 1.52 μg mL−1 for shikimate, whereas the limits of quantitation ranged between 0.084 and 0.093 μg mL−1 for amino acids and was of 4.56 μg mL−1 for shikimate. The suitability of the method was checked by application to Triticum aestivum (wheat) plants grown under controlled conditions, sprayed with different doses of glyphosate and collected at different times after exposition to the herbicide.