Analysis of major tomato xylem organic acids and PITC-derivatives of amino acids by RP-HPLC and UV detection

Major amino acids and organic acids in xylem exudates of tomato plants were separated by reversed phase high performance liquid chromatography (RP-HPLC) and quantified by UV detection. Before separation, amino acids were converted into their phenylisothiocyanate (PITC) derivatives. In a single run, Asp, Glu, Ser, Gln, His, Thr, Ala, Tyr, Val, Met, Cys, Ile, Leu, Phe, and Lys could be separated and detected down to the pmol level. Unresolved peaks were obtained for Asn and Gly and for Arg and Pro. For organic acid analysis, exudates were pre-treated by perfusion over a prepacked Adsorbex SCX cation exchange column, to eliminate exudate amino acids. Elution recoveries for organic acids were close to 100%. The exudate organic acids were separated by ion suppression RP-HPLC chromatography, and peaks could be resolved for L-malic acid, malonic acid, maleic acid, citric acid and fumaric acid, down to the pmol level. UV signals for exudate ascorbic acid, and succinic acid were below the limits of detection. Determination of oxalic acid and tartaric acid was impossible, due to the presence of the exudate salt peak in the chromatogram. The results indicate the potential of the methods applied, and show the applicability of RP-HPLC analysis for the determination of both amino acids and organic acids in xylem exudates.