Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/2007
Publication Date: 4/24/2007
Citation: Bai, C., Reilly, C.C., Wood, B.W. 2007. Identification of asparagine and citrulline using high-performance liquid chromatography (HPLC). Analytical Chemistry. 2:31-36. Interpretive Summary: Ureides are biochemicals key to the cycling of nitrogen in plants and are therefore important to plant productivity. New techniques were developed that enable the analysis of the amount of certain ureides that have heretofore proven problematic for plant sample analysis. The new technique provides an improved method for analysis of asparagine and citrulline in plant samples. These techniques facilitate the assessment of environmental or genetic factors on nitrogen metabolism and cycling in plants.
Technical Abstract: High-performance liquid chromatography (HPLC) analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates). Asparagine alone elutes earlier than citrulline alone, but when both are present in samples they may co-elute. HPLC retention times for asparagine and citrulline were influenced by other ureides in the mixture. We found that at various asparagines and citrulline rations [=3:1, 1:1, and 1:3; corresponding to 75:25, 50:50, and 25:75 (uMol/uMol)], the resulting peak exhibit different retention times. Adjustment of ureide ratios as internal standards enables peak identification and quantification. Both chemicals were quantified in xylem sap samples of pecan [Carya illinoinensis (Wangenh.) K. Koch] trees. Analysis found that tree nickel nutrition status affects relative concentrations of Urea Cycle intermediates, asparagine and citrulline, present in sap. The HPLC methods are therefore presented that enable qualitative and quantitative analysis of these metabolically important ureides.