IMPROVED UTILIZATION OF AG. PRODUCTS THROUGH IDENTIFICATION OF NITROGEN-CONTAINING BIOACTIVE COMPONENTS IMPORTANT TO QUALITY & HUMAN HEALTH

Nitrogen-containing plant metabolites are an important class of natural products that contribute to quality and utilization and span the range from essential nutrients to phytochemicals that affect mood and mental well being and, in some extreme cases, toxins. Due to complexity and difficulty in detection, past research in this area has targeted very specific compounds, resulting in most nitrogen-containing plant metabolites being largely ignored. Objective 1- Develop new or improved existing methods to detect, identify, and characterize N-containing plant metabolites. Establish extraction methods for N-containing metabolites from fruit of specialty crops (e.g., citrus, grapes, and tomatoes) and model plant species. Establish High Pressure Liquid Chromatography (HPLC) separation method for the resolution of multiple classes of nitrogen-containing metabolites in a single run. Compare existing methods for the detection and quantification of nitrogen-containing metabolites. Combine the elements of extraction, separation, identification and quantification into an optimized method for the profiling of N-containing plant metabolites. Objective 2- Screen specialty crops for their metabolomic profiles with a particular emphasis on nitrogen-containing metabolites. Initial efforts will focus on fruits from citrus, grapes, and tomatoes. Sub-Objective 2.1. Commence NMP and systematic identification and quantification of nitrogen-containing metabolites found in tissues of citrus, grapes, and tomatoes, using liquid chromatography coupled to mass spectrometer (MS & MS/MS) and nitrogen (CND and/or post column derivatization) detection systems. Sub-objective 2.2. Isolate and/or identify new or novel nitrogen- containing metabolites. This is a final report. This project has been extended as a bridging project, 2030-41430-012-00D entitled "Improved Utilization of Ag. Products through Identification of Nitrogen-containing Bioactive Components Important to Quality & Human Health." The project began with the receipt of several new and refurbished instruments purchased specifically to aid this project in meeting both objectives 1 and 2. Amongst the instruments acquired were flash and preparative chromatography systems for the isolation of compounds and a post-column reactor system that was intended to be adapted for the detection and quantification of nitrogen containing compounds. In order to capitalize on the new instrumentation, significant efforts were directed towards the introduction and adaptation of these instruments from the beginning of this reporting period and throughout the current fiscal year. To this end, existing laboratory methods for the isolation of the tomato glycoalkaloid Escueloside A, along with legacy methods for the isolation of citrus limonoids, were adapted to these instruments resulting in the rollout of streamlined methods that yielded these compounds at high purity in milligram quantities. Likewise, the post- column reactor was integrated into our existing high pressure liquid chromatography based analytical methods and evaluated for performance using para-dimethylaminobenzaldehyde as the indicator reagent. When applied to the detection and quantification of tomato glycoalkaloids the post-column reactor detection method was not as sensitive as either the chemiluminescence or mass-spectrometry detection systems. However, we found that the conditions tested were well suited for the detection and quantification of citrus limonoids. In addition to our efforts to fully integrate these instruments into our research, we continued our collaborative studies on tomatoes by expanding our metabolic profiling to include mineral content. Over 300 tomato samples gathered from commercial and research varieties were evaluated. We also initiated a collaborative study to evaluate the glycoalkaloid content found in potato tubers and to determine if the biosynthesis of glycoalkaloids is essential for tuber development. Existing and in-house methods were optimized for the analysis of potato glycoalkaloids and the resulting method applied to the analysis of over 40 lines of potatoes. Results from this exploratory study suggested that glycoalkaloids are essential for tuber development.