Purdue adheres to protocols as published in (Shanaiah et al., 2007) for identifying and quantifying novel primary metabolites not covered in target lists above. Briefly, after sample extraction according to Matson protocols (below), compounds are derivatized by 15N and 13C isotope tagging for greatly enhanced sensitivity and specificity. 2D coupling data acquisition by 1H- 15 N HSQC and 1H-13C HSQC will be performed within 15 min per sample on a Bruker Advance-III-800 spectrometer equipped with a room temperature 1H inverse detection Z- gradient probe and microcoil NMR approaches. Metabolites will be identified based on the library of the chemical shifts developed in the Raftery laboratory, and quantification will be based on ethanolamine and maleate internal standards. As NMR lends itself to absolute quantifications, NMR-based quantifications of high abundant metabolites will be used as validation method for mass spectrometry-based platforms. NMR data will be processed on an offline Redhat Linux platform using Bruker Topspin processing software and TSP as internal reference peak. Metabolites are annotated based on the NMR chemical shifts against the HMDB database and in-house spectra. Quantifications are performed using intermittent quality control samples and have been shown to yield better than 2% CV for blood plasma metabolites from replicate analyses (Ye et al., 2010) . Result data sheets will be delivered as csv documents to UC Davis using Duke sample identifiers and metabolite InChIKeys. Figure 3. Isotope tagging for 2D-NMR studies.