The ERETIC (Electronic REference To access In vivo Concentrations)2 method is a new qNMR experimental technique to measure analytes based on the signal of the reference compound without additional hardware equipment. In this study, ERETIC 2 method was validated, and we sought to identify whether it would be possible to apply this method to a specific compound analysis of metabolites in plant. The 90° pulse value (P1) and spin -lattice relaxation time ( T 1 ) of each compound were measured for ERETIC 2. The 9 1 H of 3-(trimethylsilyl) propionic -2,2,3,3 -d 4 acid (TSP) was used as a reference peak for ERETIC 2, and then, a suitable solvent and pulse sequence for each compound were selected. Under the NOESY -presat sequence, the relative accuracy e rror for quantitative analyses of primary metabolites was within the range of 5%, with the exception of glucose, which showed 5% error due to saturation. It showed excellent results for the quantification of glucose by using a 30° pulse sequence, which did not suppress the water peak. In addition, the qua ntitative accuracy for secondary metabolites was extremely accurate, with DQ HUURU ZKHQ FRQVLGHULQJ WKH SXULW\ RI WKHstandard sample. The ERETIC 2 method showed outstanding linearity, precision, and accuracy. Keywords qNMR, ERETIC , quantification Introduction Nuclear magnetic resonance (NMR) is a qualitative and quantitative analytical technique. It is the only physical method used routinely that can provide quantitative valuable information at the molecular level. Therefore NMR has found various a pplications in other fields than chemistry, such as metabolomics methodology
