Survey of ERETIC2 NMR for quantification

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, ERETIC2 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 $90^{\circ}$ pulse value (P1) and spin-lattice relaxation time ($T_1$) of each compound were measured for ERETIC2. The $9^1H$ 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 error for quantitative analyses of primary metabolites was within the range of 5%, with the exception of glucose, which showed ${\geq}$ 55% error due to saturation. It showed excellent results for the quantification of glucose by using a $30^{\circ}$ pulse sequence, which did not suppress the water peak. In addition, the quantitative accuracy for secondary metabolites was extremely accurate, with an error ${\leq}$5% when considering the purity of the standard sample. The ERETIC2 method showed outstanding linearity, precision, and accuracy.