Fresh Dual Presaturation Method for Analyzing H₂O-Rich Samples Using Quantitative ¹H NMR

A fresh dual presaturation (pre-SAT) method was developed to quantify analytes accurately near the suppressed water signal in ¹H NMR spectra obtained from H₂O-rich samples. The method includes an extra dummy pre-SAT with a suitable offset for each analyte signal in addition to the water pre-SAT. The residual HOD signal at 4.66 ppm was observed using D₂O solutions containing l-phenylalanine (Phe) or l-valine (Val) and an internal standard of 3-(trimethylsilyl)-1-propanesulfonic acid-d₆ sodium salt (DSS-d₆). When the HOD signal was suppressed using the conventional single pre-SAT method, the measured concentration of Phe from the NCH signal at 3.89 ppm decreased by a maximum of 48%, whereas the dual pre-SAT method gave a reduction in the Phe concentration measured from the NCH signal of less than 3%. The proposed dual pre-SAT method achieved accurate quantification of glycine (Gly) and maleic acid (MA) in a 10 vol % D₂O/H₂O solution. The measured concentrations of Gly of 513.5 ± 8.9 mg kg^-1 and MA of 512.2 ± 10.3 mg kg^-1 corresponded to sample preparation values of Gly of 502.9 ± 1.7 mg kg^-1 and MA of 506.7 ± 2.9 mg kg^-1 (the number after "±" indicates the expanded uncertainty (k = 2)).