Harmonized ¹H NMR Workflow Enables Quantitative Betaine Determination from Nontargeted Metabolite Profiling Using...

Nontargeted metabolite profiling prioritizes robust comparisons of the analytical outcomes rather than absolute concentration measurement. In this work, it is shown that a harmonized 1D ¹H NMR workflow, originally adopted for nontargeted NMR analysis, can also support reliable quantitative determination of betaine when spectra acquired under profiling-oriented conditions, nonideal for quantification, are anchored to gravimetrically traceable standards and corrected by a suitable factor accounting for bias in absolute concentration estimates. This study presents the results of an interlaboratory comparison designed to investigate the main factors affecting the accuracy and reproducibility of nontargeted ¹H NMR data when different spectrometers and operators are involved. The case study focused on the determination of betaine in aqueous extracts of durum wheat (cvs. Marco Aurelio and Iride) and the corresponding pasta products. A common set of samples was analyzed using a harmonized acquisition protocol across 50 spectrometers operating at magnetic field strengths ranging from 80 to 700 MHz. Two data-processing strategies were compared: operator-dependent processing (multiple operators using different software packages) and centralized processing (single operator) performed with five different software platforms. Quantification was carried out by both an internal standard method, using 3-(trimethylsilyl)-2,2,3,3-tetradeutero-propionic acid, sodium salt (TSP-d₄) as a reference, and an external standard method, employing TSP-d₄, dimethyl sulfone (DMSO₂), and betaine as references. The results demonstrated that the largest source of variability lies in operator-dependent data-processing choices rather than instrumental characteristics. TSP-d₄ systematically overestimated the betaine concentration and introduced additional variability. By contrast, DMSO₂ and betaine provided accurate and highly precise quantification with Horwitz ratios consistently below unity, indicating reproducibility superior to generic interlaboratory expectations. Internal standard method also achieved reproducibility within the accepted 0.5-2.0 HorRat range. Overall, this work shows that spectra acquired for nontargeted metabolite profiling can support quantitative determination of betaine, and potentially of other selected metabolites, provided that the same acquisition and processing protocol is maintained and that appropriate gravimetrically traceable calibration is applied.