Accuracy and Reproducibility of a 1H NMR protocol for Quantification of Betaine in Wheat and Pasta

Quantitative nuclear magnetic resonance (qNMR) spectroscopy is a powerful tool for chemical analysis. However, its accuracy and reproducibility can be influenced by various factors when different spectrometers and operators are involved. In this contribution, an extensive inter-laboratory comparison, aimed at identifying the primary sources of variability in qHNMR data processing, was presented. The target molecule chosen for ILC was the betaine, a biomarker correlated to the plant resistance to water-stress. Focusing on the quantification of betaine in aqueous extracts of durum wheat (cv. Marco Aurelio and Iride) and their corresponding pasta, the ILC involved 50 different spectrometers operating at magnetic fields ranging from 80 to 700 MHz. Two distinct data processing approaches were evaluated: multi-operator processing versus single-operator processing. Five different software platforms were compared in the single-operator approach. Additionally, quantification was conducted through both an internal standard1 and an external standard method2. This study underscores the importance of standardized correction strategies and harmonized data processing protocols, demonstrating that highly reproducible quantification methods based on 1H-NMR can be achieved even without traditional calibration curves. This research paves the way to the application of qNMR on a wider number of complex matrices, that are traditionally challenging or expensive to analyse with other methods, in multiple fields, such as nutraceuticals, pharmaceuticals, agriculture, and environmental sciences.