Harmonized <sup>1</sup> H NMR Workflow Enables Quantitative Betaine Determination from Nontargeted Metabolite Profiling Using Internal and External Standards

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.

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Journal
Analytical Chemistry
Published
2026-05-29
DOI
https://doi.org/10.1021/acs.analchem.5c05198
Primary Topic
Metabolomics and Mass Spectrometry Studies
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article
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article

Harmonized 1 H NMR Workflow Enables Quantitative Betaine Determination from Nontargeted Metabolite Profiling Using Internal and External Standards

Enrico Caneva, Marica Antonicelli, Mario Latronico, Piero Mastrorilli et al.
Analytical Chemistry
Metabolomics and Mass Spectrometry Studies
article

Harmonized 1 H NMR Workflow Enables Quantitative Betaine Determination from Nontargeted Metabolite Profiling Using Internal and External Standards

Enrico Caneva, Marica Antonicelli, Mario Latronico, Piero Mastrorilli, Mattia Spano, Luana Bontempo, Stefano Todisco, Adele Mucci, Rosa Ragone, Chiara Portesi, Roberto Gobetto, Nunzia Iaccarino, Diego Brancaccio, Dessislava Gerginova, Roland Molinié, Antonino Rizzuti, Cristina Airoldi, Marina Veronesi, Greta Petrella, Teresa Recca, Pasquale Illiano, Giacomo Di Matteo, Thomas Kuballa, Catherine Deborde, G. Hernández, Sylvie Géhanne, Algirdas Šačkus, Biagia Musio, Nicola Intini, István Timári, Valentina Petrelli, Annamaria Greco, Mihaela Bălan, Luca Goldoni, Călin Deleanu, Antonio Randazzo, Roberto Consonni, Aurimas Bieliauskas, Francesca Benevelli, Asma Bourafai-Aziez, Emanuela Callone, Cătălin Duduianu, Alina Nicolescu, Dirk W. Lachenmeier, Vito Gallo, Svetlana Simova, Maria Trisolini, Claudia Napoli, Luca Fusaro, Angeles Canales Mayordomo, Yavor Mitrev, Luca Piemontese, Pavel Solovyev, Davide Bertelli, Nicola Genna, Anatoly Petrovich Sobolev, Luca Antoniacomi, Iain J. Day, Jan Teipel, John Edwards, Mirjam Schmidt
article en

Abstract

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.

Analytical Chemistry
Fondazione Edmund Mach (IT), Agenzia Regionale per la Protezione Ambientale del Piemonte (IT), University of Rome Tor Vergata (IT), Universidad Complutense de Madrid (ES), University of Modena and Reggio Emilia (IT), University of Debrecen (HU), University of Namur (BE), Kaunas University of Technology (LT), Universidad de la República de Uruguay (UY), University of Milan (IT), Bruker (United States) (US), University of Trento (IT), University of Pavia (IT), Italian Institute of Technology (IT), Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (FR), Biologie du Fruit et Pathologie (FR), Istituto di Biologia e Biotecnologia Agraria (IT), Poughkeepsie Public Library District (US), Institute of Organic Chemistry with Centre of Phytochemistry (BG), Institute of Macromolecular Chemistry (UA), Terre des Sciences (FR), Biologie des Plantes et Innovation (FR), Presidio Ospedaliero (IT), Istituto per le Tecnologie Didattiche (IT), Genesis Biotechnologies (United States) (US), Agenzia Regionale per la Protezione dell'ambiente ligure (IT), Unité mixte de recherche Œnologie (FR), Istituto Nazionale di Ricerca Metrologica (IT), Tecnologie Avanzate (Italy) (IT), MetaboHUB (FR), Lower Saxony State Office for Consumer Protection and Food Safety (DE), Chemisches und Veterinäruntersuchungsamt Karlsruhe (DE), Innovative Scientific Solutions (United States) (US), Instituto de Química Orgánica General (ES), Universidad La República (CL), Bank of England (GB), University of Turin (IT), University of Bari Aldo Moro (IT), Chiesi (France) (FR), Romanian Academy (RO), Universitatea Națională de Știință și Tehnologie Politehnica București (RO), University of Milano-Bicocca (IT), Polytechnic University of Bari (IT), University of Naples Federico II (IT), Sapienza University of Rome (IT), Evotec (United States) (US)
Agence Nationale de la Recherche, Regione Puglia, Provincia Autonoma di Trento
Openalex Percentile: Top 9%
Metabolomics and Mass Spectrometry Studies
DOIOpen Access PDFOpenAlex
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