{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T06:13:51Z","timestamp":1780467231553,"version":"3.54.1"},"reference-count":42,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,2,26]],"date-time":"2026-02-26T00:00:00Z","timestamp":1772064000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004837","name":"Ministerio de Ciencia e Innovaci\u00f3n","doi-asserted-by":"publisher","award":["PID2022-138780OB-C21"],"award-info":[{"award-number":["PID2022-138780OB-C21"]}],"id":[{"id":"10.13039\/501100004837","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Plants"],"abstract":"<jats:p>Viticulture faces increasing challenges due to the susceptibility of Vitis vinifera L. to biotic and abiotic stresses, which trigger defense responses involving the synthesis of secondary metabolites. Untargeted metabolomics has become a powerful tool to explore these metabolic changes; however, the efficiency and reproducibility of metabolomic studies strongly depend on the extraction protocol used. Current literature shows variability in sample handling, solvent composition, and extraction conditions. This study aimed to optimize an extraction protocol for secondary metabolites in grapevine leaves to ensure high recovery of compounds relevant to untargeted metabolomics. Leaves of Vitis vinifera L. cv. Tempranillo were collected, pooled, frozen, and ground under liquid nitrogen. A factorial design was used to evaluate the effects of sample mass, sample-to-solvent ratio, and solvent type on extraction efficiency. Extracts were analyzed using UHPLC-QTOF-MS in both positive and negative ionization modes, and multivariate statistical tools (PCA and OPLS-DA) were used to identify discriminant metabolites. Optimal extraction was achieved using 750 mg of leaf powder, a sample-to-solvent ratio of 100 mg\/Ml, and methanol 80% acidified with 0.1% of formic acid. This protocol maximizes the recovery of metabolites and provides a robust basis for future untargeted metabolomics studies of grapevine responses.<\/jats:p>","DOI":"10.3390\/plants15050696","type":"journal-article","created":{"date-parts":[[2026,2,26]],"date-time":"2026-02-26T11:23:45Z","timestamp":1772105025000},"page":"696","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Optimization of an Extraction Protocol for Untargeted Metabolomics of Vitis vinifera L. Leaves"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-7119-8566","authenticated-orcid":false,"given":"Laura","family":"S\u00e1nchez-Ochoa","sequence":"first","affiliation":[{"name":"Grupo VIENAP, Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad de La Rioja, Gobierno de La Rioja), Ctra. de Burgos, Km. 6, 26007 Logro\u00f1o, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2054-9071","authenticated-orcid":false,"given":"Teresa","family":"Garde-Cerd\u00e1n","sequence":"additional","affiliation":[{"name":"Grupo VIENAP, Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad de La Rioja, Gobierno de La Rioja), Ctra. de Burgos, Km. 6, 26007 Logro\u00f1o, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Itziar","family":"S\u00e1enz de Urturi","sequence":"additional","affiliation":[{"name":"Grupo VIENAP, Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad de La Rioja, Gobierno de La Rioja), Ctra. de Burgos, Km. 6, 26007 Logro\u00f1o, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3919-2479","authenticated-orcid":false,"given":"Miriam","family":"Gonz\u00e1lez-L\u00e1zaro","sequence":"additional","affiliation":[{"name":"Grupo VIENAP, Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad de La Rioja, Gobierno de La Rioja), Ctra. de Burgos, Km. 6, 26007 Logro\u00f1o, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eva P.","family":"P\u00e9rez-\u00c1lvarez","sequence":"additional","affiliation":[{"name":"Grupo VIENAP, Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad de La Rioja, Gobierno de La Rioja), Ctra. de Burgos, Km. 6, 26007 Logro\u00f1o, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,26]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"Fungal Spores Affecting Vineyards in Montilla-Moriles Southern Spain","volume":"153","year":"2019","journal-title":"Eur. J. Plant Pathol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4837","DOI":"10.1007\/s11356-013-1841-4","article-title":"Elicitors as Alternative Strategy to Pesticides in Grapevine? Current Knowledge on Their Mode of Action from Controlled Conditions to Vineyard","volume":"21","author":"Delaunois","year":"2014","journal-title":"Environ. Sci. Pollut. Res."},{"key":"ref_3","first-page":"69","article-title":"Plant Cell Elicitation for Production of Secondary Metabolites: A Review","volume":"1","author":"Namdeo","year":"2007","journal-title":"Phcogn. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Dagher, D., Taskos, D., Mourouzidou, S., and Monokrousos, N. (2025). Microbial-Enhanced Abiotic Stress Tolerance in Grapevines: Molecular Mechanisms and Synergistic Effects of Arbuscular Mycorrhizal Fungi, Plant Growth-Promoting Rhizobacteria, and Endophytes. Horticulturae, 11.","DOI":"10.3390\/horticulturae11060592"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2731","DOI":"10.1021\/jf011429s","article-title":"Phytoalexins from the Vitaceae: Biosynthesis, Phytoalexin Gene Expression in Transgenic Plants, Antifungal Activity, and Metabolism","volume":"50","author":"Jeandet","year":"2002","journal-title":"J. Agric. Food Chem."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Krzyzaniak, Y., Trouvelot, S., Negrel, J., Cluzet, S., Valls, J., Richard, T., Bougaud, A., Jacquens, L., Klinguer, A., and Chiltz, A. (2018). A Plant Extract Acts Both as a Resistance Inducer and an Oomycide against Grapevine Downy Mildew. Front. Plant Sci., 9.","DOI":"10.3389\/fpls.2018.01085"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.pmpp.2008.07.002","article-title":"Role of Stilbenes in the Resistance of Grapevine to Powdery Mildew","volume":"72","author":"Schnee","year":"2008","journal-title":"Physiol. Mol. Plant Pathol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"101040","DOI":"10.1016\/j.genrep.2021.101040","article-title":"Secondary Metabolites in the Drought Stress Tolerance of Crop Plants: A Review","volume":"23","author":"Yadav","year":"2021","journal-title":"Gene Rep."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Adrian, M., Lucio, M., Roullier-Gall, C., H\u00e9loir, M.C., Trouvelot, S., Daire, X., Kanawati, B., Lema\u00eetre-Guillier, C., Poinssot, B., and Gougeon, R. (2017). Metabolic Fingerprint of Ps3-Induced Resistance of Grapevine Leaves against Plasmopara viticola Revealed Differences in Elicitor-Triggered Defenses. Front. Plant Sci., 8.","DOI":"10.3389\/fpls.2017.00101"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1551","DOI":"10.1016\/S1359-6446(05)03609-3","article-title":"Metabolomics: From Pattern Recognition to Biological Interpretation","volume":"10","author":"Weckwerth","year":"2005","journal-title":"Drug Discov. Today"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Chitarrini, G., Soini, E., Riccadonna, S., Franceschi, P., Zulini, L., Masuero, D., Vecchione, A., Stefanini, M., Di Gaspero, G., and Mattivi, F. (2017). Identification of Biomarkers for Defense Response to Plasmopara viticola in a Resistant Grape Variety. Front. Plant Sci., 8.","DOI":"10.3389\/fpls.2017.01524"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"20150370","DOI":"10.1098\/rsta.2015.0370","article-title":"Mass Spectrometry as a Quantitative Tool in Plant Metabolomics","volume":"374","author":"Jorge","year":"2016","journal-title":"Phil. Trans. R. Soc. A"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Goddard, M.L., Belval, L., Martin, I.R., Roth, L., Laloue, H., Degl\u00e8ne-Benbrahim, L., Valat, L., Bertsch, C., and Chong, J. (2021). Arbuscular Mycorrhizal Symbiosis Triggers Major Changes in Primary Metabolism Together with Modification of Defense Responses and Signaling in Both Roots and Leaves of Vitis vinifera. Front. Plant Sci., 12.","DOI":"10.3389\/fpls.2021.721614"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Ganugi, P., Caffi, T., Gabrielli, M., Secomandi, E., Fiorini, A., Zhang, L., Bellotti, G., Puglisi, E., Fittipaldi, M.B., and Asinari, F. (2023). A 3-Year Application of Different Mycorrhiza-Based Plant Biostimulants Distinctively Modulates Photosynthetic Performance, Leaf Metabolism, and Fruit Quality in Grapes (Vitis vinifera L.). Front. Plant Sci., 14.","DOI":"10.3389\/fpls.2023.1236199"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Avesani, S., Lazazzara, V., Robatscher, P., Oberhuber, M., and Perazzolli, M. (2023). Volatile Linalool Activates Grapevine Resistance against Downy Mildew with Changes in the Leaf Metabolome. Curr. Plant Biol., 35\u201336, 100298.","DOI":"10.1016\/j.cpb.2023.100298"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"130166","DOI":"10.1016\/j.foodchem.2021.130166","article-title":"Metabolomic Fingerprinting as a Tool for Authentication of Grapevine (Vitis vinifera L.) Biomass Used in Food Production","volume":"361","author":"Stranska","year":"2021","journal-title":"Food Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1186\/s40538-022-00299-y","article-title":"Variations of Elements, Pigments, Amino Acids and Secondary Metabolites in Vitis vinifera (L.) Cv Garganega after 501 Biodynamic Treatment","volume":"9","author":"Malagoli","year":"2022","journal-title":"Chem. Biol. Technol. Agric."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Wu, Z., Gao, T., Liang, Z., Hao, J., Liu, P., and Liu, X. (2023). Dynamic Changes in Plant Secondary Metabolites Induced by Botrytis cinerea Infection. Metabolites, 13.","DOI":"10.3390\/metabo13050654"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"110600","DOI":"10.1016\/j.plantsci.2020.110600","article-title":"Molecular Basis of Rootstock-Related Tolerance to Water Deficit in Vitis vinifera L. Cv. Sangiovese: A Physiological and Metabolomic Combined Approach","volume":"299","author":"Lucini","year":"2020","journal-title":"Plant Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"515","DOI":"10.1021\/ol990407c","article-title":"Absolute Configuration of Novel Bioactive Flavonoids from Tephrosia Purpurea","volume":"2","author":"Chang","year":"2000","journal-title":"Org. Lett."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"916","DOI":"10.1016\/S0891-5849(99)00177-X","article-title":"Glutathione and Its Role in Cellular Functions","volume":"27","author":"Sies","year":"1999","journal-title":"Free Radic. Biol. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4549","DOI":"10.1093\/jxb\/erae194","article-title":"Glutathione: A Key Modulator of Plant Defence and Metabolism through Multiple Mechanisms","volume":"75","author":"Noctor","year":"2024","journal-title":"J. Exp. Bot."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Zechmann, B. (2020). Subcellular Roles of Glutathione in Mediating Plant Defense during Biotic Stress. Plants, 9.","DOI":"10.3390\/plants9091067"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Xiao, M., Li, Z., Zhu, L., Wang, J., Zhang, B., Zheng, F., Zhao, B., Zhang, H., Wang, Y., and Zhang, Z. (2021). The Multiple Roles of Ascorbate in the Abiotic Stress Response of Plants: Antioxidant, Cofactor, and Regulator. Front. Plant Sci., 12.","DOI":"10.3389\/fpls.2021.598173"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1006\/anbo.1996.0175","article-title":"The Function and Metabolism of Ascorbic Acid in Plants","volume":"78","author":"Smirnoff","year":"1996","journal-title":"Ann. Bot."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2682","DOI":"10.1093\/jxb\/erae023","article-title":"The Ascorbate\u2013Glutathione Cycle Coming of Age","volume":"75","author":"Foyer","year":"2024","journal-title":"J. Exp. Bot."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"22684","DOI":"10.1074\/jbc.M604050200","article-title":"Separation and Identification of Major Plant Sphingolipid Classes from Leaves","volume":"281","author":"Markham","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e351","DOI":"10.1002\/pld3.351","article-title":"Phytosphingosine Induces Systemic Acquired Resistance through Activation of Sphingosine Kinase","volume":"5","author":"Seo","year":"2021","journal-title":"Plant Direct"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1081","DOI":"10.1038\/s41598-022-05083-4","article-title":"The Major Plant Sphingolipid Long Chain Base Phytosphingosine Inhibits Growth of Bacterial and Fungal Plant Pathogens","volume":"12","author":"Glenz","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"\u0160tambuk, P., \u0160ikuten, I., Karoglan Konti\u0107, J., Maleti\u0107, E., Preiner, D., and Tomaz, I. (2022). Leaf Polyphenolic Profile as a Determinant of Croatian Native Grapevine Varieties\u2019 Susceptibility to Plasmopara viticola. Front. Plant Sci., 13.","DOI":"10.3389\/fpls.2022.836318"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"14241","DOI":"10.1038\/s41598-020-70706-7","article-title":"Age- and Season-Dependent Pattern of Flavonol Glycosides in Cabernet Sauvignon Grapevine Leaves","volume":"10","author":"Bouderias","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1007\/s00425-012-1776-x","article-title":"Influence of Constitutive Phenolic Compounds on the Response of Grapevine (Vitis vinifera L.) Leaves to Infection by Plasmopara viticola","volume":"237","author":"Latouche","year":"2012","journal-title":"Planta"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Zhang, W., Qin, W., Li, H., and Wu, A.M. (2021). Biosynthesis and Transport of Nucleotide Sugars for Plant Hemicellulose. Front. Plant Sci., 12.","DOI":"10.3389\/fpls.2021.723128"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1617","DOI":"10.1093\/plphys\/kiab016","article-title":"UGT84F9 Is the Major Flavonoid UDP-Glucuronosyltransferase in Medicago Truncatula","volume":"185","author":"Adiji","year":"2021","journal-title":"Plant Physiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"uhae277.","DOI":"10.1093\/hr\/uhae277","article-title":"Grapevine Cell Response to Carbon Deficiency Requires Transcriptome and Methylome Reprogramming","volume":"12","author":"Berger","year":"2025","journal-title":"Hortic. Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"13134","DOI":"10.1038\/s41598-017-13464-3","article-title":"Insights into Grapevine Defense Response against Drought as Revealed by Biochemical, Physiological and RNA-Seq Analysis","volume":"7","author":"Haider","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"154622","DOI":"10.1016\/j.jplph.2025.154622","article-title":"Grapevine Adopts Different Strategies in Response to Drying Regimes. Procrastinator or Escaper?","volume":"314","author":"Morabito","year":"2025","journal-title":"J. Plant Physiol."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Burbidge, C.A., Ford, C.M., Melino, V.J., Wong, D.C.J., Jia, Y., Jenkins, C.L.D., Soole, K.L., Castellarin, S.D., Darriet, P., and Rienth, M. (2021). Biosynthesis and Cellular Functions of Tartaric Acid in Grapevines. Front. Plant Sci., 12.","DOI":"10.3389\/fpls.2021.643024"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"672","DOI":"10.1104\/pp.61.4.672","article-title":"Biosynthesis of (+)-Tartaric Acid from l-[4-14C] Ascorbic Acid in Grape and Geranium","volume":"61","author":"Williams","year":"1978","journal-title":"Plant Physiol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"5608","DOI":"10.1073\/pnas.0510864103","article-title":"L-Tartaric Acid Synthesis from Vitamin C in Higher Plants","volume":"103","author":"DeBolt","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Cholet, C., Claverol, S., Claisse, O., Rabot, A., Osowsky, A., Dumot, V., Ferrari, G., and G\u00e9ny, L. (2016). Tartaric Acid Pathways in Vitis vinifera L. (Cv. Ugni Blanc): A Comparative Study of Two Vintages with Contrasted Climatic Conditions. BMC Plant Biol., 16.","DOI":"10.1186\/s12870-016-0833-1"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1749","DOI":"10.1007\/s12649-021-01619-3","article-title":"Cellulose Nanofibers as Functional Biomaterial from Pineapple Stubbles via TEMPO Oxidation and Mechanical Process","volume":"13","author":"Rojas","year":"2022","journal-title":"Waste Biomass Valor."}],"container-title":["Plants"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2223-7747\/15\/5\/696\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,26]],"date-time":"2026-02-26T11:48:28Z","timestamp":1772106508000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2223-7747\/15\/5\/696"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,26]]},"references-count":42,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["plants15050696"],"URL":"https:\/\/doi.org\/10.3390\/plants15050696","relation":{},"ISSN":["2223-7747"],"issn-type":[{"value":"2223-7747","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,26]]}}}