HPLC Analysis of Grapevine Phytoalexins Coupling Photodiode Array Detection and Fluorometry

A reversed-phase HPLC method useful for the analysis of the grapevine phytoalexins resveratrol, its β-d-glucoside, ε-viniferin, and pterostilbene in leaf extracts was developed by coupling diode array detection and fluorometry. Phytoalexins were extracted from UV-irradiated grapevine leaves with met...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 1997-12, Vol.69 (24), p.5172-5177
Main Authors: Jeandet, Philippe, Breuil, Anne Céline, Adrian, Marielle, Weston, Leslie A, Debord, Sylvain, Meunier, Philippe, Maume, Gabrielle, Bessis, Roger
Format: Article
Language:English
Subjects:
Agronomy. Soil science and plant productions
Biological and medical sciences
Chemical constitution
Chemistry
Economic plant physiology
Flowers & plants
Fundamental and applied biological sciences. Psychology
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Summary:A reversed-phase HPLC method useful for the analysis of the grapevine phytoalexins resveratrol, its β-d-glucoside, ε-viniferin, and pterostilbene in leaf extracts was developed by coupling diode array detection and fluorometry. Phytoalexins were extracted from UV-irradiated grapevine leaves with methanol/water (80:20) and prepurified on C18 solid phase extraction cartridges. Separation by HPLC was achieved using a C18 column and a gradient elution with acetonitrile and water (from 10 to 85% acetonitrile). Analyses of grapevine leaf extracts were performed by injecting the equivalent of 1 mg of leaf fresh weight. Recovery was nearly 100% for the three stilbenes resveratrol, pterostilbene, and ε-viniferin (ranging from 100.2 to 104.8%), and replicate analyses gave coefficients of variation of 0.6−2.5%. Identification of each phytoalexin was accomplished by line spectral comparisons with known standards, and ε-viniferin was further characterized by MS and GC/MS. Simultaneously, stilbenes were detected by fluorometry, allowing specific identification of these compounds. This procedure provided excellent separation and enabled quantitation of all grapevine phytoalexins present in the extracts. The method can easily be extended to the analysis of wine or biological fluids.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac970582b