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 beta-D-glucoside, epsilon-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 pre-purified 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 epsilon-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 epsilon-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.