Rocket-powered high-performance liquid chromatographic analysis of plant ascorbate and glutathione.
We describe a robust procedure for the extraction and high-performance liquid chromatographic analysis of L-ascorbate (vitamin C), glutathione (gamma-glutamyl cysteinylglycine), and their respective oxidized forms from various plant tissues. Parameters such as the choice of extraction buffer, tissue disruption technique, sample stability, and separation conditions have all been optimized. In particular we found that the inclusion of the reducing agent dithiothreitol as a "stabilizer" in extracts with high phenolic content actually promoted oxidation of these antioxidants. Further, by using commercially available short "Rocket" HPLC columns in combination with high mobile-phase flow rates, analysis times were reduced to only 6min, making the method suitable for the high-resolution screening of large numbers of samples.
Davey
M W
MW
Fruitteeltcentrum, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001, Heverlee, Belgium. mark.davey@agr.kuleuven.ac.be
Dekempeneer
E
E
Keulemans
J
J
D000975:Antioxidants / Q000032:analysis; D001205:Ascorbic Acid / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003683:Dehydroascorbic Acid / Q000032:analysis; D005978:Glutathione / Q000032:analysis*; D019803:Glutathione Disulfide / Q000032:analysis; D010084:Oxidation-Reduction; D010936:Plant Extracts / Q000737:chemistry*; D013997:Time Factors
D000975:Antioxidants; D010936:Plant Extracts; D005978:Glutathione; D001205:Ascorbic Acid; D019803:Glutathione Disulfide; D003683:Dehydroascorbic Acid