Electronic paramagnetic resonance (EPR) for the study of ascorbyl radical and lipid radicals in marine organisms.

Electron paramagnetic resonance (EPR) spectroscopy detects the presence of radicals of biological interest, such as ascorbyl radical (A(•)) and lipid radicals. A(•) is easily detectable by EPR even in aqueous solution at room-temperature. Under oxidative conditions leading to changes in total ascorbate (AH(-)) content, the A(•)/AH(-) ratio could be used to estimate early oxidative stress in the hydrophilic milieu. This methodology was applied to a wide range of aquatic systems including algae, sea urchin, limpets, bivalves and fish, under physiological and oxidative stress conditions as well. The A(•)/AH(-) ratio reflected the state of one part of the oxidative defense system and provided an early and simple diagnosis of environmental stressing conditions. Oxidative damage to lipids was assessed by the EPR-sensitive adduct formation that correlates well with cell membrane damage with no interference from other biological compounds. Probe instability, tissue metabolism, and lack of spin specificity are drawback factors for employing EPR for in vivo determination of free radicals. However, the dependability of this technique, mostly by combining it with other biochemical strategies, enhances the value of these procedures as contributors to the knowledge of oxidative condition in aquatic organisms.

Monitoring of vitamin C species in aqueous solution by flow injection analysis coupled with an on-line separation with reversed-phase column.

Two vitamin C species of ascorbic acid and dehydroascorbic acid in aqueous solution were monitored by flow injection analysis. Ascorbic acid and dehydroascorbic acid were resolved by a reversed-phase column, and dehydroascorbic acid was reduced to ascorbic acid by an on-line post-column reaction with dithiothreitol. Both natural and reduced ascorbic acids were photometrically detected at 260 nm, and the two vitamin C species were simultaneously determined. The determination range was from 0 to 8 x 10(-5)M with a limit of detection of 1.7 x 10(-6)M. The proposed method was applied to the conversion monitoring of ascorbic acid and dehydroascorbic acid in weakly acidic to weakly alkaline aqueous solutions, as well as to the determination of the vitamin C in some beverage samples.

Separation of ascorbic acid, dehydroascorbic acid, diketogulonic acid and glucose by isocratic elution from a column of a hydrophilic gel.

High-performance liquid chromatography on an Asahipak GS-320 hydrophilic gel column with tartrate buffer (0.015 M, pH 3.0) containing 2 mM ethylenediaminetetraacetate and 0.05% beta-thiodiglycol as the eluent allowed the separation of glucose, diketogulonic acid, dehydroascorbic acid and ascorbic acid within 30 min. Fluorimetric monitoring of these compounds in the eluate with benzamidine at alkaline pH and at 90 degrees C in the presence of potassium sulphite allowed the determination of nanogram amounts of ascorbic acid, dehydroascorbic acid and diketogulonic acid. This method was applied to the determination of ascorbic acid in fruit juice.