Dehydroascorbic acid, determination

I. Dehydroascorbic Acid Determination by Reduction. A disadvantage of the dichlorophenolindophenol reduction method is that the dehydroascorbic acid can be determined only after its reduction to ascorbic acid. The reducing agent used may interfere in the subsequent reduction of the dye. Since ascorbic acid exists mainly in the reduced form in most tissues, this is not a serious disadvantage for the reduction method in the usual analysis. The reduction of dehydroascorbic acid to ascorbic acid is usually performed vi ith HaS by treatment for 15 min between pH 1.2 and 4.7 (R20). The completeness of the reduction was checked by the dinitrophenylhydrazine method. 

Another method that determines both ascorbic acid and dehydroascorbic acid first reduced the dehydroascorbic acid to ascorbic acid and then retains the ascorbic acid on an anionic Sephadex column (82). The ascorbic acid is oxidized on the column to dehyroascorbic acid by -benzoquinone, which simultaneously elutes the dehydroascorbic acid. The dehydroascorbic acid is reacted with 4-iiitro-l,2-phenylenediainine and absorbance of the resulting yeUow solution produced is measured at 375 nm. 

Lunec, J. and Blake, D.R, (1985). The determination of dehydroascorbic acid and ascorbic acid in the serum and synovial fluid of patients with rheumatoid arthritis. Free Rad. Res. Commun. 1, 31-39. 

However, its presence is not the only determinant of whether or not oxidative deterioration occurs. Olson and Brown (1942) showed that washed cream (free of ascorbic acid) from susceptible milk did not develop an oxidized flavor when contaminated with copper and stored for three days. Subsequently, the addition of ascorbic acid to washed cream, even in the absence of added copper, was observed to promote the development of an oxidized flavor (Pont 1952). Krukovsky and Guthrie (1945) and Krukovsky (1961) reported that 0.1 ppm added copper did not promote oxidative flavors in milk or butter depleted of their Vitamin C content by quick and complete oxidation of ascorbic acid to dehydroascorbic acid. Krukovsky (1955) and Krukovsky and Guthrie (1945) further showed that the oxidative reaction in ascorbic acid-free milk could be initiated by the addition of ascorbic acid to such milk. Accordingly, these workers and others have concluded that ascorbic acid is an essential link in a chain of reactions resulting in the development of an oxidized flavor in fluid milk. 

H Iwase, I Ono. Determination of ascorbic acid and dehydroascorbic acid in juices by high-performance liquid chromatography with electrochemical detection using L-cysteine as precolumn reduc-tant. J Chromatogr 654 215-220, 1993. 

WD Graham, D Annette. Determination of ascorbic and dehydroascorbic acid in potatoes (Solanum tuberosum) and strawberries using ion-exclusion chromatography. J Chromatogr 594 187-194, 1992. 

When dehydration occurs as a consecutive reaction, its effect on polarographic curves can be observed only, if the electrode process is reversible. In such cases, the consecutive reaction affects neither the wave-height nor the wave-shape, but causes a shift in the half-wave potentials. Such systems, apart from the oxidation of -aminophenol mentioned above, probably play a role in the oxidation of enediols, e.g. of ascorbic acid. It is assumed that the oxidation of ascorbic acid gives in a reversible step an unstable electroactive product, which is then transformed to electroinactive dehydroascorbic acid in a fast chemical reaction. Theoretical treatment predicted a dependence of the half-wave potential on drop-time, and this was confirmed, but the rate constant of the deactivation reaction cannot be determined from the shift of the half-wave potential, because the value of the true standard potential (at t — 0) is not accessible to measurement. 

V. Kmetec, Simultonous determination of acetylsalicylic, salicylic ascorbic and dehydroascorbic acid by HPLC, J. Pharm. Biomed. Anal., 70 1073 (1992). 

Sorouraddin HM, Hibara A, Proskurnin MA, Kitamori T. Integrated FIA for the determination of ascorbic acid and dehydroascorbic acid in a microfabricated glass-channel by thermal-lens microscopy. Anal Sci 2000 16 1033-1037. 

Gioia, M.G. et al. Development and validation of a liquid chromatographic method for the determination of ascorbic acid, dehydroascorbic acid and acetaminophen in pharmaceuticals. J. Pharm. Biomed. 2008, 48, 331-339. 

Chromatographic methods, notably hplc, are available for the simultaneous determination of ascorbic acid as well as dehydroascorbic acid. Some of these methods result in the separation of ascorbic acid from its isomers, eg, erythorbic acid and oxidation products such as cHketogulonic acid. Detection has been by fluorescence, uv absorption, or electrochemical methods (83—85). Polarographic methods have been used because of flieti accuracy and their ease of operation. Ion exclusion (86) and ion suppression (87) chromatography methods have recently been reported. Other methods for ascorbic acid determination include enzymatic, spectroscopic, paper, thin layer, and gas chromatographic methods. Excellent reviews of these methods have been published (73,88,89). 

The potential of PBI LC-MS in the analysis of various vitamins was explored by Careri et al. [99-100]. The fat-soluble vitamins A, D, and E were analysed in food and multivitamin preparations [99]. Absolute detection limits in SIM mode were 0.6-25 ng after fast leversed-phase separation using a 97% aqueous methanol as mobile phase. Mass spectra in El, positive-ion and negative-ion Cl were obtained and discussed. The mass-spectral and quantitative performance of PBI LC-MS in the analysis of eleven water-soluble vitamins was also explored [100]. Detection limits were determined in SIM mode under positive-ion Cl, and were below 15 ng for ascorbic acid, nicotinamide, nicotinic acid, and pyridoxal, around 100 ng for dehydroascorbic acid, panthothenic acid, and thiamine, and above 200 ng for biotin, pyridoxamime, and pyridoxine. Riboflavine was not detected. 

TLC has been widely used to determine ascorbic acid concentrations in foods,pharmaceutical preparations,and biological materials.Isomers of ascorbic acid and their oxidation product, dehydroascor-bic acid, were separated by TLC on sodium borate-impregnated silica gel and cellulose plates. This TLC method has been adapted to separate and identify ascorbic acid and dehydroascorbic acid in fresh orange and lime juices, pharmaceutical preparations (ascorbic acid), and guinea pig tissues (liver, kidney, and eye lens) and fluids (plasma and urine). 

Colorimetric Methods. The most frequently used colorimetric methods have been recently reviewed by Omaye et al. (5). Several methods of analyses are based upon the fact that ascorbic acid and dehydroascorbic acid possess certain chemical properties characteristic of sugars such as formation of osazones and conversion to furfural. Colorimetric determination of furfural, an aniline derivative, has been used to a limited extent for the estimation of ascorbic acid in certain materials. These methods have generally been found to be unsatisfactory... 

Fluorometric Methods. One of the most specific methods for the determination of ascorbic acid and its biologically active oxidation product, dehydroascorbic acid, is the fluorometric method introduced by Deutsch and Weeks (43), which is an official AOAC method (44). It is... 

Other Chemical and Physical Methods. Polarography has been tried in special investigations, such as studies of the bound form of ascorbic acid. But because of limited specificity, the procedure has not seen wide application (82,83). Ascorbic acid is oxidized at the dropping mercury electrode, the basis of the polarographic determination. Dehydroascorbic acid is not measured, however, since it is not reducible at the dropping mercury electrode. Mason et al. (84) have developed a method for the determination of ascorbic acid based on electrochemical oxidation at the tubular carbon electrode that has been modified to measure water-... 

The determination of ascorbic acid in biological materials, including foods and feeds, is beset with numerous technical problems. Hence, the methods used should be selected and conducted with extreme care with respect to reliability, specificity, sensitivity, and reproducibility. Depending upon the procedure selected, dehydroascorbic acid, hydroascorbic acid, or total ascorbic acid levels may be measured. At present, colori-... 

We have used the growth effects and pathologies associated with L-ascorbic acid deficiency as a basis for the determination of the biological potency of related compounds (Table I). At a dietary concentration of 0.5 mM, L-ascorbic acid and dehydroascorbic acid were fully active, as well as some ester derivatives including the 6-myristate and 2-phosphate compounds. The insect may be metabolically like the guinea pig because both were able to utilize those esters (17), Carboxylesterases and phosphatases probably converted those derivatives to the free vitamin (18). The 6-bromo compound was less active and apparently cannot be metabolized to L-ascorbic acid or only poorly so. 

Research Needs. Over the years L-ascorbic acid has been shown to be an essential nutrient for many insects including species of Lepidoptera, Orthoptera, Coleoptera, and Diptera. Others such as cockroaches, houseflies, and mealworms are reared on simple diets without added ascorbic acid. Perhaps those insects require very low levels of vitamin C in their diets. A sensitive analytical method is needed to measure levels of L-ascorbic acid and dehydroascorbic acid in insect tissue and food. Such a method, which is likely to be developed using HPLC with electrochemical detection, could be used to monitor vitamin C levels in feed ingredients as well as in tissues during an insect s life cycle. This information is needed to determine whether ascorbic acid is used to... 

Amplihcation factors of 8 to 12 were claimed for the determination of phenol in a FLA system by a cyclic process depicted in equations 5 (Section VI.A. 1). Phenol is converted to o-benzoquinone in contact with immobilized tyrosinase held in a fixed bed reactor the quinone reacts with ascorbic acid (91) to yield catechol and dehydroascorbic acid (136) catechol can be enzymatically oxidized again to o-benzoquinone and so forth. The accumulated dehydroascorbic acid forms with o-phenylenediamine (137) a highly fluorescent product (kex 345 nm, kfl 410 nm). LOD was ca 0.02 (xM for phenol and catechol the linear range for phenol was 0.1 to 2 p,M and for catechol 0.02 to 2... 

R18. Roe, J. H., and Kuether, C. A., The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivative of dehydroascorbic acid.. Biol. Chem. 147, 399-407 (1943). 

A local argument is going on, with one person claiming that generic labeled applesauce is poorer quality than the name brands. It is decided that a measure of the vitamin C (ascorbic acid) in each would settle the argument and because you are a chemist you have been selected to make the measurements. Vitamin C is not enough. Because dehydroascorbic acid is reduced to ascorbic acid upon standing, it is desired to determine the total amount and the amount of eaeh. 

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