1-Dehydroascorbic acid

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

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. 

Chromatographic methods, notably hplc, are available for the simultaneous deterrnination of ascorbic acid as weU 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 diketogulonic acid. Detection has been by fluorescence, uv absorption, or electrochemical methods (83—85). Polarographic methods have been used because of their accuracy and their ease of operation. Ion exclusion (86) and ion suppression (87) chromatography methods have recently been reported. Other methods for ascorbic acid deterrnination include enzymatic, spectroscopic, paper, thin layer, and gas chromatographic methods. ExceUent reviews of these methods have been pubHshed (73,88,89). 

Absorption, Transport, and Excretion. The vitamin is absorbed through the mouth, the stomach, and predominantly through the distal portion of the small intestine, and hence, penetrates into the bloodstream. Ascorbic acid is widely distributed to the cells of the body and is mainly present in the white blood cells (leukocytes). The ascorbic acid concentration in these cells is about 150 times its concentration in the plasma (150,151). Dehydroascorbic acid is the main form in the red blood cells (erythrocytes). White blood cells are involved in the destmction of bacteria. 

Ascorbic acid, for example, is oxidized to dehydroascorbic acid with reduction of the iron(III) ions. The Fe(II) ions so produced react with 2,2 -bipyridine with formation of a colored complex. 

Dehydroascorbic acid hRf 45 — 50) appears as a yellow zone on a colorless background. 

The detection limit for dehydroascorbic acid is 10 ng per chromatogram zone. 

Fig. 1 Absorption scan of a chromatogram containing 200 ng dehydroascorbic acid per chromatogram zone.

Fig. 1 Fluorescence scans of chromatogram tracks with 200 ng ascorbic acid (A) and 200 ng dehydroascorbic acid (B) Start (1), ascorbic acid (2), dehydroascorbic acid (3), -front (4).

In long-wavelength UV light (2 = 365 nm) ascorbic acid (hRf 45 — 50) and dehydroascorbic acid (hRf 50—55) yielded green fluorescent zones on an orange-colored background. The detection limits were less than 5 ng substance per chromatogram zone. 

Following the formation of dehydroascorbic acid, the reaction proceeds irreversibly the degradation process produces oxalates, formates, and carbon dioxide, depending on temperature and pH. As usual, the oxygen-scavenging reaction rate is increased by raising the pH and the temperature. 

Note Dehydroascorbic acid, the decomposition product of ascorbic acid, does not react. But it can be detected as a yellow-orange chromatogram zone (hRf 65-70) by further treatment of the chromatogram with 2,4-dinitrophenylhydrazine. This sequence... 

Fig 1 Reflectance scan of a chromatogram track with 500 ng each of ascorbic acid (1) and dehydroascorbic acid (2) per chromatogram zone. 

Note The photometric detection limits for ascorbic and dehydroascorbic acids are less than 50 ng substance per chromatogram zone. 

Ascorbic acid (h/ f 50-55) and dehydroascorbic acid (hRf 65-70) appeared as yellow chromatogram zones on a colorless background. 

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