707
V O L U M E 2 1 , NO. 6, J U N E 1 9 4 9 ducibility, with varying success. X 2-mm. analytical gap was tried with the 40-mfd. exposure, but showed very little improvement. Pulverizing the sample to pass through a 200-mesh sieve improves the reproducibility somewhat, but it is questionable whether the improvement is sufficient to justify this additional expenditure of time. LITERATURE CITED
(1) Fitz, E. J., and Murray, TIr. M., ISD. ESG. CHEM..ANAL.ED.,17, 145-7 (1945).
and Scribnei B. F., J Research A'atl. B u r . Stand(2) Helz, A . K., ards, 38, 439-47 (1947). (3) Jaycox, E. K., Soc. Applied Spectroscopy Bull., 3, No. 3, 1-10 (April 1948). and Hoagbin, J. E., J . Am. Ceram. Soc., 29, 222-8 (4) Smith, R. W., (1946).
"Statistical Methods Applied to Experiments in ( 5 ) Snedecor, G. W., Agriculture and Biology," p. 36, Ames, Iowa, Iowa State College Press, 1946. (6) Zander, J. M., and Terry, J. H.. J . Am. Ceram. SOC.,30, 366-70 11947). RECEIYED October 21, 1948.
Ascorbic Acid, Dehydroascorbic Acid, and Diketogulonic Acid In Fresh and Processed Foods 3IARY B. MILLS, CHARLOTTE RI. DAMRON, AND JOSEPH H. ROE School of Medicine, George Washington C'nirersity, Washington, D . C . Foods in various states of preservation were analyzed by the method of Roe, Mills, Damron, and Oesterling for L-ascorbic acid, dehydro- L-ascorbic acid, and diketo-L-gulonic acid. Values obtained are reported. The majority of fresh foods assayed showed less than 5 % of the total vitamin C-like compounds present as the antiscorbutically inactive diketo-
T
HE primary oxidation product of L-ascorbic acid in plant materials is dehydro-Irascorbic acid. The latter is not a stable compound; it undergoes spontaneous conversion to a second product which has been fairly well characterized as 2,3diketo-bgulonic acid, formed by the opening of the lactone ring of dehydroascorbic acid (I, 3). Dehydroascorbic acid has considerable antiscorbutic potency because it is readily reduced to ascorbic acid in the animal body (4,6, IO). The exact value for its antiscorbutic activity has not been satisfactorily determined, but it is probably about 75010 of that of ascorbic acid. Diketogulonic acid, on the other hand, has no demonstrable antiscorbutic activity ( 4 ) . The actual antiscorbutic potency of a foodstuff depends on the amount of both ascorbic acid and dehydroascorbic acid present. The oxidation-reduction methods for the determination of vitamin C are reliable only if all the antiscorbutic material present is ascorbic acid, and other reducing substances are not present in large enough concentrations to interfere. This ideal condition is usually not encountered. The Roe-Kuether ( 7 ) and Roe-Oesterling (9) methods, which use 2,4dinitrophenylhydrazine to couple with the oxidized forms of ascorbic acid, do not differentiate between dehydroascorbic acid and diketogulonic acid, both of which react with the reagent to give an identical derivative. The original dinitrophenylhydrazine methods are accurate for antiscorbutic assay only when ascorbic acid and dehydroascorbic acid occur in the food and diketogulonic acid has not been formed in appreciable amounts. This is some improvement over the oxidation-reduction methods by virtue of increased specificity and the inclusion of the dehydroascorbic acid portion, but these methods lose their usefulness when diketogulonic acid is present in the foodstuff, unless they are used as Guild, Lockhart, and Harris suggest (8) to show what the original ascorbic acid content of the foodstuff was a t the time of harvesting.
gulonic acid. Processed foods contained more of the oxidized forms of ascorbic acid, and dehydrated foods showed the greatest amount of the inactive diketogulonic acid. The rate of change of ascorbic acid into dehydroascorbic acid and of dehydroascorbic acid into diketogulonic acid in orange juice and potato slurry during storage at 2' C. wasfollowed. The new method developed by Roe, Mills, Damron, and Oesterling (8) permits the simultaneous determination of ascorbic acid, dehydroascorbic acid, and diketogulonic acid in the same tissue filtrate. It is based upon the following principles. Diketogulonic acid is the oxidation product of ascorbic acid in a metaphosphoric acid filtrate that is not reducible by hydrogen sulfide and couples with 2,4dinitrophenylhydrazine. Dehydroascorbic acid is the fraction in the filtrate that is reducible by hydrogen sulfide and couples with the reagent only before reduction. The ascorbic acid in the filtrate does not couple with 2,4dinitrophenylhydrazine under the conditions of the method until after it is oxidized by bromine. The analytical steps involved theoretically confer a high degree of specificity upon the method used. This procedure should show the closest correlation possible up to the present time between the antiscorbutic biological assay and the actual chemical analysis of a foodstuff. There are many reports in the literature of the discrepancies between biological and chemical assays for vitamin C. There are also instances of comparative analyses of stored foodstuffs wherein the indobhenol value decreased and the dinitrophenylhydrazine value remained constant (5) or increased (7) with storage. These results are consistent with the known chemistry of ascorbic acid, dehydroascorbic acid, and diketogulonic acid. Storage with the resultant oxidation of ascorbic acid to dehydroascorbic acid would decrease the indophenol value; however, the biological potency would change but little until diketogulonic acid was formed from the dehydroascorbic acid. As soon as diketogulonic acid was produced by "mutarotation" of the dehydroascorbic acid, the biological assay value would begin to decrease, but the dinitrophenylhydrazine assay value by the original methods (7, 9) would remain the same, or show an increase because of the more rapid rate of reaction of diketogulonic acid with 2,4-dinitrophenylhydrazine in the 3-hour coupling period employed.
ANALYTICAL CHEMISTRY
708 Tahle 1. Fresh Fruits iind Vegetables
E 1-00,1
DHA'
AsAO llii
3.0 17.0 50.0 36.8 50.7 25.0 41.3 7.7: i.7 17.0 33.0 8 4 . .5 42.5 7 9 . ,i 1 .o 26.2 2.3 188.0 32.8 6.0 276.0 21.8 156.0 30.0 26.0
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