Estimation of Ascorbic Acid in Food Preparations SAhIUEL A . GOLDBLITH AND ROBERT S. H.4RRIS Massachusetts Institute of Technology, Cambridge 39, Mass. The dinitrophenylhydrazine and indophenol methods are equally talid for measuring the ascorbic acid content of garden-fresh tegetables. More than 90% of the ascorbic acid in garden-fresh plant materials studied was in the reduced form. Gardenfresh vegetables, slurried with 4 parts of 0.5% oxalic acid and stored for as long as 2 weeks at room temperature (70-77' F.), may be assayed by the indophenol method to determine the ascorbic acid and
dehydroascorbic acid content at the time of assay, and by the dinitrophenylhydrazine method to determine the biologicallj acthe ascorbic acid at the time of harvesting. For longer storage times, lower temperatures of storage must be used. The ascorbic acid content of garden-fresh edible plants may be measured in a laboratory remote fromi the harvest area. Both methods may be emploj ed to establish the freshness of vegetable foods.
0
F THE various chemical methods that have btwi proposed
ures araboascorbic acid, glucoascorbic acid, and diketogulonie acid as well as I-ascorbic acid. Pijoan and Gerjowich (8) found that the lioe and Oest,erling procedure would measure ascorbic acid together with other inactive compounds, one of which Fas believed to be 2,3-diketogulonic acid. Similar observations wrre made by Guild et al. (1). The observations summarized abovc suggest that a concurrent use of these two methods niiyht give not only information on t.he biological potency of a plant food at the time of analysis, but also an indication of the original potency of this food when taken from the soil. -1series of exptirinients was designed to test the validit,y of these concepts. These experiments were also plaiintd to provide suppleniental data on the proper met.hod for the extraction and stabilization of ascorhic acid from plant, materials, with particular reference t8o oxalic acid. In a study of thirteen aeids, l'onting (9) found that 0.2y0 oxalic acid and 0.5( ;. metaphosphoric acid were most suitable for stabilizing pure solutions of ascorbic acid. He reeomniended the use of tn-ice t,his concentration of oxalic acid t,o stabilize ascorbic acid in food extract,s. In t'he present, study, 0 , 5 c ; oxalic acid was used t h r o n p h o u .
for the assay of ascorbic acid, the indophenol method with niodifications has been the most widely used. Xore recently, Roe anti Oesterling (IO) proposed a method based on the coupling of dehydroascorbic acid with 2,4-dinitrophenylhydrazine. The osazone' dissolves in sulfuric acid to form a colored complex, t8he intensity of which serves as an indicator of the amount, of ascorbic arid present. Hotwver, these methods for the determination of vitamin C in foods must be applied with a clear understanding of interfering substances occurring naturally, or those derived from ascorbic acid itself. On t,he one hand, Tuba et a!. ( I I ) , Harris and Olliver iZj, and others have shown that the indophenol technique measures biologically active ascorbic acid in all foods tested, but. that this reaction gives no indication of what the original ascorbic acid eont,ent of foods might have been in foods that are not fresh. Because it is only seldom that, garden-fresh foods are taken for analysis, this latter point may be of extreme import'ance in judging data on foods of unknown history. On the other hand, l'enney and Zilva (6) have confirmed the work of Herbert' et al. ( 3 ) ,showing that ascorbic acid, in vitro, is easily oxidized to dehydroascorbic acid, which mutarotates to biologically inactive 2,3-diketogulonie acid. Similar product,s are fornied in living plant materials by enzyme systpnis or by autoxidation. Penney and Zilva furt,her showed that dinitrophenylhydrazine couples with !-ascorbic acid, and dehydroascorbic acid, and 2,3-diketoyulonie acid, and ( 7 ) that the Roc, and Ovsterling method nic'as-
Table I.
EXPERIXlENTA L
The dinitrophenylhydrazine and indophenol methods were used in this study. [In this paper dinitrophenylhydrazine met,hod refers to the Roe and Oent,erling procedure (IO) and indophenol
.Ascorbic .kcid Content of Stored Food Slurries as IIeasured hy Dinitrophenylh?drazino and Indophenol Reactions
[C;arden-freuh foods were slurried with 0.5
