Suitability of 2,4‐dinitrophenylhydrazine Methods for Determining Dehydro‐ and Total Ascorbic Acid in Raw and Cooked Vegetables

Abstract

The 2,4-dinitrophenylhydrazine ( D N P H ) method for determining ascorbic acid of Roe and co-workers f7, 8,9) has generally yielded results which agree well with those found by indophenol methods. However, studies of the homogeneity of D N P H derivatives prepared from natural products (6) as well as analyses of foods subjected to severe heat treatments (2) have indicated the procedures may lack the desired specificity.In the present study, D N P H derivatives prepared under the usual analytical conditions for determining total ascorbic acid (AA) and dehydroascorbic acid (DHAA) in vegetable extracts were purified by chrornatographic procedures. Concentrations of AA determined from the fraction most closely resembling a reference compound prepared from crystalline AA were compared with those found by the usual D N P H methods. Studies of DHAA in unoxidized vegetable extracts are presented in Part I ; those of total AA in oxidized extracts in Part 11.Determination of DHAA presents some problems different from the ones associated with the determination of total AA. Generally, DHAA constitutes a relatively small part of the total AA content of fresh vegetables ( 3 , 4 , 5 ) . Thus, if non-AA components are present the ratio of the concentration of these components to that of DHAA would be greater than that of the components to total Ah. In addition, the possibility of interference by reduced AA exists in unoxidized extracts. METHODPreparation of Vegetable Samples. Cooking methods were those described by Halliday and Noble ( 1 ) for cooking in boiling water. Cabbage samples were blended with 8 parts by weight of 6% metaphosphoric acid; asparagus with 4 parts. Blended samples were centrifuged and filtered. Cooking waters were diluted with an equal volume of 12% metaphosphoric acid. (7,8,9) were combined into a general procedure referred to here as the D N P H method. 2,4-Dinitrophenylhydrazine (DNPH) Method. Procedures of Roe and co-workersThe DNPH reagent consisted of a 1% solution of D N P H in 4.5 M. sulfuric acid containing 1% thiourea. An incubation period of 3 hours at 37.0 2 0.5" C. was used. Following incubation, 12.5 M. sulfuric acid was employed to dissolve the D N P H derivative and the percent transmission of this solution at 520 mp was determined.Chromatographic analysis of D N P H derivatives. D N P H derivatives for chromatographic analysis were prepared under the same conditions used for the D N P H method except that the amount of each component in the reaction mixture was increased 100 times. That the difference in amounts did not affect the reaction was shown by approxi-' Scientific Journal Series Paper No. 3932, Minnesota Agricultural Experiment Station.'According to Roe, Mills, Oesterling, and Damron (8) determinations of DHAA in unoxidized extracts represent a composite of DHAA and diketogulonic acid. mately equal yields of D N P H derivative being obtained (0.189 and 0.184 mg. per ml. of original solution) regardless of whether small or large amounts of reactants were used.

SUITABILITY OF 2,4‐DINITROPHENYLHYDRAZINE METHODS FOR DETERMINING DEHYDRO‐ AND TOTAL ASCORBIC ACID IN RAW AND COOKED VEGETABLES II. TOTAL ASCORBIC ACIDa

“…The D N P H method and chromatographic analysis of DN PH derivatives were carried out as described (3), except that aliquots of the same vegetable extracts used for determining D H A A were treated with indophenol ( I ) , charcoal (7.9), bromine (8,9), or hydrogen sulfide followed by bromine (8) prior to adding the D N P H reagent. In addition, the concentration of thiourea in the D N P H reagent was reduced to 0.25%, the concentration suggested by Lowry, Lopez, and Bessey ( 5 j , since preliminary studies with cabbage indicated that the same concentration of total AA was found in the presence of 1.0 and 0.25% thiourea.…”

“…In the present study, the specificity of the 2,4-dinitrophenylhydrazine ( D N P H ) method for determining total AA in vegetables was investigated by procedures already described (3). Thus, D N P H derivatives were purified by chromatographic techniques, concentrations of total AA calculated from the fraction most closely resembling a reference coinpound prepared froin crystalline AA, and these concentrations compared with those found by the usual D N P H methods.…”

Loss of Ascorbic Acid During Estimation by the Roe‐Kuether Method

“…Since completion of this investigation, the authors came across the papers by Gordon and Noble (2,3) ; these workers showed that some vegetables contain non-AA components which react with DNPH.…”

The determination of vitamin C in evaporated and fortified sterilized milks

“…pounds, including reductones, may interfere but the specificity of the method can be greatly improved by the chromatographic separation of the DOA osazone from other DNPH derivatives (Gordon & Noble, 1959;Mapson, 1961;Vuilleumier & Nobile, 1962;Strohecker & Henning, 1965;Association of Vitamin Chemists, 1966). AA reacts with DNPH only after oxidation to DHA, and with the inclusion of a reduction step the method can be used to determine AA, DHA and DOA (Roe, Mills, Oesterling & Damron, 1948).…”