Chapter 13 Autoxidation of L-Ascorbic Acid and Its Significance in Food Processing 1
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T. Kurata , N. Miyake , E. Suzuki , and Y. Otsuka 1
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Institute of Environmental Science for Human Life, and Department of Human Biological Studies, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112, Japan Faculty of Education, Tottori University, Tottori, Japan 3
Oxidation rates of L-ascorbic acid (ASA) in a low dielectric solvent (MeOH) were determined, and initial autoxidation products were separated and identified. Mechanisms of ASA autoxidation were studied using the semi-empirical molecular orbital (MO) method. In this solvent, MeOH and in the absence of metals, ASA reacts with triplet oxygen yielding very similar to or nearly identical reaction products as those observed with singlet oxygen, suggesting the formation of ascorbate-2peroxy-anion type intermediate.Mechanisms of ASA autoxidation including the formation mechanism of superoxide anion radical were proposed, which were supported by the MO calculation.
ASA is an important antioxidant in food as well as in biological systems. In its chemical structure, ASA has an ene-diol group conjugated with a lactone carbonyl group, hence, it is a typical "aci-reductone" (1) that shows a strong reducing activity. Thus, ASA is susceptible to oxidation, and ASA present in various foods is usually very easily oxidized during processing or storage to give a mixture of complex reaction products, which strongly influence the quality of those foods. For instance, it is well known that ASA is involved in the discoloration of lemon, orange and other citrus juices and causes non-enzymatic browning reactions, and the presence of oxygen accelerates the oxidation of ASA, hence accelerates the deterioration of these fruit juices. Thus, the oxidation reaction, especially the autoxidation of ASA, i.e., the reaction of ASA with oxygen molecules, can be regarded as a marker reaction that may reflect the oxidative status of other constituent compounds in various foods. In this paper, a preliminary study to evaluate oxidation rates of ASA in both aqueous and non-aqueous systems is presented. Reaction products of the initial autoxidation process of ASA were separated and identified. To elucidate the reaction mechanism
0097-6156/96/0631-0137$15.00/0 © 1996 American Chemical Society Lee and Kim; Chemical Markers for Processed and Stored Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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CHEMICAL MARKERS FOR PROCESSED AND STORED FOODS
involved in the autoxidation process of ASA, and to obtain information on the reactivities and structures of rather unstable reaction intermediates produced in the autoxidation process, the semi-empirical molecular orbital (MO) method was employed. Some possible reaction mechanisms considered to be operative after the initial autoxidation process, involving dehydro-L-ascorbic acid (DHA), 2,3-diketo-L-gulonic acid (DKG) and 2-amino-2-deoxy-L-ascorbic acid (L-scorbamic acid; SCA) were also briefly discussed. Materials and Methods ASA was obtained from a commercial source (Wako Pure Chemical Industries, Ltd) and further purified by recrystallization. Ultra-refined water with electrical resistance of 18 Μ Ω · cm was used throughout the experiment. Commercially obtained MeOH (Wako Pure Chemical Industries, Ltd, heavy metal contents: less than 0.5 ppb) was used, and when necessary, it was further purified by distillation. A preliminary study on autoxidation rates of A S A . Very dilute ASA solutions (cone. 0.03 mg%) were prepared and incubated at 25, 30 and 35 °C for 30 min. The degradation rate of ASA was determined using HPLC with highly sensitive electrochemical detector (BAS LC-4B, 600 mV), and first order rate constants for the autoxidation of ASA were estimated. Separation and identification of initial autoxidation products of ASA in MeOH. Recrystallized ASA was dissolved in 200 ml of MeOH at a concentration of 50 β M and reacted with oxygen by bubbling oxygen gas through the solution. The solution was kept at 25"C throughout the autoxidation reaction. Oxygen gas was bubbled through the solution at a flow rate of 200 ml/min for 30 to 60 min, while the reaction chamber was kept in darkness. After the autoxidation procedure was completed, the remaining amounts of ASA were determined using HPLC under the following conditions: column, LiChrosorb-NH2; mobile phase, acetonitrile-water-acetic acid (50:20:2, v/v); flow rate, 1.4 ml/min; UV detector (at 245 nm). The amounts of DHA yielded during autoxidation of ASA were measured according to the method described in literature using 2,4-dinitrophenylhydrazine reagent (2). Analysis of the oxidation products was carried out using GC and GC-MS. After the oxidation reaction was completed, the reaction mixture was evaporated to dryness also in the darkness, and 0.5 ml of TMSI-H (GL Sciences Inc., hexamethyldisilazane : trimethylchlorosilane : pyridine = 2:1:10) or 0.25 ml of N-memyl-N-trimethylsilyl-trifluoro-acetamide (MSTFA) was added to the residue to prepare TMS derivatives of the oxidation products. A Shimadzu model GC-9A gas chromatograph equipped with an FID detector was used for the analysis of TMS derivatives. An OV-1 capillary column (25 m χ 0.25 mm i.d.) was used, and nitrogen was used as the carrier gas at the the flow rate of 0.9 ml/min. The column oven temperature was held at 60°C for 5min then programmed to 210V, with ascending temperature at a rate of 5 "C/min. The structural information of these TMS derivatives was mainly obtained using a GC-MS system comprised of JEOL model JMS-DX300 mass spectrometer equipped with Hewlett Packard model 5790 gas chromatograph. The electron impact ionization method at 70
Lee and Kim; Chemical Markers for Processed and Stored Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
13. KURATA ET AL.
Autoxidation of L-Ascorbic Acid
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eV was used to ionize TMS derivatives, with the ion source temperature maintained at ISOV.
Results and Discussion Since ASA is water soluble, it has always been believed that the antioxidant effect of ASA is confined to the hydrophilic regions of food and biological systems, and that ASA molecules do not participate in the oxidation-reduction process occurring in hydrophobic regions of those systems. Food and biological systems, however, usually have many constituents, and are essentially of multi-phase and multi-component nature. Generally speaking, therefore, the solubility of ASA and oxygen depends on the environment (hydrophobic or hydrophilic), as shown in Figure 1. Thus, highly water soluble ASA molecules might play some roles in the oxido-reduction reactions occurring in more hydrophobic regions than water. From this point of view, further evaluation is needed for the chemical behavior of ASA molecules in solvents of lower dielectric constants and higher oxygen concentrations than water. A preliminary evaluation was done for autoxidation rates of ASA in both aqueous and non-aqueous solvent systems. As shown in Figure 2, ASA was quite unstable in MeOH solution where the concentration of oxygen was much higher than the aqueous solution, and the non-dissociated form of ASA in HC1 solution was more stable than the dissociated one in water. Thus the autoxidation rates were strongly influenced by dissolved oxygen concentrations and ASA monoanion concentrations, while the nondissociated form of ASA was quite stable against oxygen. Their rate constants are given in Table I . Separation and identification of initial autoxidation products of ASA in MeOH were carried out, and formation of DHA was positively confirmed as the main oxidation product of ASA. Threonolactone was identified as its TMS derivative, 2,3-di-Otrimethylsilyl-L-threonolactone, by comparing its GC retention time with that of the authentic compound, and further confirmed by comparing the mass spectrum of the sample with the authentic mass spectral data reported in the literature (3). The formation of oxalic acid was similarly confirmed by GC and GC-MS analyses. These reaction products were also detected in the autoxidation of ASA monoanion as an ASANa salt solution in MeOH, and the formation of the same oxidation products was confirmed in the autoxidation of ASA in an aqueous solution. It was also confirmed, however, that these autoxidation reaction products of ASA were not formed from DHA. Therefore, this appears to be a new autoxidation pathway of ASA that does not proceed via DHA, and this pathway might be involved in various oxidation processes observed in food and biological systems. These experimental data obtained in the autoxidation of ASA in MeOH solution strongly suggested that, without the potent catalytic effect of heavy metal ions, ASA reacts with rather stable triplet oxygen to give very similar reaction products to those obtained in the reaction of ASA with singlet oxygen (4, 5), suggesting the formation of oxygen adduct of ASA monoanion as shown in Figure 3. In order to obtain more detailed information regarding the autoxidation mechanism of ASA, semi-empirical molecular orbital calculations on the interaction of ASA monoanion and oxygen molecule were carried out using a general molecular orbital
Lee and Kim; Chemical Markers for Processed and Stored Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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CHEMICAL MARKERS FOR PROCESSED AND STORED FOODS
Dielectric constant e
Water
Methanol Ethanol-
Έ ο JZ α ο
8 "
73
6
t
(mg%) τ ο
r
100 decrease 80 Ή 60 40 20 >» .ts υ
Solubility of oxygen
'Acetic acid ' Ethyl acetate
4 2
-Oleic acid • Hexane
increase ο
-l-ÔO
Figure 1 Solubility of oxygen and dielectric constant of various solvent
Figure 2 Autoxidation of AsA in various solution. Incubation temperature : 35t
Lee and Kim; Chemical Markers for Processed and Stored Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
13. KURATA ET AL.
Autoxidation of L-Ascorbic Acid
Table I The rate constants of AsA autoxidation 1
(min- ) 25*C
30
