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Analytica Chimica Acta 573­574 (2006) 125­132

Enzymatic methods in food analysis: determination of ascorbic acid
Tatyana N. Shekhovtsova, Svetlana V. Muginova , Julia A. Luchinina, Anna Z. Galimova
Chemistry Department, M.V. Lomonosov Moscow State University, Leninskie Gory, 119992 Moscow, Russia Received 29 November 2005; received in revised form 2 May 2006; accepted 5 May 2006 Available online 10 May 2006 4th International conference on Instrumental Methods of Analysis. Modern Trends and Applications "IMA05", Iraklion, Crete, Greece, 2­6 October 2005.

Abstract

Keywords: Horseradish and peanut peroxidases; Ascorbic acid; Food analysis

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The feasibility and expediency of enzymatic methods application in food analysis is demonstrated by the example of ascorbic acid (AsA) determination in foods. Enzymatic determination of ascorbic acid is based on its action as a second substrate of horseradish (HRP) and peanut (PNP) peroxidases in the reactions of o-dianisidine (OD) and 3,3',5,5'-tetramethylbenzidine (TMB) oxidation with hydrogen peroxide. The rates of the reactions are monitored spectrophotometrically by measuring the duration of the induction period on kinetic curves plotted in coordinates absorption-time. The proposed procedures are sensitive (cL = 0.1 M), simple, and rapid. The procedure using horseradish peroxidase and the reaction of TMB oxidation was used to determine ascorbic acid in fruit juices, milk and sour-milk products for babies' nutrition. © 2006 Elsevier B.V. All rights reserved.

1. Introduction

Enzymatic methods of analysis are closely related to chemistry and technology of food production. The determination of the catalytic activity of enzymes typical for definite foods is used to estimate the degree of their freezing, pasteurization, and sterilization, and to detect the beginning of their decomposition [1]. For instance, to ascertain the extent of thermotreatment of fish foods one should determine the content of acid phosphatase whose thermal inactivation at 60­70 C testifies to the destruction of pathogenic microbes dangerous for human health, in particular E. coli. Different native and immobilized enzymes are used to determine their substrates belonging to the compounds frequently present in foods and determining their quality. For example, many polyphenols exhibit antioxidant features and assign the quality of olive oil, fresh fish, and beverages (natural juices, tea, red wines) [2]. Changes in their content during the production and storage are used for the quality estimation and expert tasting. Electrochemical biosensors for polyphenols detection have been developed on the basis of such enzymes as tyrosinase, laccase and peroxidase using different electrode materials, flow



Corresponding author. Tel.: +7 095 9393346; fax: +7 095 9394675. E-mail address: sveta muginova@mail.ru (S.V. Muginova).

systems, and simple instrumental procedures [2­6] (phenolic compounds are the substrates of the above mentioned enzymes). To estimate the quality of alcohol beverages it is necessary to control the content of ethanol, which is a substrate of alcohol oxidase and alcohol dehydrogenase; these enzymes are widely used for its determination in such samples [7­9]. Some data related to beer and grape wines analysis obtained in our laboratory with the use of alcohol dehydrogenase were described earlier [10]. Heavy metal ions are also considered to be important chemical markers of food quality. At the same time they belong to effective inhibitors of different enzymes. Numerous highly sensitive, simple and rapid enzymatic procedures using native and immobilized enzymes, peroxidases and alkaline phosphatases isolated from various sources, have been developed for the determination of mercury, cadmium, lead and zinc [11­14] and applied for the analysis of different foods (pea, grapes, beef, pork, mineral waters, etc.). Enzymes application is prospective to control the content of different vitamins in foods. Ascorbic acid (Vitamin C), for instance, is frequently used in food industry as an antioxidant in food fats and fruit juices; to avert the formation of carcinogenic nitrosamines in meat and sausage products; to enrich milk products with vitamins [15]. Numerous instrumental methods (chromatographic, spectrophotometric, and catalytic) for the determination of ascorbic acid are known from literature [16]. At the same time ascor-

0003-2670/$ ­ see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.05.015

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bic acid is often determined by enzymatic methods [17­23]. These methods are primarily based on the determination of dehydroascorbic acid (DHAsA) as a product of AsA oxidation by oxygen or hydrogen peroxide in the presence of ascorbate oxidase [17,18] and peroxidase [19,20] (scheme).

2. Experimental

2.1. Reagents and solutions Solid preparations of plant peroxidases (EC 1.11.1.7) were used. Horseradish peroxidase was purchased from Sigma, USA (RZ = 2.2). Peanut peroxidase was isolated from the cultural medium of Arachis hypogea cells [24] (RZ = 2.0). Aqueous solutions of HRP and PNP at concentrations of 1­10 M were obtained by dissolving their preparations in 0.1 M phosphate (pH 7.0) and 0.05 M Tris­HCl (pH 7.5) buffer solutions, respectively. The concentrations of HRP and PNP stock solutions were determined by the Soret band absorbance (403 =94 [25], 405 = 112 [26] mM cm-1 , respectively). Less concentrated solutions of the enzymes were prepared daily by successive dilutions of their stock solutions with 0.05 M Tris­HCl (pH 7.5) in the case of PNP and 0.1 M phosphate buffer solution (pH 7.0) in the case of HRP. Solid preparations and solutions of the enzymes were stored in a refrigerator at +4 C.

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High sensitivity of flow-injection AsA determination (cL = 0.4 M) with spectrophotometric control of horseradish peroxidase activity was attained by use of a complicated system of two conjugated reactions with the addition of 3,5-dichloro2-hydroxyphenyl sulphate to the reaction of AsA oxidation by hydrogen peroxide [21]. The procedures of AsA determination using luminescent control of ascorbate oxidase [22] and laccase [23] catalytic activity are the most sensitive (the lower limit of determination cL = 0.1­0.8 M), but they require carrying out additional reactions between DHAsA and lucigenin [22] or ophenylenediamine [23]. The procedures for AsA determination with amperometric and potentiometric control of the enzyme activity are less sensitive (cL = 2.5­50 M) [17,18,20], but they provide the analysis of turbid an colored samples (fruit juices, wines, beer). Selectivity of the mentioned procedures is not high with the exception of the procedures, which use ascorbate oxidase since AsA is its specific substrate. In this paper we would like to demonstrate the possibility and prospects of the application of enzymatic methods in food analysis by developing the sensitive and simple procedures to determine ascorbic acid in fruit juices, milk and sour-milk products for babies' nutrition using peroxidases isolated from two sources: horseradish and peanut with spectrophotometric control of the indicator reaction rate.

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Solutions of OD and TMB (Sigma, USA) were prepared daily by dissolving accurately weighed amounts of their preparations in ethanol. Potassium dihydrophosphate and hydrophosphate, potassium biphthalate, KOH of analytical grade (Merck, Germany), and tris(hydroxymethyl)aminomethane (Tris) of analytical grade (Serva, Germany) were used to prepare 0.1 M phosphate (pH 7.0), 0.1 M phthalate (pH 4.5) and 0.05 M Tris (pH 5.0­7.5) buffer solutions, respectively. Hydrogen peroxide was purchased from Merck, Germany. Stock solution of ascorbic acid (0.5 mM) was prepared by dissolving its accurately weighed preparation of analytical grade (Sceron, Russia) in 0.1 M solution of ethylenediaminetetraacetate (EDTA) (Aldrich, USA) or 3% o-phosphoric acid of analytical grade (Chimpromtorg, Russia). Due to the instability against light this solution had to be kept in an amber glass vessel and prepared daily. Concentrated (80%) trichloroacetic acid (TCA) of analytical grade (Chimpromtorg, Russia) was used. Stock solutions of Fe(III) and Cu(II) (1 mg mL-1 ) were prepared by dissolving accurately weighed samples of spectrochemically pure metals in a minimum volume of high-purity grade HCl and H2 SO4 , respectively, and subsequent dilution with water to the required volume. Solutions of lower concentrations of metal ions were prepared by successive dilution of the stock solutions with water before operations. Solutions of sugars (glucose, fructose, sucrose) (Merck, Germany) were prepared before analysis by dissolving their accurately weighed samples in water. Deionized water with a resistance of 18.2 M cm (25 C) purified with a Simplicity Proto system (Millipore) was used in the preparation of all aqueous solutions. 2.2. Apparatus and instruments The initial rate of the reactions of peroxidase oxidation of OD and TMB was monitored spectrophotometrically by measuring an increase in the absorbance of the reaction mixture with time. The rate of the indicator reactions under their optimum conditions in the absence of peroxidases is less than 1% of the rate of the enzymatic reactions. Solution absorbance was measured using a UV-2201 Shimadzu spectrophotometer (Japan) (l = 1 cm). The pH of aqueous solutions was measured with an accuracy of ±0.005 using a potentiometer in the pH-meter mode (Econics-Expert-001, Russia). Centrifuging of milk products after their acidic decomposition was performed using centrifuge CM-6 (ELMI, Latvia), 2750 revolutions per min. All experiments were performed at room temperature without thermostatting. Note, that the rates of OD and TMB oxidation change up to 2% with a change in temperature by 1 C [27]. Graduated ground-glass stopper tubes were precleaned with concentrated high-purity grade HNO3 , thoroughly washed with deionized water, and sterilized in air-sterilizer "GP-20" (Russia). The accuracy of the results of the determination of AsA in real samples obtained by the enzymatic method was checked using two alternative techniques: oxidation­reduction titration with 2,6-dichloroindophenol (DCP) [28] and high performance liq-

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uid chromatography (HPLC) [29]. Chromatographic conditions were as follows: chromatograph "Agilent-1100", column: Phenomenex, Synergi Hydro-RP (4 mm â 250 mm); eluent­30 mM phosphate buffer; UV-detector, 254 nm. 2.3. Analytical procedure The following components were placed sequentially into a glass test-tube with a ground-glass stopper: phthalate buffer solution (with the definite pH value), solutions of the enzyme, ascorbic acid, and OD (or TMB). Finally, hydrogen peroxide solution was introduced. The total volume of the reaction mixture was 5.00 mL. At the moment when hydrogen peroxide was added and the reaction solution was mixed, a stop-watch was started and the absorbance was measured at 15 s intervals for 2­3 min. According to the obtained data, kinetic curves were plotted as absorbance (A) versus time (t, s), and the duration of the induction period ( ind , s) and the value of the slope of ascending part of a kinetic curve (tan ) were calculated. The initial rate of the indicator reactions (0 , M min-1 ) 1 was calculated as o = C = A â l = tan , where c is the t t l increase in the product concentration during the reaction time ( t), A the absorbance, l the cell length (cm), and is the molar absorbance coefficient of the product of OD oxidation at 460 nm (30 mM-1 cm-1 ) [30] or TMB oxidation at 375 and 455 nm (23.7 and 39 mM-1 cm-1 ) [31]; tan is the slope of the absorbance (A) graph versus time (t, s).

3. Results and discussion 3.1. Optimization of experimental conditions for ascorbic acid determination 3.1.1. Choice of indicator reactions Two indicator reactions were used: the oxidation of OD and TMB catalyzed by plant peroxidases. Kinetics and mechanism of these reactions were well studied. It is known [32­34] that at pH values about 7, brown bis- (3,3'-dimethoxy-4-amino)azobiphenyl (max 450­530 nm) formed from green intermediate compound (max 386 nm) featuring meriquinone structure is a stable final product of OD oxidation. The product of enzymatic single-electron oxidation of TMB is a cation-radical, existing in an equilibrium with the blue meriquinone complex (max 375 and 655 nm) [33,34]. The final product of TMB oxidation, quinonediimine, has yellow color (max 465 nm). The choice of these reactions was also prompted by the fact that the majority of the procedures for the determination of biologically active compounds ­ inhibitors of HRP, and some organic acids in particular (oxalic, tartaric, salicylic, sulfosalicylic) [35], were developed using the same indicator reactions. Besides, the optimum conditions for carrying out the oxidation of OD and TMB in the presence of horseradish and peanut peroxidases have been revealed earlier [33,34,36] (Table 1). 3.1.2. Kinetics of the peroxidase oxidation of OD and TMB in the presence of ascorbic acid Ascorbic acid was mentioned earlier to be one of the substrates of peroxidases. The sensitivity of AsA determination based on its oxidation to DHAsA catalyzed by horseradish peroxidase immobilized on the surface of a pH-sensitive field transistor is not high (cL = 0.9 mM) [20]. An alternative approach to AsA determination with the help of pH-transistors employs
Table 1 Optimum conditions of OD and TMB oxidation with hydrogen peroxide, catalyzed by horseradish (I) and peanut (II) peroxidases in the absencea and presenceb of ascorbic acid (0.1 M phthalate buffer solution) Component = 460 nm Ia [33] OD­H2 O2 Enzyme (nM) OD (mM) H2 O2 (mM) pH 0.5 0.05 0.05 5.0­5.5 (nm) 375 Ia [33] TMB­H2 O2 Enzyme (nM) TMB (mM) H2 O2 (mM) pH 0.05 0.025 1 5.0­5.5 I
b

2.4. Samples preparation

2.4.1. Juices samples To decrease the concentration of interfering components preservatives in particular, apple and pomegranate juices were diluted with water 5- and 10-fold, respectively, before introducing into the reaction mixture. 2.4.2. Milk and sour-milk products for babies' nutrition To make possible the subsequent comparison of the results of AsA determination by enzymatic and chromatographic methods, milk products were pretreated according to the procedure used in the chromatographic determination of AsA [29]. Ten milliliters of milk "Agusha" (Russia) recommended for nutrition of babies up to 8 months old, 0.75 mL of 70% ophosphoric acid, and 0.25 mL of 80% TCA were placed into the tube for centrifuging, and the mixture was centrifuged for 25 min. The obtained whey was separated from the clotted precipitate by filtration. Ten milliliters of sour-milk mixture "Agusha" (Russia) recommended for nutrition of babies up to 3 months old, and 2.00 mL of 70% o-phosphoric acid were placed into the tube for centrifuging, and the obtained mixture was centrifuged for 15 min. The whey was separated from the clotted precipitate by filtration, 1.00 mL of the whey was placed to a graduated ground-glass stopper tubes and diluted with water to 10.00 mL. In both cases the obtained whey was analyzed on the same day.

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I

b

IIa [34] 1 0.1 0.1 5.0­5.5

II

b

0.25 0.05 0.05 4.5

0.5 0.08 0.1 4.5

455 Ia [33] 0.1 0.025 0.1 5.0­5.5

375 IIa [34] 0.15 0.25 0.4 5.0­5.5 II
b

455 IIa [34] 0.5 0.25 0.03 5.0­5.5

0.03 0.0125 0.50 4.5

0.14 0.11 0.24 4.5


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Fig. 1. Kinetic curves of the reactions of OD (1­3) and TMB (1 ­4 ) oxidation catalyzed by HRP in the absence (1, 1 ) and presence (2, 3, 2 ­4 ) of ascorbic acid (optimum conditions of the reactions, AsA concentrations, M: 3 (2 ); 7 (2, 3 ); 10 (3, 4 ), 0.1 M phthalate buffer solution, pH 4.5; , nM: 440 (1­3) and 364 (1 ­4 )).

the indicator reaction o-phenylenediamine ­ AsA­HRP, where AsA acts as a second substrate of the enzyme [37]. Despite of the evident potential of significant improving the sensitivity of AsA determination, that approach has not been applied to practice. To use the possibility of AsA determination based on its action as the second substrate of plant peroxidases with spectrophotometric control of their activity, we studied the influence of AsA on the kinetics of the reactions of OD and TMB oxidation with hydrogen peroxide under their optimum conditions (Table 1). Introducing AsA with the concentration of 10­20 M, comparable with the OD and TMB concentrations, resulted in the appearance of two linear parts on kinetic curves in coordinates absorbance versus time (Fig. 1). The duration of the first flat portion, the induction period ( ind ), is directly proportional to the AsA concentration in the reaction mixture. Such an effect of AsA might be explained by its ability to be oxidized with hydrogen peroxide easier and earlier than OD or TMB, as it has a lesser redox potential (EAsA = 0.18 V, EOD = 0.26 V, ETMB = 0.30 V). Absorbance does not change during the induction period because the enzymatic oxidation of AsA to DHAA takes place, and the product of this oxidation, DHAsA, does not absorb at 375 and 460 nm, where the intermediate and final products of TMB and OD oxidation, have maximum absorbance. After the induction period the absorbance increases due to the accumulation of OD (TMB) oxidation product. The reaction rate II (tan â 102 ) after the induction period is a little bit less ( = 0.4­0.8 M min-1 or 1­2 abs. unit min-1 ), than the rate of the indicator reactions in the absence of AsA (Fig. 1). The reason for this is either a partial inhibition of the enzyme by DHAsA, or a lack of hydrogen peroxide for the oxidation of the main substrate as a result of its partial expenditure to oxidize AsA. But so far as our experiments have shown that the enhancement of hydrogen peroxide concentration does not result in the increase of OD (TMB) oxidation rate, the first suggestion seems to be more probable. An analogous experiment was performed for the reaction of TMB oxidation with monitoring its rate at 455 nm correspond-

3.1.3. Effect of pH Real redox potential of the pair AsA/DHAsA depends on pH (scheme); consequently the duration of the induction period on kinetic curves in the presence of AsA may change in depending on pH of the reaction mixture. Optimum pH values of the indicator reactions in the presence of ascorbic acid were found to lie within the pH range 4.5­6.5, and the solutions were buffered with potassium phthalate solution. Data presented in Fig. 2 show that the optimum pH value found earlier for the indicator reactions in the absence of AsA, does not change in its presence. The dependences of the duration of the induction period of OD oxidation catalyzed by HRP and PNP on pH are presented in Fig. 3. The longest induction period appears in the presence of AsA at pH value 4.5 which is close to pK1AsA 4.25. Thus, performance of the indicator reactions at pH 4.5 and the application of the induction period duration as an analytical signal might provide sufficiently sensitive determination of AsA in real samples, and foods in particularly.

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Fig. 2. Dependence of the rate of OD oxidation catalyzed by HRP (1, 2) and PNP (1 , 2 ) on pH in the absence (1, 1 ) and presence (2, 2 ) of ascorbic acid (optimum conditions of the reactions, AsA concentrations, M: 2­10; 2 ­20).

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ing to the final yellow product. It was shown that the induction period in the presence of 10­20 M AsA was not evident: the point of change of flat parts of kinetic curves to ascending parts was not clear. This fact is apparently explained by the 10 times lower concentration of hydrogen peroxide in this case in comparison with that used in the reaction which rate was monitored by the intermediate product accumulation ( 375 nm) (Table 1). This concentration was probably not enough for the subsequent oxidation of TMB. Using higher concentrations of hydrogen peroxide became complicated due to the enzyme inhibition with extra amount of the substrate. So the rate of TMB oxidation in the following experiments was monitored using the absorbance of the intermediate product ( 375 nm).


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Fig. 3. Dependence of the duration of the induction period in the reaction of OD oxidation catalyzed by HRP (1) and PNP (2) on pH in the presence of ascorbic acid (optimum conditions of the reactions, AsA concentrations, M: 1­10; 2­20).

3.2. Development of the enzymatic procedures for ascorbic acid determination using horseradish and peanut peroxidases The studied dependences of the induction period duration on logarithm of AsA concentration are shown in Fig. 5 by the example of the reaction of TMB oxidation in the presence of HRP and PNP. The obtained data show that the sensitivity of AsA determination differs for two ranges of its concentration. Gently sloping parts of the dependences ensures the AsA determination in the range of 0.1­1 M, and the range of AsA concentration correspondent to the steeper parts of the dependences is 1­10 M. Thus, it is possible to determine AsA using different calibration curves (Table 2) depending on the task. To select the appropriate linear part of the calibration plot it is necessary to measure the duration of the induction period of the kinetic curves in the pres-

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3.1.4. Effect of enzymes, OD, TMB and H2 O2 concentrations The dependences of the duration induction period on concentrations of peroxidases and substrates (OD, TMB and H2 O2 ) were studied in the presence of the same concentration of AsA, i.e. 7 M, because this concentration results in the pronounced and well reproducible induction period. The corresponding dependences are shown in Fig. 4 by the example of the reaction TMB­H2 O2 catalyzed by HRP. The selected concentrations of all components, which give the maximum analytical signal in the presence of the 7 M AsA concentration, are presented in Table 1. Note, that optimum concentrations of the substrates remain the same only in the case of OD oxidation catalyzed by HRP.

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Fig. 4. Dependence of the duration of the induction period in the reaction of TMB oxidation catalyzed by HRP on the concentrations of the enzyme (1), H2 O2 (2), TMB (3) in the presence of 7 M ascorbic acid (0.1 M phthalate buffer solution, pH 4.5).

Fig. 5. Dependence of the duration of the induction period in the reaction of TMB oxidation catalyzed by HRP (1) and PNP (2) on logarithm of the concentrations of ascorbic acid (optimum conditions of the reactions).

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Table 2 Analytical characteristics of the procedures for ascorbic acid determination using horseradish (I) and peanut (II) peroxidases (n =3, P = 0.95) Applicable concentration range ( M) Calibration graph equation, ya = A + Bx A OD-H2 O I II
2 b

RSD, %at c s(B) 1.0 0.1 0.7 0.6 7.7 1.2 r 0.9807 0.9999 7 5 21 ­ 3 ­

L

s(A) 1.9 0.3 0.4 4.4 1.0 8.1

B 8.4 11.4 64.2 69.7 71.6 73.9

2­10 0.8­10
2

5.7 5.7 6.4 11.7 11.6 14.3

I II
a b

0.1­1 1­10 0.1­1 1­10

The induction period duration ( inc. ), s. Ascorbic acid concentration, M.

3.3. Interferences

Fruit juices and milk and sour-milk products for babies' nutrition are samples of complicated composition; they contain mineral components, such as metal ions (potassium, sodium, calcium, magnesium, iron, zinc, copper, and manganese), sugars, and various vitamins (A, B, D, E, PP). Besides, fruit juices contain some organic acids (malic, oxalic, citric, etc.); fats, proteins, and biotin are the components of milk and sour-milk products for babies' nutrition. Potential interfering components of sample matrix in the conditions of ascorbic acid determination using HRP are metal ions (especially Fe(III) and Cu(II)) and sugars, glucose in particular, which have reduction properties. It is known [35], that Fe(III) being a cofactor of plant peroxidases is able to act as an effective activator of their catalytic activity at its concentrations above 1mgmL-1 . Copper(II) catalyzes ascorbic acid oxidation with air atmospheric oxygen [15]; besides at concentrations over 300 and 100 gmL-1 , respectively it inhibits the oxidation of aryldiamines, such as OD and TMB. Data on sugars effect on HRP catalytic activity in the studied indicator reactions have not been found in literature. Earlier [35] it was found that organic acids (oxalic, tartaric, citric, and some other) inhibit HRP catalytic activity in the reactions of aryldiamines oxidation only after preliminary incubation with the enzyme for 15­30 min, and this effect results in changing the slope of the ascending part of kinetic curves.

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ence of a real sample and then compare its value with the one observed in the presence of the standard solution of AsA (Fig. 5). Then the corresponding calibration curve should be selected and used to find the amount of AsA in the sample. Note, the most sensitive procedures developed for the determination of AsA are based on the application of the reaction of TMB oxidation (Table 2). Analytical characteristics of the procedures using HRP and PNP are close, though the sensitivity coefficient of the calibration curve and the reproducibility of the results of ascorbic acid determination using PNP are higher. Nevertheless we decided to use HRP to determine AsA in real samples as its preparation is commercial in contrast to the PNP preparation.

3.3.1. Influence of metal ions Concentrations of Fe(III) and Cu(II) in juices and beverages should not be higher than 26.9 mg mL-1 (0.48 mM) and 0.38 mg mL-1 (5.9 M), respectively, according to the Russian regulatory documents. Therefore their interfering effect was studied at these concentrations. The introduction of Cu(II) at its concentrations indicated above stops the reactions. On the contrary, Fe(III) activates the enzyme, and the final yellow product of TMB oxidation is formed during 10 s. The interfering effect of metal ions was eliminated with the help of EDTA. Preliminarily it was revealed that EDTA did not effect the kinetics of the indicator reaction in the presence of AsA. The data of our investigations showed that in order to mask the indicated metal ions effectively it is sufficient to add 0.7 and 6 mM of EDTA in the cases of fruit juices and milk products analysis, respectively. 3.3.2. Influence of sugars The effect of sugars on the induction period duration in the presence of ascorbic acid was studied taking as the example glucose, fructose, and sucrose. The choice of these sugars was determined primarily by the fact that they are the essential components of fruit juices and milk and sour-milk products for babies' nutrition. Sugar was considered not to interfere with the AsA determination when it did not change the induction period by more than 5%. Maximum concentration ratios sugar: ascorbic acid (7 M) resulting in less than a 5% change in the analytical signal were found to be 100, 50 and 50 for glucose, fructose, and sucrose, respectively. Note, that these sugars did not effect the catalytic activity of peroxidase neither in the absence, nor in the presence of ascorbic acid: the change in the rate of TMB oxidation was not more than 1 M min-1 . The enzymatic determination of ascorbic acid was performed with the addition method as far as the concentrations of sugars, which interfered with the determination of ascorbic acid, were 4000-fold higher than the total content of carbohydrates in the analyzed samples (according to the producers' data). 3.3.3. Influence of acid concentration Influence of the concentration of phosphoric acid and its mixture with TCA used to decompose milk products on the indicator

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TMB-H2 O

0.9999 0.9998

0.9992 0.9996


T.N. Shekhovtsova et al. / Analytica Chimica Acta 573­574 (2006) 125­132 Table 3 Results of ascorbic acid determination in fruit juices and baby dairy (n =5, P = 0.95) obtained with enzymatic (I) and alternative (II) methods Analyzed sample Method Juices Apple fresh Content (mg) in 100 g RSD (%)
a b

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Baby dairy Apple "J-7" 0.28 ± 0.01 0.29a 3 Pomegranate "RDR" 2.30 ± 0.08 2.36a 5 Milk 0.52 ± 0.06 0.56b 2 Sour-milk mixture 2.2 ± 0.8 3.0b 9

I II I

2.20 ± 0.01 2.23a 7

DCP method. Method of reversed phase HPLC.

reaction rate was studied. It was shown that in the case of sourmilk analysis it was necessary to dilute the whey by not less than 10 times in order to avoid changing of the pH value of the solution; thus, the optimum concentrations of H3 PO4 and TCA were found to be 4 and 2.2 mM, respectively. Milk analysis did not need any whey dilution. 3.4. Application of the proposed procedure 3.4.1. Determination of ascorbic acid in fruit juices The determination of ascorbic acid in fruit juices was performed according to the following procedure: 4.3 mL of a phthalate buffer solution (pH 4.5), 0.1 mL of 4.5 nM HRP solution; 0.1 mL of 35 mM EDTA solution, 0.1 mL of the sample (apple or pomegranate juices after 5- and 10-fold dilution, respectively), and 0.1 mL of 0.625 mM TMB solution. Finally, 0.1 mL of 25 mM hydrogen peroxide solution was introduced. The total volume of the reaction mixture was 5.0 mL. At the moment when hydrogen peroxide was added and the reaction solution was mixed, a stop-watch was started and the absorbance measured at 15 s intervals for 3 min ( = 375 nm). Analogous experiments were carried out in the presence of the juice and two appropriate additions of AsA. For example, in the case of fresh apple juice 1.0 and 2.0 M of AsA were added. The volume of the phthalate buffer solution was decreased taking into consideration the mentioned addition volumes. According to the obtained data, the kinetic curves of absorbance (A) versus time (t, s) were plotted and the duration of the induction period ( ind , s) calculated. Ascorbic acid concentration was calculated basing on the calibration curve plotted in the coordinates: induction period (s) ­ AsA concentration ( M). The results of ascorbic acid determination obtained with the proposed technique correlate well with the data indicated by the producers: according to the producer's data, the AsA content in pomegranate juice, for instance, is 3 mg in 100 g. Besides, these results are in a good coincidence with the results of the determination of AsA by conventional standard titration method using DCP (Table 3). It should be mentioned that the enzymatic procedure for the determination of AsA in juices and fruits is more rapid and safe than the mentioned standard method using DCP (which have a short shelf life and should be standardized immediately prior to use) and corrosive metaphosphoric acid [28].

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method according to the following procedure. The following components were placed sequentially into a glass test-tube with a ground-glass stopper: 4.3 mL of phthalate buffer solution (pH 4.5), 0.1 mL of 1.5 nM HRP solution, 0.3 mL of 0.1 M EDTA solution, 0.1 mL of the analyzed sample (in the case of milk analysis: initial whey, in the case of sour-milk mixture: 10-fold diluted whey), and 0.1 mL of 0.625 mM TMB solution. Finally, 0.1 mL of 25 mM hydrogen peroxide solution was introduced. The total volume of the reaction mixture was 5.0 mL. The following step of the procedure was the same as in the case of juice analysis. Analogous experiments were carried out in the presence of a sample and two additions of AsA ­ 1.5 and 3.0 M in the case of milk, and 0.5 and 1.0 M in the case of sour-milk mixture analysis. The results of analysis are presented in Table 3. To check the accuracy of the data on the AsA content in milk products, the same samples were analyzed by reversed phase HPLC [29]. The results obtained by enzymatic and chromatographic methods were consistent with each other (Table 3). These methods use the same procedure for pre-treatment of milk products and have the same accuracy of their analysis. But the experiment with the help of the first one takes twice less time; besides, the enzymatic method employs less complicated and expensive equipment. Both methods may be successfully used as alternative methods for the analysis of foods. 4. Conclusions The application of rather new approaches to determine the enzyme second substrate: use of two peroxidases isolated from diverse sources and fixing the duration of the induction period as the analytical signal, allowed us to develop the sensitive, rapid, and simple procedures to determine ascorbic acid in foods. To simplify these procedures and reduce their price, an available spectrophotometric method was used to control the catalytic activity of peroxidases. Acknowledgements This work was supported by the Russian Foundation for Basic Research (grant No. 04-03-33116). The authors thank A.V. Pirogov and A.A. Bendryshev from Analytical Chemistry Division of Moscow State University for the determination of ascorbic acid in milk and sour-milk products for babies' nutrition by HPLC method.

3.4.2. Determination of ascorbic acid in milk and sour-milk products for babies' nutrition Determination of ascorbic acid in milk and sour-milk products for babies' nutrition was performed with the help of addition

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