Development and validation of a differential pulse polarography method for determination of total vitamin C and dehydroascorbic acid contents in foods

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Development and validation of a differential pulse polarography method for determination of total vitamin C and dehydroascorbic acid contents in foods Artur Mazurek, Marzena Włodarczyk-Stasiak∗, Urszula Pankiewicz, Radosław Kowalski, Jerzy Jamroz Department of Analysis and Evaluation of Food Quality, University of Life Sciences, Skromna 8, 20-704, Lublin, Poland

A R T I C LE I N FO

A B S T R A C T

Keywords: L-ascorbic acid Dithiothreitol Subtraction method Analysis

The objective of the study was to develop a new method for the determination of total vitamin C and dehydroascorbic acid contents in food using the differential pulse polarography technique, and to compare achieved results to those obtained with the proposed reference method applying HPLC with spectrophotometric detection. Dithiothreitol was used to reduce dehydroascorbic acid into L-ascorbic acid. Interference due to the presence of dithiothreitol during polarographic determination of L-ascorbic acid was efficiently eliminated by clarification of the sample solution with Carrez reagent. Validation of the polarographic method indicates its usefulness for the analysis of foods products that do not contain isoascorbic acid. The coefficient of variation for the analysis of Lascorbic acid and total vitamin C content ranges from 1.86% to 6.98%, Horrat values from 0.41 to 0.89 and average recovery of L-ascorbic acid ranges from 98% to 104%. Results obtained using the developed method are characterised by high compatibility with those obtained with the HPLC reference method, indicating equivalence of both methods, and in conclusion, the method was deemed fit for purpose for measuring total vitamin C and dehydroascorbic acid in foods.

1. Introduction L-ascorbic acid (AA) and dehydroascorbic acid (DHAA) display the biological activity of vitamin C. In order to determine the total content of vitamin C, it is usually necessary to convert the oxidised form to a reduced one, or vice versa, because then it is possible to use only one technique of determination. Reduction of dehydroascorbic acid to Lascorbic acid, combined with detection of the reduced form, is the most common procedure to analyse the total content of vitamin C (Novakova, Solich, & Solichova, 2008). Simultaneous determination of total vitamin C and dehydroascorbic acid contents applying the subtraction method is also possible. In the first step, L-ascorbic acid in a sample is assayed, then quantitative reduction of dehydroascorbic acid is performed with subsequent determination of total vitamin C content. The content of dehydroascorbic acid is the difference of total vitamin C quantity and initial amount of L-ascorbic acid. The most preferred analytical approach used for vitamin C analysis is high-performance liquid chromatography (HPLC) which guarantees higher selectivity than titration, spectrophotometric or enzymatic techniques (Novakova et al., 2008). The HPLC technique is characterised by high sensitivity that greatly depends on the selection of appropriate detectors.

∗

Dehydroascorbic acid is reduced using reducing agents containing thiol moiety, such as dimercaptopropanol (Bourgeois & Mainguy, 1975; Dhariwal, Washko, & Levine, 1990; Washko, Rotrosen, & Levine, 1989), mercaptoethanol (Deutsch & Santhosh-Kumar, 1996; Schell & Bode, 1993), cysteine (Iwase & Ono, 1993), homocysteine (Chiari, Nesi, Carrea, & Righetti, 1993; Herrero-Martínez, Simó-Alfonso, Deltoro, Calatayud, & Ramis-Ramos, 1998), and most often dithiothreitol (Gibbons, Allwood, Neal, & Hardy, 2001; Gökmen, Kahraman, Demir, & Acar, 2000; Hernández, Lobo, & González, 2006; Odriozola-Serrano, Hernandez-Jover, & Martiin-Belloso, 2007; Romeu-Nadal, Castellote, & López-Sabater, 2008; Shephard, Nichols, & Braithwaite, 1999). These reagents are stable and active only under neutral or slightly acidic pH. The reducing chemical that shows high reactivity and stability in acidic solutions is tris(2-carboxyethyl)phosphine (Lykkesfeldt, 2000; Sato et al., 2010; Wechtersbach & Cigić, 2007). L-ascorbic acid is electrochemically active and gets oxidised on dropping mercury electrode. Ruiz et al. described the mechanism of that reaction and studied the influence of pH value on semi-wave potential and limiting current (Ruiz, Aldaz, & Dominguez, 1977). Along with the progress in polarography, the method was optimised for its selectivity and possibly the lowest limit of quantification. Its main

Corresponding author. E-mail address: [email protected] (M. Włodarczyk-Stasiak).

https://doi.org/10.1016/j.lwt.2019.108828 Received 4 June 2019; Received in revised form 7 November 2019; Accepted 8 November 2019 0023-6438/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

Please cite this article as: Artur Mazurek, et al., LWT - Food Science and Technology, https://doi.org/10.1016/j.lwt.2019.108828

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99.9995% pure), sodium acetate, zinc acetate dihydrate, potassium hexacyanoferrate trihydrate and glacial acetic acid from POCH S.A (Gliwice, Poland). All reagents met the quality norms required for analytical grade reagents. The certified reference material BCR-431 was obtained from the Joint Research Centre, Institute for Reference Materials and Measurements (Geel, Belgium). For the preparation of the Carrez I solution 21.95 g of zinc acetate dihydrate and 3 mL of glacial acetic acid were dissolved and made up to 100 mL with distilled water. For the preparation of the Carrez II solution 10.6 g of potassium hexacyanoferrate trihydrate were dissolved and made up to 100 mL with distilled water.

benefits are high sensitivity, selectivity, short time for a single assay and simplicity. Polarographic techniques were used to analyse L-ascorbic acid content in various types of foods (Barbera, Farre, Lagarda, & Pintor, 1993; Branca, 1980; Cherdkiatgumchai & Grant, 1987; Esteve, Farré, & Frígola, 1995; Gerhardt & Windermüller, 1981; Issa, AbdelGawad, Aly, & El-Shinawi, 1984; Kozar, Bujak, Eder-Trifunovic, & Kniewald, 1988; Lau, Shiu, & Chang, 1985; Manglano et al., 2004; Manglano, Farré, Frigola, & Lagarda, 2003; Sahbaz & Somer, 1992; Zulueta, Esteve, Frasquet, & Frigola, 2007; Zulueta, Esteve, & Frigola, 2010). Buffers of pH about 4.5 were most frequently used as a basic electrolyte. Although dehydroascorbic acid is reduced on mercury electrode, the recorded signal is well-generated only for its high concentrations due to the formation of a polarographically inactive hydrate, which makes its analytical application useless (Ruiz et al., 1977). Dehydroascorbic acid can be determined polarographically in an indirect way after derivatisation using orthophenylamine, because formed quinoxaline derivatives are reduced on mercury electrode (Rodrigues, Valente, Goncalves, Pacheco, & Barros, 2010; Wasa, Takagi, & Ono, 1961). To determine the total content of vitamin C, Lascorbic acid should be oxidised prior to polarographic measurement. Such a procedure was applied to determine vitamin C in orange juice (Davidek, Velisek, & Janicek, 1974), preserved fruits (Grundova, Davidek, Velisek, & Janicek, 1973), pharmaceuticals (Kozlov, L'vova, Tkachenko, & Karmanova, 1973), dehydrated potatoes (Jadhav, Steele, & Hadziyev, 1975; Steele, Jadhav, & Hadziyev, 1976), fruit juices and milk (Rodrigues et al., 2010). Lento et al., using homocysteine, performed dehydroascorbic acid reduction to determine total vitamin C content by polarography, then due to interference, its excess was removed by means of reaction with N-ethylmaleimide (Lento, Daugherty, & Denton, 1963). In our previous study, we confirmed the usefulness of the method of determination of the total content of vitamin C and dehydroascorbic acid applying reverse-phase high-performance liquid chromatography with spectrophotometric detection (HPLC-DAD) to assay various types of food samples (Mazurek & Jamroz, 2015), and we demonstrated the lack of statistically significant differences between results of L-ascorbic acid determination by means of differential pulse polarography (DPP) and HPLC-DAD (Mazurek, Włodarczyk-Stasiak, & Jamroz, 2018). The use of differential pulse polarography makes this method equivalent to HPLC-DAD in terms of analytical parameters, however, there is no validated method for the determination of total vitamin C and dehydroascorbic acid content using DPP and reducing chemicals. The objective of the study was to develop a novel method for the determination of total vitamin C and dehydroascorbic acid content in foods, applying differential pulse polarography technique (DPP), and to compare achieved results with those from the reference method using HPLC technique with spectrophotometric detection. Derivatisation of dehydroascorbic acid was assumed to be carried out using dithiothreitol (DTT) as the reducing agent.

2.3. Chromatographic analysis The reversed-phase high performance liquid chromatography with spectrophotometric detection (HPLC-DAD) method described in our previous paper (Mazurek & Jamroz, 2015) was used to analyse the total contents of vitamin C and L-ascorbic acid. Analyses were performed with the use of Varian (Palo Alto, USA) HPLC system equipped with a diode-array detector (DAD, type 335), an isocratic pomp (type 210), a dosing valve 7725i (Rheodyne, USA) and a column thermostat. Galaxie Chromatography Data System, version 1.9.302 (Varian, Palo Alto, USA) was used for process control and data collection. Separations were made using a column Gemini (150 × 4.6 mm, 3 μm, C18, Phenomenex, Torrance, USA) connected with a pre-column Gemini (4 × 3 mm, C18, Phenomenex, Torrance, USA). The injection volume was 20 μL. The mobile phase was a solution of orthophosphoric acid at pH 2.8 pumped at a flow of 0.6 mL/min. Chromatograms were recorded at 244 nm and temperature of 30 °C. The concentration of AA was calculated from equation of the calibration curve plotted for the standard solutions. AA identification was performed on the basis of retention time and UV absorption spectrum of the standard sample. 2.4. Polarographic analysis The sample was weighed to the nearest 1 mg (ms), then metaphosphoric acid solution (20 g/L) was added and the sample was reweighed (ms+a). The dilution factor (D) was calculated and subsequently used to determine the analyte content in the sample.

D=

ms + a ms

(1)

The sample was homogenised for 1 min using homogeniser Ultra Turrax T18 Basic, (Ika, Staufen, Germany), and then centrifuged for 5 min at 12000×g. Supernatant was filtered using a syringe filter with 0.2 μm porosity. Resulting extract was divided into two parts: L-ascorbic acid was determined in the first one (AAs), while total content of vitamin C (TC) after reduction of dehydroascorbic acid using dithiothreitol in the second. In a measuring flask of 10 mL capacity, 3 mL of 10 mmol/L dithiothreitol solution was added to 4 mL of vitamin C extract. Then pH of the solution was adjusted to about 6 using 2 mol/L Tris-acetate buffer of pH = 8 and the content was stirred for 10 min in darkness. Subsequently, 0.1 mL of Carrez I solution was added and the reagents were stirred for 3 min, after which 0.1 mL of Carrez II solution was added and adjusted to 10 mL volume using metaphosphoric acid solution (40 g/L). The flask was set aside for 10 min, then the sample was filtered through a syringe filter of 0.2 μm porosity. L-ascorbic acid determinations were performed by the method of differential pulse polarography, using a voltammetric trace analyser 746 VA (Metrohm, Herisau, Switzerland) with a 747 VA stand. A threeelectrode system was used, a dropping mercury electrode was the working electrode and a platinum electrode was the auxiliary electrode. The potential of the working electrode was measured in relation to an Ag/AgCl/KCl reference electrode. A quantitative assay was performed by the method of triple standard addition by means of an automatic dosing unit Dosino 700 (Metrohm, Herisau, Switzerland). The standard

2. Materials and methods 2.1. Food samples The following materials were used: juices from local producers (multi-vegetable, grapefruit, orange), fruits and vegetables (banana, kiwi fruit, cauliflower, broccoli, cucumber, tomato, parsley leaves), fruit cream in powder, infant milk powder, and multivitamin syrup. All samples were purchased from local supermarkets in Lublin, Poland. 2.2. Reagents and standards L-ascorbic acid, dehydroascorbic acid, dithiothreitol, N-ethylmaleimide and tris(hydroxymethyl)aminomethane (Tris) was purchased from the company Sigma-Aldrich (Saint Louis, USA), metaphosphoric acid from Merck (Darmstadt, Germany), mercury (for polarography,

2

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the certified value Xm – the mean value measured XCRM – the certified value

addition was selected during each determination in such a way that at the final stage a 3-4-fold higher mass of L-ascorbic acid was obtained in the measurement vessel. Qualitative determination of L-ascorbic acid was performed based on the comparison of the potential of its peak (obtained from sample analysis) with the potential of peaks obtained after adding the standard solution three times to the sample. Ten mL of acetate buffer with pH 4.6 and 0.5 mL of an extract of the analysed sample were added directly to the polarographic vessel. Prior to polarographic measurement, the solution was flushed with argon for 5 min to remove oxygen. The following conditions were applied during the polarographic analysis: initial potential −50 mV, final potential 200 mV, voltage step time 0.6 s, voltage step 6 mV, pulse duration 40 ms, pulse amplitude 50 mV, the rate of potential change 10 mV/s, drop size 0.38 mm2. The first step consisted in determining the L-ascorbic acid content in the sample (AAs formula 2), then quantitative reduction of dehydroascorbic acid was carried out using dithiothreitol and total vitamin C content (TC) was calculated based on formula 3. The content of dehydroascorbic acid (DHAAs) was calculated by subtracting the initial Lascorbic acid content from the total amount of vitamin C (subtraction method).

AAs =

CAA⋅D 10

Then the expanded uncertainty was calculated on the basis of uncertainty for values being compared. 2 UΔ = k um2 + uCRM

where: UΔ – expanded uncertainty of difference between the measured value and the certified value um – uncertainty of result of a measurement (expressed as the standard deviation of measurement series) uCRM – uncertainty of the certified value k – a coverage factor equals 2 at 95% level of confidence If Δm ≤ UΔ then there is no statistically significant difference between the measurement result and the certified value. Recovery of the method was estimated by addition of 50 mg/100 g acid to all analysed samples except multivitamin syrup which was supplemented with 250 mg/100 g. This step was taken before the extraction of vitamin C and then the whole procedure including reduction was conducted. The value of recovery was estimated using the following equation: L-ascorbic

(2)

where: CAA – concentration of L-ascorbic acid in extract (μg/mL)

TC =

CTA⋅2.5⋅D 10

(3)

R=

where: CTA – concentration of L-ascorbic acid after the reduction step (μg/mL). During the whole procedure, the temperature in the laboratory was maintained at 23–25 °C. All assays were performed in six replicates and the results were expressed in mg of the analyte per 100 g of fresh weight in the case of fruits and vegetables, while for other samples – per 100 g of product.

(4)

CVH = 2(1 − 0.5logC )

(5)

100%

(8)

In addition, t-Student test was applied to compare the significance of the differences between the mean recovery value and the given recovery value (100%) by calculating the parameter t from the equation:

x −μ t= ‾ s

n

(9)

where:

x‾ - the mean value, μ-the reference value, s-the standard deviation determined for the set of results used to calculate the mean value, n-the number of results.

where

The value (tcr) read from the tables of critical values of t–Student was compared with the calculated t-value. If t ≤ tcr, it can be concluded that obtained recovery results do not differ statistically significantly from 100% (Konieczka & Namieśnik, 2009). The accuracy of the polarographic method was determined by comparison of the results obtained using the polarographic method and the reference chromatographic method. For this purpose, the method of calculating the ratio of mean results (if the values did not differ between themselves, the ratio should be 1) and the uncertainty of the determination was applied (Konieczka & Namieśnik, 2009). It consists in calculating the ratio P of mean determination results and uncertainty U for the P value determined in that manner. The inference is as follows: if the interval of the determined value of calculated ratio P ± uncertainty of its determination U (P − U, P + U) includes 1, one should infer that the mean values compared do not differ in a statistically significant manner. The values of P and U were calculated using the formulae 10 and 11:

where C – mass fraction expressed as power of 10. Accuracy of the analytical method was determined by analysing the certified reference material and comparing the measured value to the certified value according to Application Note no. 1 elaborated by Institute of Reference Materials and Measurements (Linsinger, 2010). For this purpose, the absolute difference between the mean measured value and the certified value was estimated according to the equation:

Δm = Xm − XCRM

c3

c1 – the concentration measured in a sample with addition of AA c2 – the concentration measured in a sample without addition of AA c3 – the concentration added.

Precision of the method was determined by measuring repeatability based on the coefficient of variation established for six independent measurements for each sample, carried out under the same conditions. In order to assess the determined repeatability, value of the HORRAT parameter (equation (4)) defined as a ratio of coefficient of variation to coefficient estimated from the Horwitz equation (5) was calculated (Horwitz, 1982).

CV CVH

(c1 − c2)

where

2.5. Method validation

HORRAT =

(7)

(6)

where: Δm – the absolute difference between the mean measured value and 3

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P=

xDPP xHPLC − DAD

U=k

(10)

2 2 sDPP + sHPLC − DAD

(

xDPP + xHPLC − DAD 2

)

(11)

where: xDPP – mean concentration determined by the polarographic method xHPLC-DAD – mean concentration determined by the reference method k – expanded uncertainty equal to 2 (for confidence interval of ~95%) sDPP – standard deviation of the results obtained by the polarographic method sHPLC-DAD – standard deviation of the results obtained by the reference method The comparison of precision of the polarographic and chromatographic methods was performed using Snedecor's F-distribution which in the case of equinumerous sets of results is presented by the following formula:

F=

s12 s22

Fig. 1. Dependence of the degree of dehydroascorbic acid (DHAA) reduction using dithiothreitol on the process duration.

(12)

where: s1,s2 – calculated values of standard deviation of two sets of results obtained by the polarographic and chromatographic methods. The value of Fcr read from the Tables of critical values of Snedecor's F-distribution (for significance level α = 0.05 and calculated numbers of degrees of freedom f = 5) was compared with the calculated F value. If F ≤ Fcr, then it can be concluded that the calculated values of standard deviation of two sets of results obtained by the polarographic and chromatographic methods do not differ in a statistically significant manner (Konieczka & Namieśnik, 2009). Fig. 2. Polarograms of dithiothreitol solutions (DTT) and dithiothreitol with addition of: N-ethylmaleimid (DTT + NEM), Carrez I solution (DTT + C1), Carrez I and II solutions (DTT + C1+C2).

3. Results and discussion 3.1. Method development Dithiothreitol, an organic compound containing two thiol groups, is used to reduce disulphide bridges and to protect thiol groups against oxidation, e.g. to prevent the formation of intramolecular or extramolecular bonds between cysteine moieties in proteins (Hansen & Winther, 2009). Dithiothreitol is unstable, can be readily oxidised by oxygen, and is applied in molecular biology to damage the third-order protein structure and is one of the most frequently used reducing agents in vitamin C determination. Wechtersbag and Cigić described the impact of pH value of the solution on efficiency in dehydroascorbic acid reduction (Wechtersbach & Cigić, 2007). Dithiothreitol effectively reduces dehydroascorbic acid at pH above 5, in which 99% efficiency is reached after 15 min at 50-fold excess of dithiothreitol in relation to dehydroascorbic acid. Lykkesfeldt reported that at pH 6.2, the quantitative reduction occurs after 5 min using 165-fold dithiothreitol excess in relation to dehydroascorbic acid (Lykkesfeldt, 2000). In order to verify dithiothreitol solution concentration, reduction of 1 mmol/L dehydroascorbic acid was made using 20 g/L metaphosphoric acid. Such a concentration is much higher than the expected acid concentration in vitamin C extract from analysed samples. An aliquot of 4 mL of dithiothreitol solution of 10 mmol/L concentration was added to 4 mL of dehydroascorbic acid solution, then using Tris-acetate buffer of pH = 8 the acidity was adjusted to about 6. The obtained solution was analysed by means of HPLC-DAD technique to determine the time needed for the quantitative reduction. The results of these trials (Fig. 1)

Fig. 3. Polarograms of dithiothreitol solutions (DTT) and dithiothreitol with addition of: N-ethylmaleimid (DTT + NEM), Carrez I solution (DTT + C1), Carrez I and II solutions (DTT + C1+C2).

indicate that complete reduction of dehydroascorbic acid occurs after about 5 min, thus a 10-min reduction period was assumed in further procedures. The subsequent experiment dealt with the issue whether dithiothreitol shows its own polarographic peak within the range of potentials 4

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Table 1 Ascorbic acid (AAs), total vitamin C (TC) and dehydroascorbic acid (DHAAs) content in the samples obtained by differential pulse polarography and statistical characteristics of the precision of determinations and recovery (tcr = 2.571 and Fcr = 5.05 for α = 0.05 and f = 5), number of replicates = 6. Sample

Analyte

Mean (mg/100 g)

sa (mg/100 g)

CVa (%)

Horrata

Fa

Recovery ± s (%)

ta

Parsley leaves

AAs TC DHAAs

215 241 25

5 6 4

2.43 2.48 14.37

0.48 0.50 2.07

1.04 1.43 6.10

99 ±9

0.194

Tomato

AAs TC DHAAs

16.5 19.0 1.6

0.9 0.8 0.8

5.39 4.16 46.69

0.73 0.57 4.45

2.05 1.52 1.35

104 ±5

1.734

Broccoli

AAs TC DHAAs

90 99 8

2 3 3

2.38 3.10 35.40

0.41 0.55 4.29

1.74 1.05 2.09

98 ±5

1.135

Cauliflower

AAs TC DHAAs

55 59,0 4.5

2 1.6 0.6

3.84 2.71 13.06

0.62 0.44 1.45

1.27 2.18 15.76

102 ±8

0.474

Banana

AAs TC DHAAs

12.4 14.3 1.9

0.5 0.5 0.6

3.75 3.71 28.82

0.48 0.49 2.81

1.63 1.80 2.71

100 ±4

0.042

Lemon

AAs TC DHAAs

68.8 73 4

1.9 2 3

2.81 2.82 64.84

0.47 0.48 7.15

1.08 1.01 10.28

104 ±5

2.083

Kiwi fruit

AAs TC DHAAs

104 118 14

4 4 5

3.96 3.27 34.63

0.70 0.59 4.57

2.85 1.91 1.24

101 ±5

0.360

Cucumber

AAs TC DHAAs

11.0 14.6 3.6

0.8 0.9 1.1

6.98 6.45 31.22

0.89 0.85 3.35

3.78 1.92 1.85

101 ±4

0.452

Multivegetable juice

AAs TC DHAAs

39.7 41.7 2.0

1.2 1.3 1.7

3.12 3.17 87.64

0.48 0.49 8.58

1.69 1.38 3.29

101 ±3

0.893

Orange juice

AAs TC DHAAs

21.7 23.6 1.9

1.3 1.2 1.0

5.98 5.01 53.97

0.84 0.71 5.26

2.19 1.69 1.44

98 ±3

2.062

Grapefruit juice

AAs TC DHAAs

26.9 29.6 2.7

1.2 1.6 1.4

4.35 5.23 53.87

0.63 0.77 5.52

3.23 2.93 3.44

98 ±5

0.991

Fruit cream in powder

AAs TC DHAAs

87 90 2.6

3 3 0.5

3.34 3.37 19.28

0.58 0.59 1.97

1.62 1.61 4.42

99 ±4

0.628

Multivitamin syrup

AAs TC DHAAs

915 932 17

17 18 7

1.86 1.95 38.44

0.46 0.48 5.21

1.21 1.64 1.72

103 ±8

1.022

Infant milk powder

AAs TC DHAAs

77 81 4

3 3 2

3.82 4.31 55.02

0.65 0.74 6.08

1.67 2.11 14.09

100 ±6

0.155

BCR431

AAs TC DHAAs

391 470 79

12 12 15

3.17 2.65 18.38

0.69 0.59 3.14

a s – standard deviation, CV – coefficient of variation, Horrat – parameter defined as a ratio of coefficient of variation to coefficient estimated from the Horwitz equation, F – parameter of Snedecor's F-test, t – parameter of Student's t-test.

measurement. In order to mask dithiothreitol, an addition of N-ethylmaleimid was applied, which reacts with compounds containing thiol groups, then polarographic measurement could be normally performed. Figs. 2 and 3 present polarogram indicating the use up of reagents, however, in the range of potentials used for L-ascorbic acid analysis, there was a peak, which made a credible analysis impossible. N-ethylmaleimid appeared to be an inefficient masking agent for polarographic determination of L-ascorbic acid.

used during analysis of L-ascorbic acid or whether it disturbs the AA peak. A polarogram of dithiothreitol solution, within the potential range from -1 V to 0.2 V, is presented in Fig. 2, and one for a narrower potential range from −0.05 V to 0.2 V, applied during analysis of Lascorbic acid, in Fig. 3. The baseline on the polarogram within the potential range characteristic for L-ascorbic acid is considerably elevated, making it disturbed. Therefore, it appeared to be necessary to remove dithiothreitol from analysed solution prior to polarographic 5

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3.2.1. Precision Table 1 presents the results of food samples analysis by means of polarographic technique using dithiothreitol. The coefficient of variation for the analysis of L-ascorbic acid and total vitamin C content ranges from 1.86% to 6.98%, and for dehydroascorbic acid – from 13.06% to 87.64%. Horrat values for L-ascorbic acid and total vitamin C content are within the range of AOAC recommendations (AOAC, 2009), i.e. from 0.41 to 0.89. In the case of dehydroascorbic acid determination, Horrat values range from 1.45 to 8.58, indicating that subtraction analysis of this acid is imprecise, as in the chromatographic reference method. It has been shown that the DHAA analysis using the subtraction method leads to imprecise results, which is due to the assumptions of this method and to the uncertainty propagation rule (Mazurek & Jamroz, 2015). Analysis of Snedecor's F-test results for L-ascorbic acid content and total vitamin C content for all samples analysed with polarographic and chromatographic methods indicates that there are no statistically significant differences in standard deviations. The DPP method is therefore characterised by the same precision as the chromatographic reference method. When analysing dehydroascorbic acid content, Snedecor's F-test indicates the presence of statistically significant differences for samples of parsley leaves, cauliflower, lemon, and modified milk.

Table 2 Analysis of the accuracy of analytical method based on the BCR 431 certified reference material, number of replicates = 6. BCR 431

DPP

XCRM (mg/ 100 g)

uCRM (mg/ 100 g)

Xm (mg/ 100 g)

um (mg/ 100 g)

Δm

UΔ

Accuracy

483

10

470

12

13

32

yes

* XCRM – the certified value, uCRM – uncertainty of the certified value, Xm – the mean value measured, um – uncertainty of result of a measurement (expressed as the standard deviation of measurement series), Δm – the absolute difference between the mean measured value and the certified value, UΔ – expanded uncertainty of difference between the measured value and the certified value.

Krężel et al. reported that dithiothreitol forms stable complexes with some metallic ions: Zn(II), Cd(II), Pb(II), Ni(II) and Cu(I) (Krężel et al., 2001). We conducted tests using all of the mentioned metal ions in the form of nitrate salts and observed no baseline disturbance within the potential range for L-ascorbic acid, however, regardless of the concentration used, we received lower than expected results of the analysis of L-ascorbic acid. Additionally to determine the effect of zinc ions on the polarogram of dithiothreitol, an addition of zinc acetate in the form of Carrez I solution was used. Figs. 2 and 3 present the achieved polarogram where no baseline disturbance occurs within the potential range characteristic for L-ascorbic acid. Further study focused on polarographic analysis of L-ascorbic acid standard solution with an addition of dithiothreitol and Carrez I solution. Results were lower than expected, regardless of the amount of added Carrez I solution, which indicates incomplete masking of dithiothreitol by such chemical composition of the solution. In a subsequent step, clarification of dithiothreitol solution using Carrez solutions was performed. Within the range of potentials for L-ascorbic acid, the achieved polarogram (Figs. 2 and 3) is identical with that obtained after adding only Carrez I solution. In order to confirm the efficient masking of the reducing agent, after clarification with Carrez solutions, polarographic analysis of Lascorbic acid solution (100 μg/mL concentration) was carried out, with an addition of dithiothreitol. The correct results obtained (99 ± 3 μg/ mL) suggest efficient masking of dithiothreitol after clarification. We did not investigate the use of different concentrations of these solutions, because we decided that the methodology for their use is well known and it is not advisable to make changes in their concentrations. At the same time, it allowed retaining the advantages resulting from the clarification with Carrez solutions in the case of samples with different matrices. Conclusions from these experiments were used in the analysis of total vitamin C content by means of polarographic technique.

3.2.2. Accuracy The accuracy of the obtained results was demonstrated by analysing a sample of the BCR 431 certified reference material and comparing the obtained mean value with the certified value. Table 2 shows data that reveal no statistically significant differences. Table 3 provides a comparison of results for L-ascorbic acid, total vitamin C and dehydroascorbic acid obtained with the polarographic method using the dithiothreitol with the reference chromatography. Achieved results do not show statistically significant differences, but attention should be paid to the high uncertainty of dehydroascorbic acid results due to imprecise analysis. This results in the absence of statistically significant differences in results that are significantly different from each other, e.g. 29 mg/100 g and 17 mg/100 g in the case of multivitamin syrup. 3.2.3. Recovery The results of L-ascorbic acid recovery from polarographic method for vitamin C determination using dithiothreitol are shown in Table 1. Average values range from 98% to 104% and are in accordance with AOAC requirements for analytical recovery (AOAC, 2009). The Student's t-test indicates that there is no statistically significant difference between the achieved average recovery value and the 100% value. 4. Conclusions

3.2. Method validation This is the first article describing the development of a novel method for the determination of total vitamin C and dehydroascorbic acid content in foods, applying differential pulse polarography technique. Validation of the developed polarographic method for the determination of total vitamin C content using dithiothreitol indicates its usefulness in the analysis of food samples that do not contain isoascorbic acid. It should be noted that isoascorbic acid does not occur naturally in food (Mazurek et al., 2018). When analysing dehydroascorbic acid content, imprecise results are obtained as a consequence of the subtraction method (Mazurek & Jamroz, 2015). The time needed to prepare a sample for polarographic or chromatographic analysis is comparable. The polarographic measurement lasts from 7 to 10 min depending on the calibration technique used, and the use of autosampler for fully automatic polarographic determination allows obtaining high throughput of the assays. The main advantage of using the polarographic method for the determination of vitamin C, especially in relation to the spectrophotometric method, is its high selectivity comparable to the chromatographic method. The use of differential

The selectivity and linearity of the developed analytical method are associated with the polarographic technique used for L-ascorbic acid determinations. The way of determining the lower detection limit based on the standard curve parameters allows to calculate the detection and determination limits of a polarograph. Therefore, values of the above parameters are common with the previous validation parameters obtained for polarographic method of L-ascorbic acid determination (the limits of detection and quantitation were 0.84 μg/mL and 2.52 μg/mL, respectively) (Mazurek et al., 2018). The modification of the method, making it possible to analyse the total vitamin C and dehydroascorbic acid contents, consists in carrying out the reduction of dehydroascorbic acid during sample preparation for polarographic analysis of L-ascorbic acid and has no impact on the above validation parameters. Therefore, only precision, accuracy and recovery were determined in undertaken study upon developing the method for the total vitamin C and dehydroascorbic acid contents determination applying the differential pulse polarography technique. 6

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Table 3 Comparison of ascorbic acid (AAs), total vitamin C (TC) and dehydroascorbic acid (DHAAs) values obtained by polarographic (DPP) and chromatographic methods (HPLC-DAD), number of replicates = 6. Sample

Analyte

HPLC-DAD

DPP

Mean (mg/100 g)

sa (mg/100 g)

Mean (mg/100 g)

sa (mg/100 g)

Pa

Ua

Accuracy

Parsley leaves

AAs TC DHAAs

226 255 30

5 5 9

215 241 25

5 6 4

1.05 1.06 1.2

0.07 0.06 0.7

yes yes yes

Tomato

AAs TC DHAAs

18.2 20.5 2.3

0.6 0.6 0.9

16.5 19.0 1.6

0.9 0.8 0.8

1.10 1.08 1.4

0.13 0.10 1.2

yes yes yes

Broccoli

AAs TC DHAAs

94 104 10

3 3 4

90 99 8

2 3 3

1.04 1.06 1.3

0.08 0.08 1.10

yes yes yes

Cauliflower

AAs TC DHAAs

57.5 64 6

1.9 2 2

55 59.0 4.5

2 1.6 0.6

1.06 1.08 1.4

0.10 0.09 0.9

yes yes yes

Banana

AAs TC DHAAs

13.0 15.9 2.9

0.6 0.7 0.9

12.4 14.3 1.9

0.5 0.5 0.6

1.05 1.12 1.5

0.12 0.12 0.9

yes yes yes

Lemon

AAs TC DHAAs

71.4 74.0 2.6

1.9 2.0 0.9

68.8 73 4

1.9 2 3

1.04 1.01 0.6

0.08 0.08 1.7

yes yes yes

Kiwi fruit

AAs TC DHAAs

109 125 16

2 3 4

104 118 14

4 4 5

1.05 1.05 1.1

0.09 0.08 0.9

yes yes yes

Cucumber

AAs TC DHAAs

11.5 16.1 4.6

0.4 0.7 0.8

11.0 14.6 3.6

0.8 0.9 1.1

1.05 1.10 1.3

0.15 0.15 0.7

yes yes yes

Multivegetable juice

AAs TC DHAAs

41.0 43.5 2.5

1.0 1.6 1.0

39.7 41.7 2.0

1.2 1.3 1.7

1.03 1.04 1.2

0.08 0.10 1.8

yes yes yes

Orange juice

AAs TC DHAAs

23.0 25.2 2.2

0.9 0.9 1.2

21.7 23.6 1.9

1.3 1.2 1.0

1.06 1.06 1.2

0.14 0.12 1.6

yes yes yes

Grapefruit juice

AAs TC DHAAs

27.1 30.2 3.1

0.6 0.9 0.8

26.9 29.6 2.7

1.2 1.6 1.4

1.01 1.02 1.2

0.10 0.12 1.1

yes yes yes

Fruit cream in powder

AAs TC DHAAs

92 96 3.3

2 2 1.1

87 90 2.6

3 3 0.5

1.06 1.07 1.2

0.08 0.08 0.8

yes yes yes

Multivitamin syrup

AAs TC DHAAs

939 969 29

19 14 9

915 932 17.

17 18 7

1.03 1.04 1.7

0.05 0.05 0.9

yes yes yes

Infant milk powder

AAs TC DHAAs

82 85 2.4

2 2 0.6

77 81 4

3 3 2

1.07 1.04 0.6

0.09 0.10 1.5

yes yes yes

a

s – standard deviation, P – the ratio of means of determination results, U – uncertainty for P value.

Declaration of competing interest

pulse polarography shows that this method is equivalent to high-performance liquid chromatography with diode-array detection in terms of analytical parameters, allowing easier and faster determination of vitamin C. The results obtained with the developed DPP method are highly consistent with those achieved by means of reference chromatography, indicating the equivalence of both methods, and in conclusion, the method was deemed fit for purpose for measuring total vitamin C in foods. The polarographic method is more suitable for routine analyses, owing to lower costs, shorter time and simplicity of the analysis.

None.

Acknowledgements The work was financed by a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Food Science and Biotechnology of University of Life Sciences in Lublin.

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