Letter pubs.acs.org/ac
Simultaneous Electrochemical Analysis of Hydrophilic and Lipophilic Antioxidants in Bicontinuous Microemulsion Eisuke Kuraya,†,‡ Shota Nagatomo,‡ Kouhei Sakata,‡ Dai Kato,§ Osamu Niwa,§ Taisei Nishimi,∥ and Masashi Kunitake*,‡ †
Science and Technology Division, Okinawa National College of Technology, 905 Henoko, Okinawa 905-2192, Japan Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan § National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan ∥ Fujifilm Corporation, 577 Ushijima, Kaisei-machi, Ashigarakami-gun, Kanagawa 258-9577, Japan ‡
S Supporting Information *
ABSTRACT: Qualitative and quantitative analyses of hydrophilic and lipophilic antioxidants, such as polyphenols, by simple electrochemical measurements were conducted in a bicontinuous microemulsion (BME), in which water and oil phases coexisted bicontinuously on a microscopic scale. Hydrophilic and lipophilic antioxidants were individually monitored in the same BME solution using a hydrophilic indium tin oxide (ITO) electrode and a lipophilic fluorinated nanocarbon film electrode (F-ECR), respectively. The combination of well-balanced BME and extremely biased electrodes, such as ITO and F-ECR, in terms of hydrophilic−lipophilic balance allowed us to achieve individual monitoring of hydrophilic and lipophilic antioxidants in the same BME solution without extraction. Furthermore, the antioxidant activities of functional liquid foods, such as coffee and olive oil, were also evaluated by means of electrochemical measurements in BME solutions containing analytes in concentrations of several percent. The technique we propose provides a very simple, rapid, easily serviceable, and highly reproducible analysis and can be extended to a wide range of analytes and media.
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method using acetone. Antioxidant activity can be greatly affected by the solvents used in the extraction process.7 Therefore, simple and simultaneous analytical techniques for the qualitative and quantitative investigation of antioxidant materials in foods, especially fat-soluble species, are eagerly sought after.8−10 Antioxidants in foods and biological samples were also investigated by electrochemical analyses11−16 in aqueous media or organic solvents, such as isopropanol15 and ethanol.16 In this letter, we introduce the novel qualitative and quantitative electrochemical analysis of hydrophilic, lipophilic, and amphiphilic antioxidants in bicontinuous microemulsions (BMEs) simultaneously. BMEs, in which water and oil phases coexist bicontinuously on a microscopic scale, can dissolve hydrophilic, lipophilic, and amphiphilic compounds simultaneously. Rusling and coworkers pioneered work on the electrochemistry of BMEs in the 1990s.17 In our previous research, we reported that it is possible to separately measure the oxidation−reduction power of hydrophilic and lipophilic compounds in a BME. We made use of the fact that the microemulsion structure at a solid/
n recent years, functional foods and their role in human health have received increasing attention. Many biochemical studies have indicated that antioxidants inhibit or delay the oxidation of other molecules by interrupting the initiation or propagation of the oxidizing chain reaction.1 These beneficial effects have been partly attributed to biological compounds that possess antioxidant activity. The major antioxidants in foods are vitamins C and E, carotenoids, flavonoids, and phenolic compounds.2 Plant-derived polyphenols, such as flavonoids, anthocyanins, and catechins, are well-known antioxidants. The analysis of bioactive polyphenols in functional foods is crucial for the establishment of evaluation criteria. The Folin− Ciocalteu method, based on the reduction of the phenolic hydroxyl group using a phenol reagent, is a popular analysis technique.3 It is used to evaluate the antioxidant activity of compounds by comparing their reducing power with that of gallic acid. Other popular methods include the DPPH (2,2diphenyl-1-picrylhydrazyl) method,4 the oxygen radical absorbance capacity (ORAC) method, and the TEAC (trolox equivalent antioxidant capacity, trolox is 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) method.5,6 A method for the evaluation of the antioxidant activity of fat-soluble species, such as α-tocopherol, which inhibits lipid peroxidation, is in demand, although it could be measured by the ORAC © XXXX American Chemical Society
Received: December 1, 2014 Accepted: January 13, 2015
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DOI: 10.1021/ac5044576 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
electrodes contact predominantly with hydrophilic and lipophilic antioxidants in the micro saline and oil phases, respectively. Figure 2 shows CVs of gallic acid, ascorbic acid, trolox, and α-tocopherol measured using ITO (A) and F-ECR (B)
liquid/liquid interface changes according to the hydrophilicity or lipophilicity of the surface.18−20 Electrochemical contact with the micro saline and oil solution phases in a BME can be alternately or simultaneously achieved by controlling the hydrophilicity and lipophilicity of the electrode surfaces (Figure 1). Therefore, the individual antioxidant activities of hydrophilic, lipophilic, or amphiphilic compounds in the same BME solution can be measured simultaneously.
Figure 1. Schematic representation of electrochemical analysis in a bicontinuous microemulsion.
BME solutions comprising phosphate buffer (pH = 7.0), saline, sodium dodecyl sulfate surfactant, 2-butanol cosurfactant, toluene, and antioxidants were prepared for cyclic voltammetry (CV) analysis. Gallic acid, trolox, α-tocopherol (vitamin E), and ascorbic acid were chosen as typical representatives of phenolic, amphiphilic, lipophilic, and hydrophilic antioxidants, respectively. All the hydrophilic, amphiphilic, and lipophilic compounds we used were soluble in the BME (Table S1 in the Supporting Information). The CV measurements were conducted in the BME solution at 25 °C without degassing using an ordinary three-electrode system. A platinum wire and a saturated calomel electrode (SCE, BAS Inc., Japan) were used in the BME solution as counter and reference electrodes, respectively. An indium tin oxide (ITO) electrode18−21 and a fluorinated nanocarbon film (F-ECR) electrode22,23 were used as hydrophilic and lipophilic working electrodes, respectively. As pretreatment prior to use, the ITO electrodes were flushed with methanol and pure water, and the F-ECR electrodes were flushed with toluene. Gold and glassy carbon (GC) disc electrodes (BAS Inc., Japan) were polished and flushed with pure water prior to use. The geometrical electrode areas of ITO and F-ECR were regulated by an O-ring (inside diameter 5 mm). The F-ECR electrode was prepared by electron cyclotron resonance (ECR) sputtering with a short period of CF4 plasma treatment. The nanocarbon film on a F-ECR electrode has a nanocrystalline sp2 and sp3 mixed-bond structure with an atomically flat surface. The fluorinated surface is easily prepared without losing the high activity and surface flatness of the electrode, which exhibits excellent electrochemical performance when used to study various biomolecules.24−28 The surfaces of the hydrophilic ITO and lipophilic or hydrophobic F-ECR
Figure 2. CVs (A and B) at 0.1 V/s and plots of peak currents against the square root of scan rates (C and D) of BME solutions in the presence of 1 mM gallic acid, ascorbic acid, trolox, or α-tocopherol measured using an ITO electrode (A and C) and an F-ECR electrode (B and D).
electrodes in the BME solution. In this letter, the concentrations of denoted antioxidants corresponded to each concentration in saline or toluene solution prior to mixing for preparation of BME. Using the ITO electrode, gallic acid, ascorbic acid, and trolox were found to have irreversible anodic oxidation peaks at 0.61, 0.41, and 0.72 V, respectively (Figure 2A). In contrast, no oxidation peak at all was observed for αtocopherol, indicating that hydrophilic ITO electrode surfaces cannot contact the micro oil phase in which α-tocopherol dissolves. Complementary results were obtained in the above same BME solution with the lipophilic F-ECR electrode, as shown in Figure 2B. Using the F-ECR electrode with amphiphilic trolox and lipophilic α-tocopherol, irreversible oxidation peaks were observed at 0.90 and 0.69 V, respectively. No electrochemical responses at all were observed for hydrophilic gallic or ascorbic acids using the F-ECR electrode. Gallic acid and ascorbic acid were not detectable (0.996) in the concentration range of roughly 0.01−10 mM (see also Figures S7 and S8 in the Supporting Information for low concentration regions). We were able to evaluate the concentration of antioxidants in each phase by measuring oxidation currents. As we expected, the hydrophilic ITO electrode gave an oxidative peak for hydrophilic ascorbic acid but no peak at all for lipophilic α-tocopherol, even in the mixed solution of ascorbic acid and α-tocopherol (Figure 5A). Conversely, the FECR electrode gave an oxidation peak for α-tocopherol but none for ascorbic acid (Figure 5B). The peak currents for ascorbic acid with the ITO electrode and for α-tocopherol with the F-ECR electrode in the mixed solutions were in good agreement with those for the individual solutions. These results indicate that there is no interference. Furthermore, it was found that electrochemical analysis in a BME with hydrophilic and lipophilic electrodes has great potential for the evaluation of actual antioxidant activities in functional liquid foods. As a demonstration of its application, Figure 6 shows typical CVs of the BME solutions to which were
Figure 3. Repeating measurements of CVs of the BME solutions in the presence of 1 mM trolox with (A) ITO and (B) F-ECR electrodes at 0.1 V/s. Prior to each CV measurement, flushing with methanol, pure water, and toluene was conducted. C
DOI: 10.1021/ac5044576 Anal. Chem. XXXX, XXX, XXX−XXX
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the potential range between 0.2 and 1.0 V using an ITO electrode and an exponential oxidative current using an F-ECR electrode. In the case of EVOO, a broad oxidative peak was observed using an F-ECR electrode, but no oxidative current was observed using an ITO electrode, because of the lack of hydrophilic antioxidants. Moreover, canned coffee containing 0.25 mM caffeic acid and EVOO containing 0.1 mM αtocopherol as additives were also examined to prove the analytical potential of this method. Addition of caffeic acid produced a broad peak current at 0.72 V using an ITO electrode. Judging by the similarity between the peak shapes for the two BME solutions containing coffee, including the reductive current at 0 V, the oxidative peak observed for canned coffee was predominantly attributed to caffeic acid. The concentration of the caffeic acid in the canned coffee was estimated to be 0.56 mM by means of proportionality calculation with currents monitored using an ITO electrode at 0.72 V. As the BME in the presence of caffeic acid alone gave an oxidative shoulder peak at 0.68 V (see Figure S9 in the Supporting Information), the origin of the oxidative current obtained for coffee was also attributed to caffeic acid. The fact that caffeic acid was detected by both electrodes, ITO and FECR, demonstrates the amphiphilic property of caffeic acid. The CV of the BME solution in the presence of EVOO and α-tocopherol revealed an obvious oxidative current peak at 0.63 V using an F-ECR electrode. No oxidation was observed using an ITO electrode for the EVOO with or without α-tocopherol. The concentration of α-tocopherol in the EVOO was estimated to be 0.031 mM from the oxidation current obtained at the FECR electrode. These results prove the analytical potential for functional foods. The oxidative peak area would be used to evaluate the overall antioxidant activity of each hydrophilic and lipophilic species simultaneously. Generally, antioxidant activity in a liquid food is estimated as the apparent concentration of a typical antioxidant, such as α-tocophenol. Thus, this method permits us not merely to monitor the absorption of a specific antioxidant but as well to measure the antioxidant activity in a liquid food from the quantity of electricity (oxidation peak area). In conclusion, the antioxidant activities of several compounds were investigated individually, without extraction, by simple cyclic voltammetry analysis using a combination of wellbalanced BME and extremely biased electrodes. Electrochemistry in BME using various electrode combinations paves the way for simultaneous multimode analysis and may have a wide variety of analytic applications beyond the antioxidant activity analysis of functional liquid foods.
Figure 5. CVs of a mixed BME solution in the presence of 1 mM ascorbic acid and 1 mM α-tocopherol using (A) an ITO and (B) an FECR electrode at 0.1 V/s.
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ASSOCIATED CONTENT
S Supporting Information *
Details of preparation of the BME solution and procedure of electrochemical analysis with ITO and F-ECR electrodes and the CV curves with HOPG, polished Au, and polished GC electrodes. This material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 6. Typical CVs of the BME solutions in the absence and presence of 5 v/v% EVOO, 5 v/v% EVOO with 0.1 mM α-tocopherol, 5 v/v% canned coffee, and 5 v/v% canned coffee with 0.25 mM caffeic acid using an ITO electrode (A) and an F-ECR electrode (B) at 0.1 V/ s.
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-96-342-3674. Fax: +81-96-342-3679. E-mail:
[email protected].
added canned coffee and extra virgin olive oil (EVOO).9,29−31 The canned coffee in the BME gave a broad oxidative peak at
Notes
The authors declare no competing financial interest. D
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(28) Kato, D.; Sumimoto, M.; Ueda, A.; Hirono, S.; Niwa, O. Anal. Chem. 2012, 84, 10607−10613. (29) Matos, L.; Pereira, J.; Andrade, P.; Seabra, R.; Oliveira, M. B. P. P. Food Chem. 2007, 102, 976−983. (30) Cämmerer, B.; Kroh, L. W. Eur. Food Res. Technol. 2006, 223, 469−474. (31) Risso, É. M.; Péres, R. G.; Amaya-Farfan, J. Food Chem. 2007, 105, 1578−1582.
ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas: “New Polymeric Materials Based on Element-Blocks (no. 2401)” (Grant 24102006) from MEXT and A-STEP (Adaptable & Seamless Technology Transfer Program) of JST, Japan. The work was also conducted in part at the Nano-Processing Facility, AIST, Japan.
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