Food Chemistry 123 (2010) 886891

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Simultaneous voltammetric determination of phenolic antioxidants in food using a boron-doped diamond electrode
Roberta Antigo Medeiros, Romeu C. Rocha-Filho, Orlando Fatibello-Filho *
Departamento de Qumica, Universidade Federal de So Carlos, C.P. 676, 13560-970 So Carlos, SP, Brazil

a r t i c l e

i n f o

a b s t r a c t
A method for the simultaneous determination of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) in food was developed using square-wave voltammetry (SWV). The determination of these phenolic antioxidants was carried out using a cathodically pre-treated boron-doped diamond electrode (BDD) and an aqueous-ethanolic (30% ethanol, v/v) 10 mmol L1 KNO3 solution (pHcond. 1.5) as supporting electrolyte. In the SWV measurements using the BDD electrode, the oxidation peak potentials of BHA and BHT present in binary mixtures were separated by about 0.3 V. The attained detection limits for the simultaneous determination of BHA and BHT (0.14 and 0.25 lmol L1, respectively) are lower than the ones by voltammetric techniques reported in the literature. The proposed method was successfully applied in the simultaneous determination of BHA and BHT in food products, with results similar to those obtained using a high-performance liquid chromatography method, at a 95% condence level. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 4 August 2009 Received in revised form 9 February 2010 Accepted 1 May 2010

Keywords: Phenolic antioxidants Butylated hydroxyanisole Butylated hydroxytoluene Simultaneous determination Square-wave voltammetry Boron-doped diamond electrode

1. Introduction Antioxidants (natural and synthetic) play a signicant role in retarding lipid oxidation reactions in food products. Thus, currently there are around 30 types of synthetic antioxidants whose addition to food directly or indirectly is allowed. The phenolic compounds BHA and BHT are among the primary synthetic antioxidants widely used to interrupt the chain of free radicals involved in the autoxidation that constitutes the most common form of deterioration of fats used in the food industry. They have been used both alone and in mixtures in oils, margarine, and mayonnaise (Delgado-Zamarreno, Gonzalez-Maza, Sanchez-Perez, & Martinez, 2007; Diaz, Cabanillas, Franco, Salinas, & Vire, 1998), but their use is not a problem-less solution. Since BHA and BHT are suspected of being responsible for liver damage and carcinogenesis in laboratory animals, their potential harmful effects on health have been extensively discussed and studied. Therefore, in several countries the use of these additives is subject to regulations, which dene specic approved antioxidants, establish permitted use levels, and include labelling requirements. However, there are differences among the individual countries, i.e., antioxidants permitted in one country may be prohibited in another. Internationally, the JECFA (Joint FAO/WHO Expert Committee on Food Additives) periodically considers food additives, including synthetic phenolic antioxidants (SPAs), on the basis of all available scientic data, to
* Corresponding author. Tel.: +55 16 33518098; fax: +55 16 33518350. E-mail address: bello@ufscar.br (O. Fatibello-Filho). 0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.05.010

establish acceptable daily intake levels and specications of identity and purity for them (FAO/WHO, 1995; Guan, Chu, Fu, Wu, & Ye, 2006). In the European Union, for example, the amount of synthetic antioxidants in food is limited to 0.01% (100 mg kg1) for each antioxidant, if used individually, and to 0.02% as total fraction, if the antioxidants are used in mixtures (Delgado-Zamarreno et al., 2007). In Brazil, the use of these antioxidants is controlled by The National Health Surveillance Agency (ANVISA), which limits the amount to 200 mg kg1, for BHA, and to 100 mg kg1, for BHT (ANVISA, 2005). Thus, the determination of SPAs in foods is necessary to ensure the fullment of legal requirements as well as quality-control procedures in the food industry. Many methods for determining BHA and BHT individually or simultaneously have been recently reported, based on spectrophotometry (Capitan-Vallvey, Valencia, & Nicolas, 2004), liquid and gas chromatography (Guo, Xie, Yan, Wan, & Wu, 2006; Perrin & Meyer, 2002; Saad et al., 2007), micellar electrokinetic chromatography (Delgado-Zamarreno et al., 2007; Guan et al., 2006), and ow injection and HPLC with amperometric detection (Luque, Rios, & Valcarcel, 1999; Riber, de la Fuente, Vazquez, Tascon, & Batanero, 2000; Ruiz, Garcia-Moreno, Barbas, & Pingarron, 1999). But they are prone to many drawbacks, such as expensiveness, complicated and lengthy procedures, and unsuitability for eld use. Electrochemical techniques, such as the voltammetric ones, are a promising alternative to classical approaches due to their relatively low operational cost, good miniaturization potential, and rapid and sensitive detection procedures, which are suitable for faster analyses. Some methods for determining BHA and BHT by

R.A. Medeiros et al. / Food Chemistry 123 (2010) 886891

887

voltammetric techniques were already reported (Ag, Reviejo, Yanezsedeno, & Pingarron, 1995; Ceballos, 2006; Ceballos & Fernandez, 2000a; Ceballos & Fernandez, 2000b; De la Fuente, Acuna, Vazquez, Tascon, & Batanero, 1999; Diaz et al., 1998; Kumar & Narayanan, 2008; Ni, Wang, & Kokot, 2000; Raymundo, Paula, Franco, & Fett, 2007). Diaz et al. (1998) studied the voltammetric behaviour of propyl gallate (PG), BHA, and BHT at a glassy-carbon (GC) electrode (static and rotating) in an acetonitrilewater medium; they used a chemometric procedure for the determination of these antioxidants in different spiked samples of packet soup. Ni et al. (2000) studied the voltammetric behaviour of BHA, BHT, PG, and tert-butylhydroquinone, at a GC electrode in a 0.1 mol L1 perchloric acid solution containing 1% methanol, using chemometric approaches such as classical least squares, principal-component regression, and partial least squares. Linear calibration plots were obtained in the concentration ranges 2.883 lmol L1, for BHA, and 2.836 lmol L1, for BHT, with detection limits of 1.0 and 0.68 lmol L1, respectively. The method was applied to determine the four antioxidants in a set of synthetic mixtures as well as in several commercial food samples. Ceballos and Fernandez (2000b) used SWV with carbon-disk ultramicroelectrodes to determine BHA and BHT in vegetable oils. The determinations were carried out directly in benzene/ethanol/H2SO4 solutions or in acetonitrile after an extractive procedure, with better results in the latter case. Ag et al. (1995) used cylindrical carbon-bre microelectrodes in the simultaneous determination of BHA and BHT by SWV, obtaining detection limits of 4.0 lmol L1, for BHA, and 0.37 lmol L1, for BHT; however, the supporting electrolyte used contained acetonitrile, a high-cost reagent. Thin lms of BDD have emerged as excellent electrode materials for several electrochemical applications, especially electroanalytical ones, mainly due to properties such as: a wide potential window in aqueous solutions (up to 3 V), low background currents, long term stability, and low sensitivity to dissolved oxygen (Hupert et al., 2003; Panizza & Cerisola, 2005). The properties of BDD are commonly affected by the quantity and kind of the doping agent, morphologic factors and defects in the lm, presence of impurities (sp2 carbon), crystallographic orientation, and surface terminations (hydrogen or oxygen) that may be markedly determined by electrochemical pre-treatments (Salazar-Banda et al., 2006; Suffredini et al., 2004). Suffredini et al. (2004) called to attention that a cathodic pre-treatment of a BDD electrode dramatically increased the electroanalytical detection limit for chlorophenols, indicating that the analytical performance of BDD electrodes greatly depends on their surface termination, i.e., whether they are hydrogen or oxygen terminated. Recently, in our research group cathodically pre-treated BDD electrodes were used for the determination of aspartame and cyclamate in dietary products, individually (Medeiros, de Carvalho, Rocha-Filho, & Fatibello-Filho, 2007; Medeiros, de Carvalho, Rocha-Filho, & Fatibello-Filho, 2008b) or simultaneously (Medeiros, de Carvalho, Rocha-Filho, & Fatibello-Filho, 2008a), acetylsalicylic acid (Sartori, Medeiros, Rocha-Filho, & Fatibello-Filho, 2009), paracetamol and caffeine (Loureno, Medeiros, Rocha-Filho, Mazo, & Fatibello-Fiho, 2009) or sulfamethoxazole and trimethoprim (Andrade, Rocha-Filho, Cass, & Fatibello-Filho, 2009) simultaneously, and sildenal citrate Viagra (Batista, Sartori, Medeiros, RochaFilho, & Fatibello-Filho, 2010) in pharmaceutical formulations. In this paper, we report on the coupling of voltammetric techniques with the unique properties of the BDD electrode for the development and optimisation of a method for the simultaneous determination of BHA and BHT in several food products. The practical use of the method is demonstrated by determining the concentration of BHA and BHT in commercial margarine and mayonnaise samples and by comparing the obtained results with those from a high-performance liquid chromatography (HPLC) method.

2. Materials and methods 2.1. Apparatus The cyclic (CV), differential pulse (DPV), and square-wave (SWV) voltammetric experiments at a stationary BDD electrode were performed using an Autolab PGSTAT-30 (Ecochemie) potentiostat/galvanostat controlled with the GPES 4.0 software. A three-electrode cell system was also used: a BDD working electrode, a Pt-wire auxiliary electrode, and an Ag/AgCl (3.0 mol L1 KCl) reference electrode to which all electrode potentials hereinafter are referred. The boron-doped (8000 ppm) diamond (0.72 cm2 exposed area) lm on a silicon wafer was obtained from the Centre Suisse de Electronique et de Microtechnique SA (CSEM), Neuchatl, Switzerland (Salazar-Banda et al., 2006). Prior to use, the BDD electrode was cathodically or anodically pre-treated in a 0.5 M H2SO4 solution by applying 1.0 A cm2 or 1.0 A cm2, respectively, during 120 s. A GC electrode (0.2 cm2) was also used for comparative purposes. Prior to use, this electrode was pre-treated by sequential polishing with alumina (1 and 0.05 lm)/water slurries on felt pads, followed by rinsing with ultra-pure water. The BHA and BHT determinations by HPLC were carried out using an LC-10 AT Shimadzu system, with an ultravioletvis detector (SPD-M10-AVP) set at 290 and 278 nm. A Shim-Pack CLC-ODS (6.0 mm 250 mm, 5 lm) chromatographic column was used. The mobile phase was an acetonitrile/methanol mixture (50/50, v/v) at a ow rate of 1.0 mL min1, while the injection volume was 30 lL (Perrin & Meyer, 2002). 2.2. Reagents, supporting electrolyte and standards All reagents were of analytical grade: BHA and BHT (Sigma), KNO3 (Aldrich), and ethanol (Quemis, Brazil). An aqueous-ethanolic (30% ethanol, v/v) 10 mmol L1 KNO3 solution (pHcond 1.5 adjusted with 1.0 mol L1 HNO3) was used as supporting electrolyte. Standard 1.0 mmol L1 BHA and BHT solutions were prepared in this supporting electrolyte. All solutions were prepared using ultra-puried water supplied by a Milli-Q system (Millipore) with a resistivity greater than 18 MO cm. 2.3. Measurement procedures After optimising the experimental parameters for the proposed methods, the analytical curves were constructed by adding small and equal volumes of the concentrated standard solutions of the two analytes to the supporting electrolyte in order to have the following concentrations: 0.6, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 lmol L1. The detection limit was calculated as three times the standard deviation for the blank solution divided by the slope of the analytical curve. 2.4. Inuence of voltammetric techniques in the determination of the antioxidants The electrochemical behaviour of the antioxidants was investigated using three different voltammetric techniques. CV was used for preliminary studies, such as the choosing of supporting electrolyte and pH. DPV and SWV were used for investigating the determination of the antioxidants and nding the best conditions. 2.5. Treatment of commercial food samples A procedure similar to that proposed by Luque et al. (1999) and Raymundo et al. (2007) was followed for the determination of BHA

888

R.A. Medeiros et al. / Food Chemistry 123 (2010) 886891

and BHT in commercial margarine and mayonnaise samples purchased at a local supermarket. A sample of about 1.0 g was dissolved in 1 mL of ethanol contained in a large test tube. After shaking for 5 min, this mixture was centrifuged at 5000 rpm for 10 min. This extraction procedure was repeated twice; the extracts were collected and then diluted to 5 mL with the supporting electrolyte. A 500 lL aliquot was transferred to the electrochemical cell already containing 9.5 mL of the supporting electrolyte, where BHA and BHT were simultaneously determined by using the standard addition method. For the HPLC measurements, after the extraction procedure, the extracts were diluted in 5 mL of the mobile phase; previous to injection, a 500 lL aliquot was further diluted to 10 mL with the mobile phase. 3. Results and discussion 3.1. Investigation of the electrochemical behaviour of BHA and BHT Firstly, CV was used to investigate the electrochemical behaviour of both compounds in various supporting electrolytes: phosphate buffer, BrittonRobinson buffer, acetate buffer, and potassium nitrate, all prepared with an aqueous-ethanolic (30% ethanol, v/v) mixture as solvent. The best results were obtained with a 10 mmol L1 KNO3 solution (pHcond. 1.5 adjusted with 1.0 mol L1 HNO3). Using this supporting electrolyte, peak oxidation potentials of 0.65 and 0.93 V were obtained for BHA and BHT, respectively, and the obtained voltammograms presented an irreversible behaviour (results not shown), in agreement with data previously reported in the literature (Ag et al., 1995). A linear plot of the peak current vs. the square root of the scan rate was obtained for both antioxidants (r = 0.999 and 0.996 for BHA and BHT, respectively), indicating that the electrode process is controlled by mass transport. Secondly, electroanalytical procedures were developed for BHA and BHT individually using SWV and DPV. The optimisation of the experimental parameters that affect the SWV and DPV responses was carried out. The optimum values obtained for SWV were: frequency, 100 Hz; amplitude, 50 mV; scan increment, 4 mV. For DPV, they were: pulse amplitude, 60 mV; scan rate, 60 mV s1; Modulation time, 10 ms. According to the obtained results, the electroanalytical procedure developed using SWV yielded the best values for the gures of merit, for both antioxidants. Therefore, SWV was the technique chosen for the subsequent development of an electroanalytical procedure for the simultaneous determination of BHA and BHT. 3.2. Simultaneous determination of BHA and BHT Fig. 1 shows the square-wave voltammetric curves obtained at BDD and GC electrodes for 10 lmol L1 BHA and 10 lmol L1 BHT simultaneously in the supporting electrolyte. Well-dened peak currents were obtained for both antioxidants; however, the voltammogram obtained with the BDD electrode yielded higher peak-current values, especially the one for BHT. Hence, further studies were carried out only with the BDD electrode. The square-wave voltammograms obtained for both antioxidants with the BDD electrode after anodic and cathodic pre-treatments are shown in Fig. 2. As it can be seen, when the cathodically pre-treated electrode is used, two well-dened oxidation waves can be observed. The rst (Ep = 0.65 V) and the second (Ep = 0.93 V) waves correspond to the oxidation of BHA and BHT, respectively. When the anodically pre-treated electrode was used, the magnitude of these two waves decreased and their peak potentials became more positive. Consequently, all subsequent experiments were carried out using a cathodically pre-treated BDD electrode.

2.0 1.5 j(A cm-2) 1.0 0.5 0.0 0.4 0.6 0.8 1.0 E / V vs. Ag/AgCl (3.0 M KCl)

Fig. 1. SWV curves for 10 lmol L1 BHA and 10 lmol L1 BHT on a CG electrode (dashed lines) and a BDD electrode (solid line). Supporting electrolyte: aqueousethanolic (30% ethanol, v/v) 10 mmol L1 KNO3 solution (pHcond. 1.5).

1.6 1.2 I / 0.8 0.4 0.0 0.4 0.6 0.8 1.0 1.2 E / V vs. Ag/AgCl (3.0 M KCl)

Fig. 2. SWV curves obtained on an anodically (dashed line) and a cathodically (solid line) pre-treated BDD electrode, using a mixture of 10 lmol L1 BHA and 10 lmol L1 BHT. Supporting electrolyte: aqueous-ethanolic (30% ethanol, v/v) 10 mmol L1 KNO3 solution (pHcond. 1.5).

According to the accepted SWV theories (Scholz, 2005), the number of electrons transferred in the redox process can be investigated using the relationship:

DEp =D log f 2:3RT =anF

where a is the transfer coefcient and n the number of electrons involved in the redox reaction, the other terms having their usual meaning. The slopes obtained from the Ep vs. log f plots were 0.0608, for BHA, and 0.0591, for BHT; thus, by means of Eq. (1), values equal to 0.970 V (BHA) and 0.993 V (BHT) were determined for an. If the value of a is assumed as equal to 0.5, a common feature for organic molecules, these results indicate that the oxidation of BHA and BHT involves two electrons per molecule. From these results and considering the proposed oxidation mechanisms for compounds BHA and BHT (Ceballos, 2006; de la Fuente et al., 1999), the electrochemical oxidation of BHA or BHT is believed to occur by a two-electron mechanism (Fig. 3). The approximate surface concentration (C) of the adsorbed species was also calculated according to the accepted SWV theories, using the following equation (Scholz, 2005):

Ip 5 1 102 Aan2 FfaDEs C

where A is the electrode geometric area. The calculated values for C were 0.10 nmol cm2, for BHA, and 11 nmol cm2, for BHT.

R.A. Medeiros et al. / Food Chemistry 123 (2010) 886891

889

OH C(CH3)3 H2O

O C(CH3)3 H3O
+

O C(CH3)3 2 e2H2O CH3OH H3O+

OCH3 BHA

OCH3

OH (CH3)3C C(CH3)3 H2O (CH3)3C

O C(CH3)3 + H3O+ 2 e-

CH3 BHT

CH3

Fig. 3. BHA and BHT oxidation mechanisms at the BDD electrode surface.

The SWV curves presented a good peak-potential separation (about 0.3 V), which clearly allows the simultaneous determination of the compounds. Firstly, the separate determination of BHA in the concentration range 0.6010 lmol L1 was accomplished in solutions containing BHT at the xed concentration of 7.0 lmol L1 (Fig. 4). An examination of Fig. 4A allows concluding that the peak oxidation current for BHA increases regularly as its concentration is increased at a xed concentration of BHA (its peak oxidation current remained constant RSD = 3.0%). Similarly, as shown in Fig. 4B, the peak oxidation current for BHT increases regularly as its concentration is increased at a xed concentration of BHA of 1.0 lmol L1 (its peak oxidation current remained fairly constant RSD = 8.1%). After this previous study, both antioxidants were determined by simultaneously changing their equal concentrations (see Fig. 5). By analyzing the insets in this gure one can conclude that the respective analytical curves presented a good linearity in the investigated concentration range (0.6010 lmol L1, for both BHA and BHT). The corresponding calibration equations are (r = 0.9996, for both):

2.5 2.0 1.5 I / 1.0 0.5 0.0

(A)

9
1

0.5

0.6 0.7 0.8 0.9 1.0 E / V vs. Ag/AgCl (3.0 M KCl)

1.1

1.2 0.9 I / 0.6 0.3 0.0

(B)

Ipa =lA 0:00179 0:227c=lmol L for BHA Ipa =lA 0:0244 0:0973c=lmol L for BHT
The detection-limit values calculated for BHA and BHT were 0.14 and 0.25 lmol L1, respectively, which are lower than those reported by both Ag et al. (1995) and Ni et al. (2000). In the case of BHA, the obtained detection limit is almost 30 or seven times lower than the one reported by Ag et al. (1995) or Ni et al. (2000), respectively. For BHT, the detection limit here reported is slightly lower than the ones reported by these authors. The intra- and inter-day repeatabilities were determined by successive measurements (n = 5) of BHA and BHT in different concentrations; the obtained RSD values were: for BHA, intra-day, 1.4%, and inter-day, 2.4%; for BHT, intra-day, 2.4%, and inter-day, 2.7%. Next, the selectivity of the proposed method was evaluated by the addition of possible interferents (NaCl, soluble starch, EDTA, citric acid, and acetic acid) in a standard solution containing 10 lmol L1 BHA and 10 lmol L1 BHT, in the concentration ratios (standard solution:interferent) 10:1, 1:1, and 1:10; the obtained current signals were compared with those in the absence of each possible interferent. The analysis of the obtained responses allowed concluding that these compounds do not signicantly interfere with the here proposed method. The highest deviation was
1

12 1

0.5

0.6 0.7 0.8 0.9 1.0 E / V vs. Ag/AgCl (3.0 M KCl)

1.1

Fig. 4. (A) SWV curves for various concentrations of BHA at a xed concentration of BHT (7.0 106 mol L1). BHA concentrations (19): 0.6010 lmol L1. (B) SWV curves for various concentrations of BHT at a xed concentration of BHA (1.0 lmol L1). BHT concentrations (212): 0.6010 lmol L1. Supporting electrolyte: aqueous-ethanolic (30% ethanol, v/v) 10 mmol L1 KNO3 solution (pHcond. 1.5).

found for EDTA at the concentration ratio 1:10, but it amounted to only 5.7%. Finally, BHA and BHT were determined in seven different commercial products (ve mayonnaises and two margarines) by the standard addition method. Recovery experiments carried out to

890

R.A. Medeiros et al. / Food Chemistry 123 (2010) 886891

2.5

2.0
Ip /

2.0 1.5 1.0 0.5 0.0 0

(A)

1.5 I / 1.0
Ip /

6
-6

10
-1

[BHA] / 10
1.0 0.8 0.6 0.4 0.2 0.0 0 2 4

mol L

(B)

(0.14 lmol L1) and BHT (0.25 lmol L1); these values are lower than the ones previously reported in the literature using voltammetric methods. Besides, addition and recovery studies allowed concluding that the matrix effect did not present any signicant interference. The concentration values obtained for BHA and BHT are similar to those obtained using a HPLC method. Hence, the SWV method here reported is effective for the simultaneous determination of BHA and BHT in food products; furthermore, it is a very simple, inexpensive, and rapid method.
12

0.5 0.0 0.0

6
-6

8
-1

10

Acknowledgements The authors gratefully acknowledge nancial support from the Brazilian funding agencies FAPESP, CNPq, and CAPES. References
Ag, M. L., Reviejo, A. J., Yanezsedeno, P., & Pingarron, J. M. (1995). Analytical applications of cylindrical carbon-ber microelectrodes Simultaneous voltammetric determination of phenolic antioxidants in food. Analytical Chemistry, 67(13), 21952200. Andrade, L. S., Rocha-Filho, R. C., Cass, Q. B., & Fatibello-Filho, O. (2009). Simultaneous differential pulse voltammetric determination of sulfamethoxazole and trimethoprim on a boron-doped diamond electrode. Electroanalysis, 21(13), 14751480. ANVISA (2005). Collegial board resolution RDC no. 23, of 15 February 2005. <http:// www.anvisa.gov.br>. Batista, E. F., Sartori, E. R., Medeiros, R. A., Rocha-Filho, R. C., & Fatibello-Filho, O. (2010). Differential pulse voltammetric determination of sildenal citrate (Viagra) in pharmaceutical formulations using a boron-doped diamond electrode. Analytical Letters, 43(6), 10461054. Capitan-Vallvey, L. F., Valencia, M. C., & Nicolas, E. A. (2004). Solid-phase ultraviolet absorbance spectrophotometric multisensor for the simultaneous determination of butylated hydroxytoluene and co-existing antioxidants. Analytica Chimica Acta, 503(2), 179186. Ceballos, C. D. (2006). Using square wave voltammetry on ultramicroelectrodes to determine synthetic antioxidants in vegetable oils. Journal of Chemical Education, 83(9), 13521394. Ceballos, C., & Fernandez, H. (2000a). Synthetic antioxidants determination in lard and vegetable oils by the use of voltammetric methods on disk ultramicroelectrodes. Food Research International, 33(5), 357365. Ceballos, C., & Fernandez, H. (2000b). Synthetic antioxidants in edible oils by square-wave voltammetry on ultramicroelectrodes. Journal of the American Oil Chemists Society, 77(7), 731735. De la Fuente, C., Acuna, J. A., Vazquez, M. D., Tascon, M. L., & Batanero, P. S. (1999). Voltammetric determination of the phenolic antioxidants 3-tert-butyl-4hydroxyanisole and tert-butylhydroquinone at a polypyrrole electrode modied with a nickel phthalocyanine complex. Talanta, 49(2), 441452. Delgado-Zamarreno, M. M., Gonzalez-Maza, I., Sanchez-Perez, A., & Martinez, R. C. (2007). Analysis of synthetic phenolic antioxidants in edible oils by micellar electrokinetic capillary chromatography. Food Chemistry, 100(4), 17221727. Diaz, T. G., Cabanillas, A. G., Franco, M. F. A., Salinas, F., & Vire, J. C. (1998). Voltammetric behavior and simultaneous determination of the antioxidants propyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene in acidic acetonitrilewater medium using PLS calibration. Electroanalysis, 10(7), 497505. FAO/WHO, J. (1995). Codex general standard for food additives. <http://www.who.int/ ipcs/food/jecfa/en/index.html>. Guan, Y. Q., Chu, Q. C., Fu, L., Wu, T., & Ye, J. N. (2006). Determination of phenolic antioxidants by micellar electrokinetic capillary chromatography with electrochemical detection. Food Chemistry, 94(1), 157162. Guo, L., Xie, M. Y., Yan, A. P., Wan, Y. Q., & Wu, Y. M. (2006). Simultaneous determination of ve synthetic antioxidants in edible vegetable oil by GCMS. Analytical and Bioanalytical Chemistry, 386(6), 18811887. Hupert, M., Muck, A., Wang, J., Stotter, J., Cvackova, Z., Haymond, S., et al. (2003). Conductive diamond thin-lms in electrochemistry. Diamond and Related Materials, 12, 19401949. Kumar, S. S., & Narayanan, S. S. (2008). Mechanically immobilized nickel aquapentacyanoferrate modied electrode as an amperometric sensor for the determination of BHA. Talanta, 76(1), 5459. Loureno, B. C., Medeiros, R. A., Rocha-Filho, R. C., Mazo, L. H., & Fatibello-Fiho, O. (2009). Simultaneous voltammetric determination of paracetamol and caffeine in pharmaceutical formulations using a boron-doped diamond electrode. Talanta, 78, 748752. Luque, M., Rios, A., & Valcarcel, M. (1999). A poly(vinyl choloride) graphite composite electrode for ow-injection amperometric determination of antioxidants. Analytica Chimica Acta, 395(12), 217223. Medeiros, R. A., de Carvalho, A. E., Rocha-Filho, R. C., & Fatibello-Filho, O. (2007). Square-wave voltammetry determination of aspartame in dietary products using a boron-doped diamond electrode. Analytical Letters, 40(1618), 31953207.

[BHT] / 10 mol L

0.2 0.4 0.6 0.8 1.0 E / V vs. Ag/AgCl (3.0 M KCl)

Fig. 5. SWV curves obtained for the oxidation of BHA and BHT. The concentrations of both BHA and BHT were equal and changed simultaneously (212): 0.60 10 lmol L1. Inset: the respective analytical curves for BHA (A) and BHT (B). Supporting electrolyte: aqueous-ethanolic (30% ethanol, v/v) 10 mmol L1 KNO3 solution (pHcond. 1.5).

Table 1 Results obtained in the simultaneous determination of BHA and BHT in food products by HPLC and the proposed method (SWV). Samples Mayonnaise 1 2 3 4 5 Margarine 6 7
a b c

BHA (mg/100 g) HPLCa 2.0 0.1 1.7 0.2 2.3 0.2 2.3 0.1 1.9 0.1 n.d.c n.d.c SWVa 1.9 0.1 1.7 0.1 2.2 0.2 2.3 0.1 1.8 0.1 n.d.c n.d.c

BHT (mg/100 g) HPLCa 1.1 0.1 1.3 0.2 1.8 0.2 1.4 0.2 1.6 0.2 4.2 0.2 3.8 0.2 SWVa 1.1 0.1 1.2 0.2 1.7 0.2 1.5 0.1 1.6 0.2 4.0 0.2 3.7 0.1

BHA Error (%)b 5.0 0 4.3 0 5.2

BHT Error (%)b 0 7.7 5.5 7.4 0 4.8 2.6

Average of three measurements. Error (%) = 100 (SWV value HPLC value)/HPLC value. n.d. = not detected.

evaluate matrix effects after standard-solution additions yielded excellent average recoveries for both substances (101% for BHA and 99% for BHT), indicating that there were no important matrix interferences for the samples analyzed by the proposed SWV method. Table 1 presents the BHA and BHT concentrations determined simultaneously in the analyzed food products employing the proposed SWV method and an HPLC method (Perrin et al., 2002). By analyzing the results obtained for the seven commercial food products (Table 1), one can conclude that the values obtained by the proposed method agree quite well with those obtained by the reference HPLC method. Applying the paired t-test to the results obtained by both methods, the resulting t values (0.128 for BHA and 0.232 for BHT) are smaller than the critical one (2.31, a = 0.05), indicating that there is no difference between the obtained results, at a condence level of 95%. 4. Conclusions The obtained results allow concluding that SWV along with a cathodically pre-treated BDD electrode can be used with some benets for the quantitative determination of BHA and BHT, alone or mixed as commonly found in food products. Very low detection limits were obtained in the simultaneous determination of BHA

R.A. Medeiros et al. / Food Chemistry 123 (2010) 886891 Medeiros, R. A., de Carvalho, A. E., Rocha-Filho, R. C., & Fatibello-Filho, O. (2008a). Simultaneous square-wave voltammetric determination of aspartame and cyclamate using a boron-doped diamond electrode. Talanta, 76(3), 685689. Medeiros, R. A., de Carvalho, A. E., Rocha-Filho, R. C., & Fatibello-Filho, O. (2008b). Voltammetric determination of sodium cyclamate in dietary products using a boron-doped diamond electrode. Quimica Nova, 31(6), 14051409. Ni, Y. N., Wang, L., & Kokot, S. (2000). Voltammetric determination of butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate and tertbutylhydroquinone by use of chemometric approaches. Analytica Chimica Acta, 412(12), 185193. Panizza, M., & Cerisola, G. (2005). Application of diamond electrodes to electrochemical processes. Electrochimica Acta, 51(2), 191199. Perrin, C., & Meyer, L. (2002). Quantication of synthetic phenolic antioxidants in dry foods by reversed-phase HPLC with photodiode array detection. Food Chemistry, 77(1), 93100. Raymundo, M. D., Paula, M. M. D., Franco, C., & Fett, R. (2007). Quantitative determination of the phenolic antioxidants using voltammetric techniques. LWT Food Science and Technology, 40(7), 11331139. Riber, J., de la Fuente, C., Vazquez, M. D., Tascon, M. L., & Batanero, P. S. (2000). Electrochemical study of antioxidants at a polypyrrole electrode modied by a nickel phthalocyanine complex. Application to their HPLC separation and to their FIA system detections. Talanta, 52(2), 241252.

891

Ruiz, M. A., Garcia-Moreno, E., Barbas, C., & Pingarron, J. M. (1999). Determination of phenolic antioxidants by HPLC with amperometric detection at a nickel phthalocyanine polymer modied electrode. Electroanalysis, 11(7), 470474. Saad, B., Sing, Y. Y., Nawi, M. A., Hashim, N., Ali, A. S. M., Saleh, M. I., et al. (2007). Determination of synthetic phenolic antioxidants in food items using reversedphase HPLC. Food Chemistry, 105, 389394. Salazar-Banda, G. R., Andrade, L. S., Nascente, P. A. P., Pizani, P. S., Rocha-Filho, R. C., & Avaca, L. A. (2006). On the changing electrochemical behaviour of borondoped diamond surfaces with time after cathodic pre-treatments. Electrochimica Acta, 51(22), 46124619. Scholz, F. (Ed.). (2005). Electroanalytical methods. New York: Springer. Sartori, E. R., Medeiros, R. A., Rocha-Filho, R. C., & Fatibello-Filho, O. (2009). Squarewave voltammetric determination of acetylsalicylic acid in pharmaceutical formulations using a boron-doped diamond electrode without the need of previous alkaline hydrolysis step. Journal of the Brazilian Chemical Society, 360, 366. Suffredini, H. B., Pedrosa, V. A., Codognoto, L., Machado, S. A. S., Rocha-Filho, R. C., & Avaca, L. A. (2004). Enhanced electrochemical response of boron-doped diamond electrodes brought on by a cathodic surface pre-treatment. Electrochimica Acta, 49(2223), 40214026.