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doi:10.1016/j.lw

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(M. dos Santos

LWT 40 (2007) 1133–1139

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Quantitative determination of the phenolic antioxidants usingvoltammetric techniques

Melissa dos Santos Raymundoa,�, Marcos Marques da Silva Paulab,Cesar Francoc, Roseane Fetta

aDepartment of Science and Technology Food, CAL, Center of Agricultural Science, University of Santa Catarina, Rod. Admar Gonzaga, 1346,

Itacorubi, Florianopolis CEP 88034-001, BrazilbDepartment of Material Science, UNESC, University of Extremo Sul Catarinense, Av. Universitaria, 1105, Bairro Universitario, Caixa Postal 3167,

CEP: 88806-000, BrazilcDepartment of Chemistry, Center of Mathematical and Physics Sciences, CFM, University of Santa Catarina, UFSC, Trindade, Florianopolis,

Santa Catarina, Brasil, Caixa Postal 476, CEP 88040-900, Brazil

Received 25 April 2006; received in revised form 26 June 2006; accepted 4 July 2006

Abstract

Synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone

(TBHQ), show sensitivity to voltammetric waves. The waves of these antioxidants, however, are seriously overlapped and it is difficult to

determine them simultaneously. The influence of different parameters (working electrode, supporting electrolyte, pH, voltammetric

technique) was evaluated in a quantitative simultaneous determination of three antioxidants in alcoholic mixtures and real sample foods.

Glassy carbon (GC) and platinum (Pt) working electrodes were investigated as mediators of oxireduction reactions. Two supporting

electrolytes were investigated: Britton–Robinson 0.1mol l�1 buffer (pH 2.0) and HCl 0.1mol l�1 (pH 2.0) both with 2 g l�1 (p/v) of

methanol. In this paper, voltammetric conditions for the analysis of up to three-component mixtures of antioxidant present at levels:

2.0–100.0mg l�1 for BHA, 4.0–100.0mg l�1 for TBHQ and 2.0–20.0mg l�1 for BHT at GC in HCl 0.1mol l�1 and 8.0–120.0, 10.0–130.0

and 4.0–30.0mg l�1 for BHA, TBHQ and BHT, respectively, at Pt in the same supporting electrolyte. The results show that for real food

samples, the parameters investigated were satisfactory for quantitative determination using square wave voltammetry (SWV) without

chemometric approaches and without suffering overlapping problems.

r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Voltammetry; Synthetic antioxidants; Food; Analysis

1. Introduction

Antioxidant compounds continue to play a veryimportant role in many biological processes where freeradicals are present (Cui, Luo, Xu, & Ven Murth, 2004;Mariani, Polidori, Cherubini, & Mecocci, 2005). Theprimary antioxidants, such as butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT) and tert-butylhy-droquinone (TBHQ), act by blocking free radicals,converting them into stable products via redox reactions

0 r 2006 Swiss Society of Food Science and Technology. Pu

t.2006.07.001

ing author. Tel.: +55 48 3331 5374; fax: +55 48 3331 9943.

esses: melissa@qmc.ufsc.br, melissasrbr@yahoo.com.br

Raymundo).

(Madhavi, 1995). They are commonly used, alone ortogether in commercial mixtures as additives in oils or fats,in order to prevent oxidative rancidity (Bruggemann,Visnjevski, Burch, & Patel, 2004; Pinho, Ferreira, Oliveira,& Ferreira, 2000). One of the foods that most containsthese additives is mayonnaise, a combination of lemonjuice or vinegar with egg yolks and oil and with one ormore synthetic antioxidants in its formulation (Jacobsen,Schwarz, Stockmann, Meyer, & Adler-Nissen, 1999).Colorimetry and spectrophotometry methods were firstused for the determination of these additives in differentfood samples and today it is possible to find differentvariations (Capitan-Vallvey, Valencia, & Nicolas, 2004;Cruces-Blanco, Segura Carretero, Merino Boyle, &

blished by Elsevier Ltd. All rights reserved.

ARTICLE IN PRESSM. dos Santos Raymundo et al. / LWT 40 (2007) 1133–11391134

Fernandez Gutierrez, 1999; Viplava Prasad, Divakar,Hariprasad, & Sastry, 1987). Later chromatographicmethods were developed (Karovicova & Simko, 2000;Perrin & Meyer, 2002 ;Yanez-Sedeno, Pingarron, & PoloDıez, 1991; Yang, Lin, & Choong, 2002; Zhang, Wu,Wang, Yang, & Liu, 2002). Electrochemical methods ortechniques, for example, polarographic and voltammetrictechniques (differential pulse polarography, absorptivevoltammetry and linear sweep voltammetry, etc.), generallypossess high sensitivity and are extensively used inanalytical chemistry (Campanella, Bonanni, Bellantoni,Favero, & Tomassetti, 2004; Cheng, Ren, Li, Chang, &Chen, 2002; Korotkova, Korbainov, & Shevchuk, 2002;Pournaghi-Azar, Hydarpour, & Dastangoo, 2003; Yang &Huang, 2002). Studies of electrochemical behaviour ofthese antioxidants using amperometric techniques havebeen reviewed and tested at bare electrode and modifiedelectrode (Chevion, Roberts, & Chevion, 2000; Fuente,Batanero, Tascon, Vasquez, & Acuna, 1999; Gonzalez,Ruiz, Yanez-Sedeno, & Pingarron,1994; Ni, Wang, &Kokot, 2000; Riber, Fuente, Vazquez, Tascon, & SanchezBatanero, 2000; Surareungchai & Kasiwat, 2000); besidesthe agreed methods, chromatography with electrochemicaldetectors is also used (King, Joseph & Kissinger, 1980;Baldwin & Thomsen, 1991; Bianchi et al., 1997). In the caseof the electrochemical methods, when the mixture has morethan two antioxidant components present, the voltam-metric waves of these antioxidants are closely overlappedand it is difficult to analyse them individually (Cheng et al.,2002; Ni et al., 2000). Recently some chemometricapproaches, such as principal component regression(PCR) and partial least square regression (PLSR), havebeen introduced for the determination of mixtures ofsynthetic antioxidants with three or more components(Galeano Diaz, Guiberteau Cabanillas, Alexandre Franco,Salinas, & Vire (1998); Richards, Bessant, & Saini, 2002).Even though they are strictly limited in use (Shahidi, 2000),it is important to analyse their contents in foods. Thus, inthe present work, the results of voltammetric quantitativeanalyses in different conditions for use in the determinationof these antioxidants using a rapid, clean technique arepresented.

2. Materials and methods

2.1. Reagents

Tert-butylhydroxyanisole (BHA) from Fluka, Germany;TBHQ and 2,6-di-t-butyl-p-hydroxytoluene (BHT) fromAldrich, Germany. All reagents were of analytical gradeand were used without previous purification. The waterwas obtained by Mili-Q purification system. The standardsolutions of antioxidants mixed and in isolation, wereprepared in methanol at 500mg l�1. To modify the pH ofthe two supporting electrolytes, a 2mol l�1 NaOH solutionwas added in sufficient quantity to achieve the pH valuesrequired.

2.2. Apparatus

An EG&G PARC potentiostat (model 273A, USA) wasused. The working electrodes (WE) tested were a glassycarbon disk (GC, 0.950 cm2, Pine Instrumentation) and aPt disk (Pt, 0.502 cm2, Pine Instrumentation); as reference,Hg/Hg2Cl2/KCl(sat.) electrodes (SCE) and one home madePt plate auxiliary electrode were used. All the experimentswere carried out at a constant temperature (20.070.05) 1Cunder a controlled atmosphere of argon.

2.3. Procedure

2.3.1. Influence of techniques voltammetric in determination

of antioxidants synthetics

The electrochemistry activity of antioxidants wereinvestigated with different voltammetric techniques, cyclicvoltammetry (CV) (ub ¼ 50mV s�1), differential pulsevoltammetry (DPV) (DE ¼ 25mV; ub ¼ 50mV s�1) andsquare wave voltammetry (SWV) (DE ¼ 25mV;f ¼ 60Hz). The electrochemical behavior of the syntheticantioxidants was investigated both in mixture and inisolation under the same analytical conditions, with theobjective of obtaining more information regarding over-lapping waves. Two supporting electrolytes were used:Britton–Robinson buffer 0.1mol l�1 (A) and solution HCl0.1mol l�1 (B), both containing 20ml l�1 methanol. Inaddition to each measurement, cleaning of the electrodewas realized by polishing with aluminium, cleaning withdistilled water, clean stirring with methanol for 30 s and theapplication of range potential 1.3–0mV at 30mV s�1 usinglinear sweep voltammetry.

2.3.2. Quantitative determination of TBHQ, BHA and BHT

in food samples

Between 10.0 and 20.0 g of mayonnaise samples,accurately weighed to 0.0001 g, were individually dissolvedin 5ml pure methanol and transferred to a 100mlErlenmeyer flask. After shaking for 5min, this mixturewas centrifuged at 3000 rpm for 10min. The extractionprocedure was repeated twice, all the extracts werecollected and then the solution was diluted with methanol.One 10.0ml aliquot of this sample solution was analysed bySWV, as described in Section 2.3.1 and applying thestandard addition method. Five aliquots of standardmixture at 25mg l�1 had been used for construction ofthe addition curve current (Ipa) vs. concentration (mg l�1)and after interpolation. The SWV technique was chosen forall experiments due to its improved performance incomparison with the other amperometric techniques testedin 2.3.1.

2.3.3. Statistical analysis

Data are presented as mean7standard deviation. Theresults were statistically analysed by analysis of variance(ANOVA) followed by Duncan’s test. Differences wereconsidered significant for Po0.05.

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Fig. 2. Square wave voltammogram SWV ( ), cyclic voltammogram

CV (—), differential pulse voltammogram DPV (?) and linear sweep

voltammogram SLV (- - -) in supporting electrolyte Britton–Robinson

buffer 0.1mol l�1 with methanol 20ml l�1 for 50mg l�1 BHA, BHT and

TBHQ, pH 2.0, n ¼ 5 at Pt working electrode.

M. dos Santos Raymundo et al. / LWT 40 (2007) 1133–1139 1135

3. Results and discussion

3.1. Influence of voltammetric techniques in the

determination of synthetic antioxidants

In the voltammetric measurement of antioxidants, thereis a proportional relationship between the peak current andthe concentration of electroactive species. The systemstudied here is characterized by the exchange of only oneelectron (Cheng et al., 2002). Although it is well knownthat in voltammetric techniques, differential pulse is moresensitive than classic voltammetry, in this work theantioxidant determination was performed using differentvoltammetric techniques, as presented in Fig. 1 (GC) andFig. 2 (Pt). The SWV technique is both more rapid andmore sensitive than DPV. The DPV at GC WE was betterthan that at Pt with well defined TBHQ and BHA waves.Figs. 1 and 2 also show that CV and LSV techniques wereless adequate with only two waves for GC and Pt WEs. CVis very favourable for investigating the electrochemicalbehaviour of analyte, however, it is of limited use forquantitative analysis (Gonzalez et al., 1994). SWV was theonly technique that presented three clear waves for bothWEs: 385, 555 and 790 for TBHQ, BHA and BHT,respectively, for GC; and 382, 540 and 736mV for TBHQ,BHA and BHT, respectively, for Pt.

3.2. Influence of working electrode

Measurements were realized for each antioxidant sepa-rately and for the antioxidant mixture, a square wavevoltammogram of the mixture in supporting electrolyte Aat pH 2.0 is presented in Fig. 3. The GC WE is morefavourable to BHA and TBHQ (greater Ipa values) than

Fig. 1. Square wave voltammogram SWV ( ), cyclic voltammogram

CV (—), differential pulse voltammogram DPV (?) and linear sweep

voltammogram SLV(- - -) in supporting electrolyte Britton–Robinson

buffer 0.1mol l�1 with methanol 20ml l�1 for 50mg l�1 BHA, BHT and

TBHQ, pH 2.0, n ¼ 5 at GC working electrode.

Fig. 3. Square wave voltammograms in supporting electrolyte Britton–

Robinson buffer 0.1mol l�1 with methanol 20ml l�1 for 50mg l�1 BHA,

BHT and TBHQ, pH 2.0, n ¼ 5 at Pt work electrode (?) and GC work

electrode (—).

BHT, though both working electrodes allowed for thesimultaneous determination of three antioxidants in arange of interest for food analysis. However, the Pt WE hasa less absorptive surface and is therefore more susceptibleto contamination.

3.3. Influence of pH and supporting electrolyte

The influence of pH (2.0; 4.0, 6.0 and 8.0) on the mixtureoxidation signal was studied by SWV using supporting

ARTICLE IN PRESSM. dos Santos Raymundo et al. / LWT 40 (2007) 1133–11391136

electrolytes A and B. The Ipa (mAcm�2) values decreasedlinearly with pH increase for all synthetic antioxidants forboth working electrodes (Pt and GC) and for bothsupporting electrolytes (A and B). pH 2.0 was the onlysolution that allowed for the detection of all threeantioxidants. The oxidation of TBHQ was not detectedat pH 6.0 and 8.0 (A) and pH 8.0 (B). The oxidation ofBHT was not detected at pH 6.0 and 8.0 (B). In relation tothe performance of the supporting electrolyte, both weresatisfactory and similar. The oxidation of BHA at GC, forexample (y ¼ 317.26–22.83x, r2 ¼ 0.99 for A and y ¼

389.02–37.36x, r2 ¼ 0.93 for B) and at Pt (y ¼ 640.8–34.797x, r2 ¼ 0.99 for A and y ¼ 247.76–12,248x, r2 ¼ 0.77for B). TBHQ and BHT showed similar behaviour(data not shown). Table 1 shows the results for Epa (mV)and Ipa (mAcm�2) at GC and Pt working electrodesat pH 2.0 for an antioxidant mixture at a concentration

Table 1

Epa (mV) and Ipa (mAcm�2) for synthetic antioxidants mixture in different su

Antioxidant Working electrode Supporting electrolyt

TBHQ GC A

GC B

Pt A

Pt B

BHA GC A

GC B

Pt A

Pt B

BHT GC A

GC B

Pt A

Pt B

A—Britton–Robson 0.1mol l�1 buffer.

B—solution HCl 0.1mol l�1.aEp and jp are anodic peak potential (mV) and anodic peak current (mA), r

Table 2

Linear relation and ranges obtained for mixtures of BHA, BHT and TBHQ a

Range (mg l�1) r2 Slope (mA lmg�

Supporting electrolyte A

BHA S 2.0–80.0 0.9710

TBHQ S 4.0–80.0 0.9112

BHT S 4.0–30.0 0.9941

BHA M 2.0–100.0 0.9274

TBHQ M 4.0–100.0 0.9521

BHT M 2.0–40.0 0.9563

Supporting electrolyte B

BHA S 2.0–100.0 0.9814

TBHQ S 1.0–80.0 0.7895

BHT S 8.0–20.0 0.9762

BHA M 2.0–100.0 0.9853

TBHQ M 4.0–100.0 0.9682

BHT M 2.0–20.0 0.7222

S—separated antioxidant, M—mixture of antioxidants.

of 25mg l�1 and likewise, for the highest signal-to-back-ground ratio for SWV. The mean values of Epa (mV)presented more deviation for BHT (Epa ¼ 650 mV (A) andEpa ¼ 720 mV (B).

3.4. Influence of antioxidant concentration

Using the SWV technique, the voltammograms of Ipa vs.concentration at pH 2.0 were evaluated for the mixture ofantioxidants and for each in isolation, with both electrolyte(A and B) and both WE (GC and Pt). The concentrationranges, slopes and intercepts for GC are shown in Table 2and for Pt in Table 3. The results show a good relationbetween the ranges for the mixture of antioxidants andeach antioxidant in isolation. The linear concentrationrange for the same supporting electrolyte appears to becorrelated with the relative solubility of each antioxidant,

pporting electrolytes, n ¼ 5 at pH 2.0

e Epa (mV) jp

a (mAcm�2) cv

440.00710.23 122.2874.34 3.54

350.00712.56 189.0274.39 2.32

430.0078.34 211.00714.56 6.91

415.00710.98 80.8072.72 3.36

575.0079.03 274.0675.25 1.91

530.00712.13 292.3476.96 2.38

575.00710.44 433.06740.06 9.25

540.00714.43 155.2879.79 6.30

835.0077.45 72.7773.12 4.28

825.00711.67 141.8273.21 2.26

840.0077.78 149.4277.29 4.87

650.0075.21 121.86710.96 8.99

espectively.

t GC working electrode using square wave voltammetry (SWV), n ¼ 5

1) Intercept (mA) Epa (mV)

7.8172.11 259.08714.51 440.078.11

3.3271.28 152.97712.59 570.079.04

4.3770.94 73.5375.80 830.077.65

4.9070.71 178.68711.16 570.074.61

2.6570.52 166.3179.43 440.074.72

1.6170.27 107.0579.21 830.079.52

6.2371.59 154.67710.43 535.078.21

3.4976.34 110.78710.32 350.075.25

7.2171.07 49.2075.24 825.077.32

5.0870.79 167.83711.04 520.073.32

3.6070.83 77.2279.23 330.075.67

2.8670.66 111.77717.19 820.075.83

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Table 3

Linear relation and ranges obtained for mixtures of BHA, BHT and TBHQ at Pt working electrode using square wave voltammetry (SWV), n ¼ 5

Range (mg l�1) r2 Slope (mA lmg�1) Intercept (mA) Epa (mV)

Supporting electrolyte A

BHA S 1.0–80.0 0.9919 6.7871.02 120,54711.80 580.075.17

TBHQ S 8.0–40.0 0.8842 8.0272.11 0 410.073.28

BHT S 10.0–30.0 0.9218 5.6570.98 354.59715.86 750.076.11

BHA M 10.0–60.0 0.9915 5.6171.16 122.92717.38 600.075.63

TBHQ M 12.0–30.0 0.9968 3.8170.72 66.7179.77 450.076.81

BHT M 10.0–40.0 0.9034 3.2270.59 243.63725.85 770.075.63

Supporting electrolyte B

BHA S 4.0–80.0 0.9619 3.2071.25 212.15711.74 540.078.23

TBHQ S 12.0–120.0 0.9968 5.0974.23 112.7479.53 415.077.55

BHT S 12.0–40.0 0.9823 3.3970.76 177.6577.15 630.077.21

BHA M 8.0–120.0 0.9919 6.0971.84 92.1077.34 530.077.27

TBHQ M 10.0–130.0 0.9887 4.3970.81 123.5279.23 420.077.75

BHT M 4.0–30.0 0.9609 5.2870.95 103.01710.03 650.0710.23

Table 4

Determination of BHA, BHT and TBHQ in commercial mayonese samples by SWV in supporting electrolyte HCl 0.1mol l�1, n ¼ 5

Mayonnaise Values in mg/100 g7SD

Pt working electrode GC working electrode

S L TBHQ BHA BHT TBHQ BHA BHT

B 1 — 3.4770.81 — — 2.7870.29 2.1170.12

B 2 — 4.3670.53 — — 3.1970.76 2.4570.21

B 3 — 4.3270.45 — — 3.0570.51 2.6770.18

C 1 — 4.2470.69 2.3270.32 — 3.1170.54 2.3470.22

C 2 — 3.7870.41 1.9770.54 — 2.6970.32 2.5570.16

C 3 — 5.8170.79 1.7670.42 — 2.1370.67 2.6470.25

D 1 5.8970.62 — — 5.6770.44 — —

D 2 6.1170.74 — — 5.8870.65 — —

D 3 7.3870.56 — — 7.0170.68 — —

E 1 — 2.8470.38 2.1370.28 — 3.6570,45 2.4370.25

E 2 — 3.3670.31 2.1870.26 — 4.957 0,23 2.5670.19

E 3 — 3.1570.55 2.0270.31 — 3.2570.17 2.2370.43

S—sample, L—different lot.

M. dos Santos Raymundo et al. / LWT 40 (2007) 1133–1139 1137

for example, BHT is the least soluble of the three andshows the least range.

3.5. Food samples analysis

The procedure described in the experimental section forSWV applying the standard addition method was used todetermine BHA, BHT and TBHQ in commercial mayon-naise. One of the advantages of this method is thereduction of the matrix effect for complex food samples,as mayonnaise. The results obtained are summarized inTable 4. The mean value obtained for BHA, BHT andTBHQ agrees with the expected concentration levels forthese antioxidants in this kind of sample (legislation—BRAZIL, 1998). The SWV technique was favourable forthe detection of synthetic antioxidants in the mixture.However, BHT probably has a low solubility in both thesupporting electrolytes tested and cannot be detected with

precision. The sample concentration of BHT was outsidethe concentration range for this antioxidant, thereforeBHT was quantified in another more diluted solution. BothBHA and TBHQ showed greater linear range concentra-tion in the course of this study.

3.6. Interferences

Possible interferences between the antioxidant mixtureand mayonnaise components were investigated. Analyseswere realized for lactic acid, citric acid, EDTA andpotassium sorbate, because these are commonly used inthis kind of product. The different concentrations testedwere: 1:1 (p/p), 1:2 (p/p), 1:5 (p/p), 1:10 (p/p) and 1:20(p/p) or 25–500mg l�1 of each interferent cited. For thecitric acid at concentrations above of 125mg l�1 it wasobserved, especially about the BHT, one change of peakvalue (dislocation) and increasing reduction of Ipa value

ARTICLE IN PRESSM. dos Santos Raymundo et al. / LWT 40 (2007) 1133–11391138

until complete overlapping at 500mg l�1 of citric acid. Forthe lactic acid, even so the values of current (Ipa) very werelittle affected and at concentrations above of 125mg l�1

already were possible to observed overlapping of the threepeaks. For the potassium sorbate the overlapping occurredit only at concentration of 500mg l�1 and nothing itoccurred for EDTA. It is interesting to observe that theoverlapping caused by interferent occurred at concentra-tions greater than 125mg l�1 (ratio 1:5 (p/p)) and theseratio is improbable in sample food real. Yanez-Sedeno,Pingarron, & Polo Dıez (1991) tested possible interferencesof citric acid on BHT and BHA at a concentration of0.5mg l�1 in the presence of acid at a concentrationranging from 0.5 to 50.0mg l�1 without observing inter-ference.

Gonzalez et al. (1994) also studied the effects of citricacid, BHT and TBHQ on the electrochemical determina-tion of BHA at pH 2.0 (GC work electrode in Britton–Robinson buffer 0.2mol l�1) without finding significantinterferences up to a relation of 1:200 (p/p). Observationsmade in the present study showed the same outcome.

4. Conclusions

The electrochemical determination of a mixture contain-ing BHA, BHT and TBHQ using different techniques, pH,working electrodes and distinct supporting electrolytesshowed that some simultaneous determination was possiblewithout chemometric approaches or prior treatments. Thesynthetic antioxidants showed different detection ranges inthis study. BHT showed a smaller concentration rangeprobably due to its lower solubility, a behaviour thatdemanded an isolated determination at a lower concentra-tion and that made the simultaneous determination ofthree antioxidants in one measurement impossible. Theoption of selecting between working electrodes andsupporting electrolytes make a reduction in the costs ofanalysis possible. All the systems tested showed goodperformance and applicability in food sample analysiswithout the use of chemometric approaches to bypass thewave overlapped problem.

Acknowledgements

The authors are grateful to CAPES for their financialsupport of this work.

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