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Analytica Chimica Acta 674 (2010) 79–88

Contents lists available at ScienceDirect

Analytica Chimica Acta

journa l homepage: www.e lsev ier .com/ locate /aca

etermination of antioxidants by a novel on-line HPLC-cupric reducingntioxidant capacity (CUPRAC) assay with post-column detection

aliha Esin Celik, Mustafa Özyürek, Kubilay Güclü, Resat Apak ∗

epartment of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey

r t i c l e i n f o

rticle history:eceived 26 March 2010eceived in revised form 31 May 2010ccepted 9 June 2010vailable online 16 June 2010

eywords:n-line HPLC-cupric reducing antioxidantapacity (CUPRAC)

a b s t r a c t

A novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) method was developed for theselective determination of polyphenols (flavonoids, simple phenolic and hydroxycinnamic acids) incomplex plant matrices. The method combines chromatographic separation, constituent analysis, andpost-column identification of antioxidants in plant extracts. The separation of polyphenols was per-formed on a C18 column using gradient elution with two different mobile phase solutions, i.e., MeOH and0.2% o-phosphoric acid. The HPLC-separated antioxidant polyphenols in the extracts react with copper(II)-neocuproine (Cu(II)-Nc) reagent in a post-column reaction coil to form a derivative. The reagent is reducedby antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm.The negative peaks of antioxidant constituents were monitored by measuring the increase in absorbance

ost-column detection

pectrophotometric cupric reducingntioxidant capacity assayolyphenols

due to Cu(I)-Nc. The detection limits of polyphenols at 450 nm (in the range of 0.17–3.46 �M) afterpost-column derivatization were comparable to those at 280 nm UV detection without derivatization.The developed method was successfully applied to the identification of antioxidant compounds in crudeextracts of Camellia sinensis, Origanum marjorana and Mentha. The method is rapid, inexpensive, versatile,non-laborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of

of com

antioxidant constituents

. Introduction

The measurement of the antioxidant capacity of food prod-cts is a matter of growing interest in diagnosis and therapy ofxidative stress-related diseases such as muscle and tissue degen-ration, heart disease, diabetes, and cancer. Many assays haveeen developed to determine the antioxidant capacity of variousatrices such as fruits, vegetables, beverages, and biological fluids

1,2]. Efforts directed to individual identification and quantifica-ion of antioxidant compounds in plant matrices may give rise toroblems, because the activities of antioxidant compounds mayecrease during their isolation and purification due to decompo-ition. Thus procedures for the separation and quantification ofntioxidants should be performed simultaneously. More recently,ertain assays have been modified for post-column-coupled on-ine high performance liquid chromatographic (HPLC) applications3–7]. The most widely used assays in post-column applications

re free radical decolorization methods, based on the scavenging ofhromogenic free radicals DPPH (2,2′-diphenyl-1-picryl hydrazyl)6] or ABTS (2,2′-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid)7]. It is difficult to precisely quantify antioxidant activity because

∗ Corresponding author. Tel.: +90 212 473 7028; fax: +90 212 473 7180.E-mail address: rapak@istanbul.edu.tr (R. Apak).

003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2010.06.013

plex plant samples.© 2010 Elsevier B.V. All rights reserved.

of the short lifetimes of these radicals. Moreover, reaction kinet-ics may vary with these radicalic reagents as a function of phenolicsteric effects, solvent composition, and pH [3–7]. A method combin-ing separation of components in the complex matrix and evaluationof antioxidant capacity provides significant advantages for suchinvestigations [6].

According to the on-line HPLC analysis scheme developed byKoleva et al., the HPLC-separated analytes react in the post-column mode with DPPH solution, and the induced bleachingis detected as a negative peak at 517 nm [6]. Bandoniene andMurkovic improved the on-line HPLC–DPPH screening method forphenolic antioxidants in apple extracts based on a decrease inabsorbance after post-column reaction of HPLC-separated antiox-idants with the DPPH radical [8]. Some drawbacks of the DPPHpost-column detection system are; (i) requirement of purgingof DPPH• solution before use to reduce baseline drift caused bydissolved O2, (ii) variations in analyte detection caused by theinstability of DPPH• in daylight, (iii) reduced areas of negativepeaks caused by slow and complex kinetics of phenolic–DPPH•

reactions, (iv) possible clogging in the reaction coil caused by

deviations from neutral conditions in the post-column mixturesolution, (v) increased noise level beyond a certain DPPH• con-centration [4,7]. By using the DPPH and ABTS radicalic detectionsystems, Exarchou et al. [9] showed the possibility of on-line rapidscreening of antioxidant constituents in methanolic extracts of

80 S.E. Celik et al. / Analytica Chimic

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The other polyphenolic antioxidants were analyzed using these

Fig. 1. Instrumental configuration for on-line HPLC-CUPRAC detection system.

harmaceutically active plants such as nettle, rosehip, and linden.¸ apanoglu et al. [10] used HPLC-ABTS on-line detection to deter-

ine the effect of processing steps in the industrial productionhain for tomato starting from fresh fruit to paste. The signifi-ant advantages of these on-line methods are that the antioxidantctivity of a single compound can be measured and its contribu-ion to the overall activity of a complex mixture be calculated,nd also the activity of a single compound can be compared tohose of other constituents in the matrix. Thus, it is no longerecessary to purify every single constituent for off-line assays,

eading to very significant reduction of costs and time to obtainesults [6]. Although instrumental hardware and related param-ters have not changed much since the first reports of Koleva etl. [6,7], high-resolution antioxidant screening using HPLC post-olumn detection has now become a more efficient tool for theapid identification of antioxidants in plant extracts, foods and bev-rages [11].

The aim of this study is to develop a novel post-columnerivatization technique using cupric reducing antioxidant capac-

ty (CUPRAC) methodology for individual antioxidant compoundsn synthetic and sample mixtures. Compared to other post-columneagents of similar properties, the CUPRAC reagent has been showno be much less dependent on phenolic lipophilicity, steric effects,olvent (mobile phase) composition, pH, dissolved oxygen, andaylight [12,13]. The instrumental configuration showing the on-

ine system is given in Fig. 1. In this system, the HPLC-separatedntioxidants react with the Cu(II)-Nc reagent in the reaction coil.he reagent is reduced by the antioxidants to the yellow-coloredu(I)-Nc complex having an absorption maximum at 450 nm. It wasbserved that the antioxidant capacity of each substance is showny an increase in the area of negative peaks which is proportionalo increasing concentration.

. Experimental

.1. Reagents, materials and apparatus

The following chemicals of analytical reagent grade were sup-lied from the corresponding sources: quercetin (QR), feruliccid (FRA), p-coumaric acid (COU), caffeic acid (CFA), gallic acidGA), luteolin (LT), chlorogenic acid (CA), rosmarinic acid (ROS),pigenin (APG), epicatechin (EC), epigallocatechin (EGC), epigal-ocatechingallate (EGCG), epicatechin gallate (ECG), neocuproine,

ethanol (MeOH) were purchased from Sigma Co.; trolox (TR)as purchased from Aldrich Chemicals Co. (Steinheim, Germany);

atechin (CT), myricetin (MR) were purchased from Fluka Co.Buchs, Switzerland); copper(II) chloride, absolute ethyl alco-ol (EtOH), ammonium acetate (NH Ac) and o-phosphoric acid

4H3PO4) were purchased from Merck (Darmstadt, Germany). Theeagents were ‘analytical reagent’ grade unless otherwise stated.reen tea (Camellia sinensis), mint (Mentha), and sweet marjoram

Origanum marjorana) were purchased from Malatya Pazari A.S.

a Acta 674 (2010) 79–88

(Istanbul, Turkey), black tea (C. sinensis) was purchased from CaykurA.S. (Rize, Turkey).

The extraction of plant material was carried out in anUltra-Turrax CAT X-620 model homogenizer apparatus (Staufen,Germany). The spectra and absorption measurements wererecorded in matched quartz cuvettes using a Varian CARY Bio 100UV–vis spectrophotometer (Mulgrave, Victoria, Australia).

A Waters BreezeTM 2 Model HPLC system (Milford, MA, USA)equipped with a 1525 binary pump, a column oven with thermo-stat, a 2998 photo-diode array detector (Chelmsford, MA, USA), anda Hamilton 25 �L-syringe (Reno, NV, USA) was used for chromato-graphic measurements. Data acquisition was accomplished usingEmpower PRO (Waters Associates, Milford, MA). The HPLC-coupledpost-column system consisted of a reaction module placed after thechromatographic column equipped with an RXN-1000 reaction coil(volume 1 mL, approximate length 5 m, Waters), and the mobilephase stream from the HPLC pump mixed with the post-columnreagent flowing through a 0.50-mm i.d. knitted PTFE (Teflon) tubingwith reaction time ≥1 min. The analytical wavelengths of detectionwere 280 and 450 nm for conventional and on-line HPLC post-column applications, respectively.

2.2. Preparation of solutions

The standard solutions of antioxidants were all prepared inmethanol at a concentration of 10 mM with the exception of api-genin which was dissolved in 1 M potassium hydroxide and dilutedwith ethanol. All standard solutions were stored at −20 ◦C prior toanalysis. The CuCl2 solution (10 mM) and ammonium acetate buffersolution (1 M, pH 7) were prepared in pure distilled water (MilliporeSimpak1 Synergy 185, USA), and neocuproine solution (7.5 mM) inabsolute ethanol.

2.3. Solvent extraction of plant materials

The dried plant specimens were crushed in a mill, and 2-g sam-ples were taken for each plant species. These samples were soakedin 20 mL of 80% MeOH overnight, and homogenized in an Ultra-Turrax apparatus by gradually increasing the number of cyclesper unit time. The extracts were transferred to centrifuge tubes,centrifuged for 10 min (at 5000 rpm), and subsequently filteredthrough a blue-band Whatman filter paper into 100-mL flasks. Theplant material was re-extracted another three times with 25 mL of80% MeOH each. All filtered extracts were combined, and diluted to100 mL using the extraction solvent [14]. The extracts could be ana-lyzed for their antioxidant capacities on the next day if preservedby storing under N2 in stoppered flasks in a freezer at −20 ◦C. Plantextracts were filtered through a 0.45 �m micro-filter (Whatman,UK) before HPLC analysis.

2.4. Chromatographic separation – conventional HPLC assay

The analyses were carried out using a reverse-phase ACE C18column (4.6 mm × 250 mm, 5 �m particle size) (Milford, MA, USA).Three different HPLC elution programs were used for tea antioxi-dants, other polyphenolic compounds, and trolox. The mobile phaseconsisted of two solvents, i.e., methanol (A) and 0.2% of o-H3PO4 inbidistilled water (B). The following parameters and gradient wereused for the analysis of tea antioxidants [15]: (Vsample= 20 �L; flowrate = 0.8 mL min−1; � = 280 nm): 1 min 0% A–100% B (slope 1.0);20 min 70% A–30% B (slope 1.0); 25 min 0% A–100% B (slope 1.0).

parameters and gradient program [16]: (Vsample = 20 �L; flowrate = 0.8 mL min−1; � = 280 nm): 8 min 7% A–93% B (slope 1.0);13 min 30% A–70% B (slope 1.0); 45 min 66% A–34% B (slope 1.0);50 min 7% A–93% B (slope 1.0).

Chimica Acta 674 (2010) 79–88 81

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Table 1TEACCUPRAC coefficients of several antioxidant compounds in methanol (expressedas mean ± standard deviation).

Antioxidant TEACCUPRAC

Trolox (TR) 1.00 ± 0.05Gallic acid (GA) 2.98 ± 0.06Catechin (CT) 3.70 ± 0.12Epicatechin (EC) 3.80 ± 0.12Epigallocatechin (EGC) 3.96 ± 0.17Epicatechingallate (ECG) 5.60 ± 0.13Epigallocatechingallate (EGCG) 6.33 ± 0.21Chlorogenic acid (CA) 2.88 ± 0.03Caffeic acid (CFA) 3.40 ± 0.08p-Coumaric acid (COU) 1.12 ± 0.04Ferulic acid (FRA) 1.38 ± 0.08Rosmarinic acid (ROS) 4.95 ± 0.33Myricetin (MR) 4.10 ± 0.27Quercetin (QR) 5.54 ± 0.32Luteolin (LT) 3.27 ± 0.13

S.E. Celik et al. / Analytica

Trolox was analyzed using these parameters and gradient pro-ram: (Vsample = 20 �L; flow rate = 1.0 mL min−1; � = 280 nm): 5 min0% A–50% B (slope 1.0); 13 min 80% A–20% B (slope 1.0); 15 min0% A–50%B (slope 1.0).

Using the above working modes, the calibration curves wereonstructed and linear equations of peak area versus concentrationound for the antioxidants of interest.

.5. Conventional CUPRAC spectrophotometric assay

The CUPRAC method, as described by Apak et al. [12], is basedn the reduction of a cupric neocuproine complex (Cu(II)-Nc) byntioxidants to the cuprous form (Cu(I)-Nc). To a test tube weredded 1 mL each of Cu(II), Nc, and NH4Ac buffer solutions. Antioxi-ant standard solution or samples (x mL) and H2O (1.1 − x)mL weredded to the initial mixture so as to make the final volume: 4.1 mL.he tubes were stoppered, and after 30 min, the absorbance at50 nm (A450) was recorded against a reagent blank. The molarbsorptivity for each antioxidant pertaining to the CUPRAC methodas calculated from the slope of the calibration line drawn as

bsorbance versus concentration.

.6. On-line HPLC-CUPRAC assay with post-column detection

An on-line method is described for the rapid detection andapacity determination of antioxidant compounds. In this method,u(II)-Nc complex in pH 7 ammonium acetate medium wassed as the chromogenic reagent. The HPLC-separated com-ounds reacted in the post-column reaction coil with the CUPRACeagent. The detector extracted the 450 nm wavelength. CUPRACeagent was freshly prepared from the corresponding solutions ofu(II):Nc:NH4Ac at a ratio of 1:1:1 (v/v/v) prior to analysis, androtected from daylight. The flow rate of the CUPRAC reagent was.5 mL min−1.

.7. Statistical analysis

Descriptive statistical analyses were performed using Exceloftware (Microsoft Office 2002) for calculating the means andhe standard error of the mean. Results were expressed as the

ean ± standard deviation (SD). Using SPSS software for Windowsversion 13), the data were evaluated by two-way Analysis of vari-nce (ANOVA) [17].

. Results and discussion

The antioxidant capacity of individual antioxidants (galliccid, catechin and derivatives, ferulic acid, chlorogenic acid,uercetin, luteolin, etc.), synthetic mixtures and crude plantxtracts (C. sinensis, O. marjorana and Mentha) were assessedy a novel on-line HPLC-CUPRAC method. The chromatographiceparation and antioxidant capacity analyses can be carried outimultaneously using the developed method. Following HPLCnalysis with UV detection at 280 nm, the eluate was mixedith CUPRAC solution (Cu(II):Nc:NH4Ac at a ratio of 1:1:1,

/v/v) using the pump in the post-column reactor. The efflu-nt was directed to a PDA detector monitoring absorbance at50 nm. Our simple, low-cost, and widely applicable CUPRACntioxidant capacity assay for dietary polyphenols, flavonoids,itamins C and E [12], and plasma antioxidants [13] utilizeshe Cu(II)-Nc reagent as the chromogenic oxidant in which phe-

olic hydroxyls are oxidized to the corresponding quinones,roducing a yellow-colored chromophore of Cu(I)-Nc absorbingt 450 nm. Cu(I)-Nc complex produced as a result of the redoxeaction with antioxidants is reflected by negative peaks moni-ored at 450 nm in the post-column chromatogram while positive

Apigenina (APG) 0.13 ± 0.01

a Apigenin was dissolved in ethanolic KOH.

peaks show absorption at 280 nm. Three different gradient elu-tion programs were used to identify tea antioxidants, phenolicantioxidants and the reference standard (trolox), as described inSection 2.4.

3.1. CUPRAC antioxidant capacities of individual antioxidants

The trolox-equivalent antioxidant capacities (TEAC) – definedas the mM trolox-equivalent capacity in reducing power of 1 mMsolution of the compound – of the antioxidants in MeOH were cal-culated by dividing the molar absorptivity of each antioxidant tothe molar absorptivity of trolox (εTR being 1.58 × 104 L mol−1 cm−1

under the specified conditions) (Table 1).In general, in an electron transfer-based antioxidant assay like

CUPRAC, the molar absorptivities for antioxidant compounds mayshow certain variations depending on the composition and polarityof the solvent medium [18]. The DPPH radical scavenging method,again basically an electron transfer-based antioxidant assay, hasbeen demonstrated to show slow kinetics in pure methanolic solu-tion, whereas in a more aqueous solution, the assay results aregoverned by thermodynamics rather than kinetics due to the rapidestablishment of a dynamic equilibrium, meaning that water inthe reaction medium showed a huge effect on reaction kinet-ics [11]. For example, the measured TEACCUPRAC value of 6.33 forEGCG in the methanolic medium of this work is higher than the4.89 value in ethanol [19] probably due to facilitated electrontransfer in ionizing solvents capable of phenolate anion solva-tion, since MeOH is the alcohol that best supports ionization[20].

3.2. Conventional HPLC results of the antioxidants

Synthetic mixtures of antioxidant standards were analyzedby reference HPLC methods [15,16] with minor modifications.Retention times and linear working ranges of the antioxidantsat a detection wavelength of 280 nm were given in Table 2.This table also shows the linear calibration equations of antiox-idants (tea antioxidants, phenolic antioxidants, and trolox) aschromatographic peak area (denoted by the symbol y) versus

molar concentration (denoted as c). Antioxidants were quantifiedusing the equations with the correlation coefficients (r) rangingbetween 0.9994 and 0.9999, generally closer to the upper value(Table 2).

82 S.E. Celik et al. / Analytica Chimica Acta 674 (2010) 79–88

Table 2Conventional HPLC calibration equations, retention times and linear ranges of trolox, tea antioxidants and phenolic antioxidants.

Antioxidant tR (min) Linear range (mol L−1) Calibration equationd andcorrelation coefficient (r)

Referencea

Trolox 11.57 1 × 10−5 to 1 × 10−3 y = 3.18 × 109c − 7.95 × 103

r = 0.9999

Tea antioxidantsb

Gallic acid 9.88 4 × 10−5 to 2 × 10−4 y = 1.38 × 1010c + 1.43 × 104

r = 0.9999(−) Epigallocatechin 13.74 4 × 10−5 to 2 × 10−4 y = 1.46 × 109c + 1.75 × 103

r = 0.9999

(+) Catechin 14.13 4 × 10−5 to 2 × 10−4 y = 6.88 × 109c − 1.30 × 104

r = 0.9998

(−) Epigallocatechin gallate 15.09 4 × 10−5 to 2 × 10−4 y = 1.73 × 1010c − 2.29 × 104

r = 0.9999

(−) Epicatechin 16.04 4 × 10−5 to 2 × 10−4 y = 7.79 × 109c + 4.05 × 103

r = 0.9999

(−) Epicatechin gallate 17.11 4 × 10−5 to 2 × 10−4 y = 8.77 × 109c + 1.41 × 104

r = 0.9999

Phenolic antioxidantsc

Catechin 17.87 2 × 10−5 to 2 × 10−4 y = 6.50 × 109c + 7.46 × 103

r = 0.9999Chlorogenic acid 19.08 2 × 10−5 to 2 × 10−4 y = 1.58 × 1010c + 1.54 × 104

r = 0.9999Caffeic acid 21.15 2 × 10−5 to 2 × 10−4 y = 1.82 × 1010c + 1.62 × 104

r = 0.9999p-Coumaric acid 25.40 2 × 10−5 to 2 × 10−4 y = 2.31 × 1010c − 1.66 × 104

r = 0.9995Ferulic acid 26.25 2 × 10−5 to 2 × 10−4 y = 1.72 × 1010c + 6.41 × 103

r = 0.9999Rosmarinic acid 32.00 2 × 10−5 to 2 × 10−4 y = 1.78 × 1010c + 6.97 × 103

r = 0.9999Myricetin 32.61 2 × 10−5 to 2 × 10−4 y = 1.06 × 1010c + 7.43 × 103

r = 0.9999Quercetin 38.16 2 × 10−5 to 2 × 10−4 y = 1.29 × 1010c + 3.93 × 102

r = 0.9999Luteolin 39.56 2 × 10−5 to 2 × 10−4 y = 1.49 × 1010c − 3.16 × 104

r = 0.9999Apigenin 43.79 2 × 10−5 to 2 × 10−4 y = 1.84 × 1010c − 1.46 × 104

r = 0.9994

( ograms (see Section 2.4).( on (denoted as c).

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a–c) These classes of compounds were analyzed with different gradient elution prd) Chromatographic peak area (denoted by the symbol y) versus molar concentrati

.3. Identification and quantification of antioxidants by then-line HPLC-CUPRAC method with optimal instrumentalarameters

The standard mixtures of antioxidants were separated on aeverse-phase column and identified at 280 nm. HPLC-separatedntioxidants were mixed with Cu(II)-Nc reagent in a post-columneaction coil resulting in a redox reaction. The post-column eluateontaining the Cu(I)-Nc chelate was detected at 450 nm. The chro-atograms for the synthetic mixtures of tea antioxidants (Fig. 2)

nd other polyphenolic antioxidants (Fig. 3) were simultaneouslyecorded as positive peaks (monitored at 280 nm) and negativeeaks (as a result of the CUPRAC reaction, monitored at 450 nm).

Some instrumental parameters of the post-column methodere optimized using a fixed length reaction coil (used as sold). In

eneral, the noise in a post-column HPLC system decreased as theerivatizing reagent concentration was decreased, but at the sameime the signal decreased as well [21]. Unnecessarily high concen-

rations of the derivatizing reagent may give rise to clogging of theeaction coil in nonpolar media [4]. Thus, both the concentration ofhe CUPRAC reagent fed to the reaction coil and the flow rate of theost-column pump were optimized by minimizing the signal-to-oise (S/N) ratio. The pump flow rate was chosen as 0.5 mL min−1

Fig. 2. The chromatograms of a synthetic mixture of tea antioxidants at 280 (positivetrace) and 450 nm (negative trace).

S.E. Celik et al. / Analytica Chimica Acta 674 (2010) 79–88 83

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ig. 3. The chromatograms of a synthetic mixture of other polyphenolic antioxi-ants at 280 (positive trace) and 450 nm (negative trace).

o provide the higher S/N value (Fig. 4), and by fixing this value,he final concentration of the CUPRAC reagent was optimized forhe assay of trolox (2.0 × 10−4 M). The higher S/N value (i.e., 29.6)as observed for 3.33 mM Cu(II)/2.5 mM Nc/333 mM NH4Ac con-

entrations compared to that (i.e., 22.1) for 1.66 mM Cu(II)/1.25 mMc/166 mM NH4Ac.

It should be mentioned here that there are only two post-columnntioxidant assays in on-line HPLC literature that may competeith CUPRAC, namely post-column DPPH and ABTS assays. Both

ssays are based on the bleaching of colored radicals, therefore thenitial concentrations of the reagents of both assays need to be care-ully optimized, since too concentrated antioxidant solutions mayause total bleaching. For example, it has been reported that thePPH concentration can vary by a factor of 10, and higher concen-

rations should be used when analyte concentrations are high to

void too much quenching of the colored radical DPPH• [11]. This isot a problem with post-column CUPRAC as long as the absorbancet 450 nm remains within the linear working range, becauseUPRAC is based on color formation as opposed to bleaching.

able 3imits of detection (LOD) values for antioxidants measured with conventional HPLC-UV d

Antioxidant tR (280 nm) tR (450 nm)

(min) (min)

Referencea

Trolox 11.57 12.49

Tea antioxidantsb

Gallic acid 9.88 11.32(−) Epigallocatechin 13.74 15.06(+) Catechin 14.13 15.39(−) Epigallocatechin gallate 15.09 16.38(−) Epicatechin 16.04 17.32(−) Epicatechin gallate 17.11 18.34

Phenolic antioxidantsc

Catechin 17.87 19.29Chlorogenic acid 19.08 20.64Caffeic acid 21.15 22.87p-Coumaric acid 25.40 27.36Ferulic acid 26.25 28.09Rosmarinic acid 32.00 33.97Myricetin 32.61 34.85Quercetin 38.16 40.34Luteolin 39.56 42.32Apigenin 43.79 46.05

a–c) These classes of compounds have different gradient elution modes (see Section 2.4)

Fig. 4. Signal-to-noise (S/N) ratios determined for trolox (2.0 × 10−4 M) at differentflow rates of the CUPRAC reagent.

The relative standard deviation (RSD) of three measurementsof (negative) peak areas in the on-line HPLC-CUPRAC assay fordifferent concentrations of polyphenols varied between 1.8% and4.4%, from the higher to the lower end of the concentration range.The limit of detections (LOD = 3�bl/m), where �bl denotes the stan-dard deviation of a blank and m is the slope of the calibration line(A450 = mC + n), for various polyphenolic antioxidants measured byboth conventional HPLC and on-line HPLC-CUPRAC are depicted inTable 3. These data show that polyphenols can be assayed with highprecision by both conventional HPLC and on-line HPLC with post-column detection, and quite low concentrations of polyphenols canbe detected. Since the LOD values of the on-line HPLC-CUPRACmethod (Table 3) were lower – up to two orders of magnitude –than those of the on-line HPLC–DPPH assay previously reported

to be more sensitive than the analogic DPPH assay.The calibration curves of tea antioxidants measured by the

post-column CUPRAC assay are shown in (Table 4). Satisfactorylinear correlations were observed between the areas of negative

etection, on-line HPLC-CUPRAC, and on-line HPLC–DPPH methods.

280 nm 450 nm LOD(DPPH)

LOD (�M) LOD (�M) (�M) [6,8]

3.37 1.14 6.00

0.47 0.480.54 1.422.55 1.981.79 1.030.23 0.73 10.690.72 0.82 2.49

0.52 1.78 32.460.44 1.89 21.200.40 2.25 10.600.97 3.460.17 0.39 39.690.18 0.77 2.300.32 0.470.04 0.17 0.982.86 0.971.07 2.21

.

84 S.E. Celik et al. / Analytica Chimica Acta 674 (2010) 79–88

Table 4On-line HPLC-CUPRAC calibration equations (as negative peak area versus concentration), retention times (tR), and linear concentration ranges for trolox, tea antioxidantsand phenolic antioxidants.

Antioxidant tR (min) Linear concentration range (mol L−1) Calibration equationd andcorrelation coefficient (r)

Referencea

Trolox 12.49 1 × 10−5 to 1 × 10−3 y = 1.32 × 1010c + 3.33 × 104

r = 0.9999

Tea antioxidantsb

Gallic acid 11.32 4 × 10−5 to 2 × 10−4 y = 3.07 × 1010c + 3.21 × 104

r = 0.9999(−) Epigallocatechin 15.06 4 × 10−5 to 2 × 10−4 y = 3.37 × 1010c + 1.06 × 105

r = 0.9999(+) Catechin 15.39 4 × 10−5 to 2 × 10−4 y = 1.92 × 1010c + 8.57 × 104

r = 0.9997(−) Epigallocatechin gallate 16.38 4 × 10−5 to 2 × 10−4 y = 5.69 × 1010c + 1.30 × 105

r = 0.9998(−) Epicatechin 17.32 4 × 10−5 to 2 × 10−4 y = 2.20 × 1010c + 6.56 × 104

r = 0.9996(−) Epicatechin gallate 18.34 4 × 10−5 to 2 × 10−4 y = 4.31 × 1010c + 7.81 × 104

r = 0.9999

Phenolic antioxidantsc

Catechin 19.29 4 × 10−5 to 2 × 10−4 y = 1.88 × 1010c + 7.46 × 104

r = 0.9992Chlorogenic acid 20.64 4 × 10−5 to 2 × 10−4 y = 1.66 × 1010c + 6.99 × 104

r = 0.9996Caffeic acid 22.87 4 × 10−5 to 2 × 10−4 y = 1.83 × 1010c + 9.17 × 104

r = 0.9992p-Coumaric acid 27.36 4 × 10−5 to 2 × 10−4 y = 1.60 × 109c + 1.23 × 104

r = 0.9990Ferulic acid 28.09 4 × 10−5 to 2 × 10−4 y = 1.19 × 1010c + 1.03 × 104

r = 0.9993Rosmarinic acid 33.97 4 × 10−5 to 2 × 10−4 y = 3.40 × 1010c + 5.82 × 104

r = 0.9999Myricetin 34.85 4 × 10−5 to 2 × 10−4 y = 3.10 × 1010c + 3.26 × 104

r = 0.9995Quercetin 40.34 4 × 10−5 to 2 × 10−4 y = 5.84 × 1010c + 2.15 × 104

r = 0.9999Luteolin 42.32 4 × 10−5 to 2 × 10−4 y = 3.87 × 1010c + 8.38 × 104

r = 0.9998Apigenin 46.05 4 × 10−5 to 2 × 10−4 y = 1.12 × 109c − 5.49 × 103

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pOhpogai(gd[fr[tddaachhMao

a–c) These classes of compounds were analyzed with different gradient elution prd) Chromatographic peak area (“negative peak area”, denoted by the symbol y) ver

eaks and concentrations of the antioxidants (r = 0.9990–0.9999).f the catechin derivatives, epigallocatechingallate had theighest antioxidant capacity, possibly due to the number andosition of the –OH groups on the molecule. Antioxidant capacityf catechin derivatives decreased in the following order: epi-allocatechingallate > epicatechingallate > epigallocatechin > galliccid > epicatechin > catechin. Among phenolic antioxidants (includ-ng flavonoids), quercetin exhibited the highest capacity, due toi) the o-dihydroxy (catechol) structure in the B-ring, imparting areater stability to the aryloxy radicals formed by flavonoid oxi-ation, possibly through H-bonding and electron-delocalization22]; (ii) the 2,3-double bond, in conjugation with the 4-oxounction, and the resulting planar geometry which delocalizes theadical throughout the entire molecule enhancing electron transfer23,24]; (iii) the presence of both 3- and 5-OH groups, enablinghe formation of stable quinonic structures upon flavonoid oxi-ation [25]. Antioxidant capacity among phenolic antioxidantsecreased in the following order: quercetin > luteolin > rosmariniccid > myricetin > catechin > caffeic acid > chlorogenic acid > feruliccid > p-coumaric acid > apigenin. Rosmarinic acid, the dimer ofaffeic acid, exhibited the highest antioxidant capacity among

ydroxycinnamic acids, because it possesses four phenolicydroxyl groups which correlate with antioxidant strength [23].oreover, it has a conjugated structure, further stabilizing the

ryloxy radicals produced during the course of rosmarinic acidxidation. It is known that as the redox potential of the aryloxy

r = 0.9999

s (see Section 2.4).olar concentration (denoted as c).

radical/phenol couple is lowered as a result of stabilization ofthe 1-e oxidation product of phenol, e.g., through intra-molecularhydrogen-bonding, the corresponding phenolic molecule becomesa stronger antioxidant in an electron transfer-based antioxidantassay like CUPRAC. With the exception of gallic acid and luteolin,the observed capacity order for these antioxidants was in accor-dance with those of the conventional CUPRAC assay in methanolicmedium [18]. The kinetics of the reaction of gallic acid and luteolinwith the CUPRAC reagent may be faster in the post-column modethan in the conventional CUPRAC spectrophotometric assay. Thedifferent kinetic behavior of antioxidants is a critical point in theevaluation of antioxidant activity, as the post-column reaction hasa time limit proportional to the length of reaction capillary [8].

Fig. 5 shows that CUPRAC is a relatively fast assay, and mostof the tested antioxidants reach ≥80% of their maximal molarabsorptivities within ≥1 min (spent in the post-column reactioncoil). When the antioxidant assay reagent is a coordinatively satu-rated metal complex species involving different oxidation statesof a given metal ion in the same ligand environment such asbis(neocuproine)copper(II,I) capable of fast outer-sphere e-transferwith the polyphenol, then a negligible reorientation of the already

existing ligands around the central metal ion may be expectedin the formation of the transient intermediate during e-transfer,and consequently, the rate of e-transfer may only be affected to alimited extent by solvent polarity [18]. Naturally, the aim here isnot to reach the maximal molar absorptivities, but to obtain opti-

S.E. Celik et al. / Analytica Chimic

Fr

mccod9i

3m

aatate

TC

o

ig. 5. Reaction kinetics for oxidation of phenolic antioxidants with the CUPRACeagent: cupric neocuproine. Final concentration of each antioxidant was 9.76 �M.

al absorptivities within ≥1 min so as to precisely calculate theoncentrations of antioxidant constituents at the end of the post-olumn reaction. On the other hand, other post-column methods ofn-line HPLC antioxidant assay such as DPPH and phosphomolyb-enum blue may require higher temperatures of 60 ◦C [26] and5 ◦C [27], respectively, to overcome kinetic problems (i.e., increas-

ng the extent of reaction completion within ≥1 min).

.4. Total antioxidant capacity (TAC) measurements of syntheticixture solutions

Four mixtures of antioxidants were prepared (Cfinal: 0.1 mM),nd the solutions were analyzed for total antioxidant capacity (TAC)s mM trolox (TR) equivalents using (i) conventional CUPRAC spec-rophotometry, (ii) conventional HPLC with CUPRAC calculation,nd (iii) on-line HPLC-CUPRAC assay with post-column detec-ion. Calculations of TAC values according to the three assays arexplained below:

(i) The experimentally found TACCUPRAC of the synthetic mix-tures or samples were calculated by dividing the observedabsorbance (A450) to the molar absorptivity of trolox

able 5omparison of CUPRAC, HPLC, and on-line HPLC-CUPRAC TAC values in the synthetic anti

Sample Method Mean concentration (mM)

Mixture 1b On-line HPLC-CUPRAC 1.37Conventional CUPRAC 1.47

Mixture 1b On-line HPLC-CUPRAC 1.37HPLCd 1.45

Mixture 2c On-line HPLC-CUPRAC 1.67Conventional CUPRAC 1.71

Mixture 2c On-line HPLC-CUPRAC 1.67HPLCd 1.69

a S = [((n1 − 1) · s21 + (n2 − 1) · s2

2)/(n1 + n2 − 2)]1/2

and texp = (a1 − a2)/[S · (1/n1 + 1/n2

f the two populations with sample sizes of n1 and n2, and sample means of a1 and a2, reb Mixture 1: QR + FRA + ROS + CA; final concentration of each constituent = 0.1 mM.c Mixture 2: GA + EGCG + EC + EGC; final concentration of each constituent = 0.1 mM.d Conventional HPLC with CUPRAC calculation; values calculated using Eq. (3.2).

a Acta 674 (2010) 79–88 85

(A450 = εTRCTR − 0.01 where εTR = 1.58 × 104 L mol−1 cm−1 andr = 0.9995) according to the spectrophotometric CUPRAC assay.

TACCUPRAC = Absorbance (total)εTR

× 103 (3.1)

(ii) TACHPLC values of the synthetic mixtures or samples were cal-culated by multiplying the concentration with the TEAC valueof each HPLC-identified antioxidant, and summing the prod-ucts.

TACHPLC =n∑

i=1

ci (TEAC)i (3.2)

where TACHPLC is the total antioxidant capacity in mM trolox(TR)-equivalents, ci is the final concentration of antioxidantcomponent: i and (TEAC)i is its TEAC coefficient of the CUPRACmethod.

(iii) TAC values of synthetic mixtures or real samples from the on-line HPLC-CUPRAC method with post-column detection werecalculated by summing the areas of negative peaks of the indi-vidually identified antioxidants and dividing the total peakarea to the slope of the calibration equation of trolox at 450 nm(y = 1.32 × 1010 CTR + 3.31 × 104 where y = area of negative peakand CTR = molar concentration of TR):

TACon-line HPLC-CUPRAC =

n∑

i=1

yi

slope× 103 (3.3)

Binary synthetic mixtures of phenolic antioxidants were pre-pared, and TAC values (as mM TR equivalent) were evaluated withdetection of constituents at 280 and 450 nm. The calibration equa-tion was found using these TAC values:

TACon-line HPLC-CUPRAC = 0.68 TACHPLC − 1.0 × 10−5 (r = 0.9947)

This means that the values obtained from the on-line HPLC-CUPRAC method are ≈68% of values found from conventional HPLCwith CUPRAC calculation. The former values were smaller (i.e., theconversion factor was less than unity) possibly because of dilutioneffects in the reaction coil and incomplete reaction within the fixedtime period of the assay. So (1/0.68 = 1.47) was chosen as the con-stant for calculations to find the actual total antioxidant capacity(i.e., TACHPLC = 1.47 TACon-line HPLC-CUPRAC).

Two synthetic mixture solutions of different compositionand concentrations were analyzed thrice (N = 3) by conventionalCUPRAC, HPLC with CUPRAC calculation, and on-line HPLC-CUPRACwith post-column detection. It was found that the experimentalvalues (i.e., |t| calculated from the pooled-estimate of standard devi-

oxidant mixtures.

SD (s) Sa t ttable F Ftable

0.05 – – – – –0.04 0.04 2.70 2.78 1.56 19.00

0.05 – – – – –0.03 0.04 2.37 2.78 2.78 19.00

0.06 – – – – –0.03 0.05 1.03 2.78 4.00 19.00

0.06 – – – – –0.03 0.05 0.51 2.78 4.00 19.00

)1/2], where S is the pooled standard deviation, s1 and s2 are the standard deviationsspectively, and t = (n1 + n2 − 2) degrees of freedom (n1 = n2 = 3).

86 S.E. Celik et al. / Analytica Chimica Acta 674 (2010) 79–88

Table 6TAC values of plant extracts evaluated by the conventional CUPRAC, conventional HPLC with CUPRAC calculation, and on-line HPLC-CUPRAC methods.

Plant sample Botanical name Conventional CUPRACspectrophotometric assay(mmol TR g−1)

HPLC assay with CUPRACcalculation (mmol TR g−1)

On-line HPLC-CUPRACassay with post-columndetection (mmol TR g−1)

Green tea Camellia sinensis 1.19 ± 0.03 1.03 ± 0.04 1.12 ± 0.05Black tea Camellia sinensis 0.27 ± 0.04 0.044 ± 0.001 0.046 ± 0.002Mint Mentha 0.85 ± 0.04 0.47 ± 0.03 0.49 ± 0.04

0.28 ± 0.04 0.26 ± 0.03

( PRAC assays: P = 0.05, Fexp = 0.25, Fcrit (table) = 10.13, Fexp < Fcrit (table). (ii) Data presented as(

avr

3

aaav

t(Tttc(mC(oa

ottrkaw

FC

Sweet marjoram Origanum marjorana 0.51 ± 0.01

i) The ANOVA comparison between the conventional HPLC and on-line HPLC-CUmean ± SD), N = 3

tion, and F from the ratio of variances) did not exceed the criticalalues, indicating that there were no significant differences in accu-acy and precision between the methods of determination (Table 5).

.5. TAC measurements of crude plant extracts

In Table 6, the TAC values found by conventional CUPRAC, HPLC,nd on-line CUPRAC-HPLC procedures of crude plant extracts suchs green tea, black tea, mint, and sweet marjoram were reporteds trolox equivalents (mmol TR g−1 solid matter) in MeOH:H2O sol-ent mixture (4:1, v/v).

The two-way analysis of variance (ANOVA) comparison byhe aid of F-test of the mean-squares of ‘between treatments’i.e., conventional HPLC and on-line HPLC-CUPRAC procedures inable 6) and of residuals [17] for a number of real samples enabledhe conclusion that there was no significant difference betweenreatments. In other words, the TAC values found with the two pro-edures for a given plant extract were alike at 95% confidence levelFexp = 0.25, Fcrit = 10.13, Fexp < Fcrit at P = 0.05). Thus, the proposed

ethodology was validated for real samples. The conventionalUPRAC assay gave higher values than the HPLC-based assaysTable 6), because the latter required the individual identificationf all antioxidant constituents in the plant extract, which is notlways possible.

The conventional HPLC with CUPRAC calculation – with the usef Eq. (3.2) – was capable of estimating the following percentages ofhe experimental CUPRAC capacities of plant samples: 87% of green

ea extract, 16% of black tea extract, 55% of mint and sweet marjo-am extract. Since most of the green tea antioxidants are definitelynown, they can easily be identified in the HPLC chromatograms,nd their contribution to the overall TAC can easily be calculatedith Eq. (3.2). Thus HPLC-based assays were capable of estimating

ig. 6. The chromatogram of green tea extract showing HPLC and on-line HPLC-UPRAC assays (C: caffeine).

Fig. 7. The chromatogram of black tea extract showing HPLC and on-line HPLC-CUPRAC assays (C: caffeine).

TAC values for green tea – using Eqs. (3.2) and (3.3) – close to thatexperimentally found by the conventional CUPRAC assay (Table 6).The chromatograms of plant extracts detected at 280 nm (positivetrace) and 450 nm (negative trace) are given in Figs. 6–9. Accordingto HPLC analysis, the antioxidant components of tea were gal-

lic acid, catechin, epicatechin, epigallocatechin, epicatechin gallateand epigallocatechin gallate. EGCG is the most abundant antiox-idant in green tea and its contribution to the observed TAC washighest.

Fig. 8. The chromatogram of sweet marjoram extract showing HPLC and on-lineHPLC-CUPRAC assays.

S.E. Celik et al. / Analytica Chimic

Fa

1acorpcnbcdabbIstdeapr

mattrl

3C

arwatmw(rt

ig. 9. The chromatogram of mint extract showing HPLC and on-line HPLC-CUPRACssays.

It is noteworthy that although caffeine (C): 1,3,7-trimethyl-H-purine-2,6(3H,7H)-dione is a major constituent of both greennd black tea extracts, it appears only in the positive trace ofhromatograms whereas it is non-existent in the negative tracef post-column detection (Figs. 6 and 7), because it lacks theeducing phenolic hydroxyl groups, and consequently does notossess any antioxidant behavior. This is an advantage of the post-olumn CUPRAC method as the non-antioxidant constituents areot detected, simplifying chromatograms of complex samples. Inlack tea, as opposed to the major constituent, caffeine, the con-entration and contribution to the observed TAC of other catechinerivatives were low (Fig. 7). HPLC-based CUPRAC assays could onlyccount for a low percentage of the experimental TAC (Table 6),ecause black tea has certain phenolic compounds such as thearu-igin and theaflavins that cannot be separated by this HPLC method.

n the manufacture of black tea from green tea, flavan-3-ols areignificantly reduced during the fermentation process and tendedo have slightly lower TAC than calculated for green tea [28]. Theifference between the TAC values of black tea found with thexperimental CUPRAC and HPLC-based procedures (Table 6) prob-bly represents the loss in antioxidant capacity of thearubigins asolymeric proanthocyanidins, which appear not to elute from theeverse-phase HPLC column [29].

In the sweet marjoram extract, the major components were ros-arinic acid and myricetin, and the minor constituents were caffeic

cid, p-coumaric acid, luteolin and apigenin (Fig. 8). The contribu-ion of these components to the observed TAC was about 55%. Inhe mint extract, the greatest antioxidant contribution came fromosmarinic acid, and contributions of myricetin and quercetin wereower (Fig. 9).

.6. Advantages, selectivity, and baseline drift of the post-columnUPRAC method

The major advantage of the proposed method is that it is anntioxidant capacity assay on-line with HPLC; it involves high-esolution screening combining a separation technique like HPLCith fast post-column chemical detection that can rapidly pinpoint

ctive compounds in complex mixtures [11]. The chromatogram ofhis post-column HPLC procedure for antioxidant analysis is much

ore informative than usual, because it can be directly observedhich chromatographic bands arise from antioxidant constituents

i.e., those constituents not showing antioxidant character are noteflected in the negative peak counterparts), bringing the advan-age of added specificity to antioxidant detection. For example, in

a Acta 674 (2010) 79–88 87

this work, the positive peak of caffeine (detected at 280 nm) in thechromatograms is not reflected at a negative peak (detected afterCUPRAC reaction at 450 nm), because caffeine is not an antioxi-dant (Figs. 6 and 7). In a similar on-line HPLC method reported inthe literature, constituents not showing antioxidant activity (likecaffeine) did not yield negative peaks in the post-column ABTSchromatogram [28]. The dual analysis of samples – conventional(with UV–vis detection) and post-column CUPRAC (at 450 nm) –provided valuable on-line information about the correspondencebetween the presence of a determined compound and its possibleantioxidant activity [30]. Moreover, the LODs in the current post-column CUPRAC assay are – up to two orders of magnitude – lowerthan those of post-column DPPH assay.

The CUPRAC reagent is selective, because it has a lower redoxpotential (≈0.6 V) than those of Folin or ferric ion-based oxidativereagents, close to that of ABTS+•/ABTS redox couple. There-fore, simple sugars and citric acid – which are not classified astrue antioxidants – are not oxidized with the CUPRAC reagentwhereas most phenolic antioxidants are easily oxidized due to theirfavourable redox potentials. Some antioxidants remaining inerttoward iron(III)-based reagents like FRAP (such as thiols) are easilyoxidized with the CUPRAC reagent [13]. The optimal pH (i.e., pH7.0) of the CUPRAC method is close to physiological pH, simulatingantioxidant action under real conditions, and the method is capableof measuring both hydrophilic and lipophilic antioxidants [19].

From previous works on the post-column HPLC assays of antiox-idants, it is known that both reagent concentration and reactiontime influence the sensitivity and baseline noise of the method [21].Baseline drift has been proposed to be a common problem in HPLC-chemiluminescence (CL) detection with gradient elution, becauseCL intensity is sensitive to a variety of environmental factors such assolvent, pH, and ionic strength [31]. Post-column HPLC luminol CL-detection with alkaline H2O2 produced an unacceptable baselinedrift, probably caused by rising back-pressure during gradient runs,which in turn was derived from viscosity differences of organicsolvent (MeOH)-water mixtures [4]. On the other hand, baselinestability in the DPPH radical scavenging assay was provided byhelium purging (O2 elimination) of DPPH solution and by dimin-ished pressure variations during gradient runs when acetonitrilewas preferred over methanol, whereas post-column neutralizationusing buffered DPPH solution in 96% acetonitrile gave rise to clog-ging of the reaction coil [4]. Other researchers believe that if thechanges in mobile phase pH of online HPLC–DPPH analyses arerestricted with the use of a suitable buffer, a non-drifting baselinewith minimal noise can be maintained [32].

There is no consensus among literature reports on a single rea-son for baseline drifts in post-column HPLC with derivatization. Itmay be envisaged in this work that the reason for baseline drifts isthe possible accumulation of Cu(I)-neocuproine colored complex inthe reaction coil accompanied by viscosity and polarity differencesof the solvent media in gradient runs. As opposed to the DPPH rad-ical scavenging method of antioxidant assay sensitive to dissolvedoxygen, CUPRAC is free of such interference which may otherwisegive rise to baseline distortions. The relatively small baseline dis-tortion observed in this work (compared to that in on-line DPPHanalyses) did not affect the peak area determination of chromato-graphic bands because of the calculation procedure used [33].

4. Conclusions

The developed on-line HPLC method with post-column CUPRACdetection of antioxidants is rapid, inexpensive, versatile, and non-laborious. The CUPRAC reagent is relatively insensitive to a numberof parameters adversely affecting certain radicalic reagents such asDPPH, i.e., air, sunlight, solvent type, suspension formation at neu-

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8 S.E. Celik et al. / Analytica

ral pH, and slow and complex kinetics for phenolics oxidation [34].he reagent is much more stable and easily accessible than thether chromatographic chromogenic radical reagents (e.g., ABTS,PPH). Colored Cu(I)-Nc chelate formation in the post-column

eaction coil is usually sufficient for the quantification of most phe-olic antioxidants within the fixed time of the assay. The method

s applicable to conventional laboratories not using more costlyquipment such as LC/MS/MS. Simple sugars and citric acid, whichre not true antioxidants, are not oxidized in the CUPRAC methodnd therefore do not yield negative peaks in the post-columnhromatogram. This on-line HPLC method has the advantage ofensitive determination of individual antioxidant constituents inomplex matrices like plant extracts. In addition to the capabilityf quantification by UV detection, antioxidative capacity of a singleonstituent can be measured on-line, and its contribution to theAC can be evaluated. Moreover, the determination of a substancehich possesses no antioxidant activity can be observed simulta-eously, by the absence of a negative peak at 450 nm as opposedo the presence of a positive peak detected at 280 nm. In this work,he novel on-line method has been applied to the identificationnd quantification of antioxidants in synthetic mixtures and crudelant extracts, and the aim is to be diverse enough to include otherood and biological samples.

cknowledgments

Author S. Esin Celik would like to thank Istanbul Univer-ity Research Fund, Bilimsel Arastirma Projeleri (BAP) Yurutucuekreterligi (Project T-1450/11092007), and to Istanbul Univer-ity, Institute of Pure and Applied Sciences (I.U. Fen Bilimlerinstitüsü), for the support given to her Ph.D. thesis entitled “Mod-fied CUPRAC Antioxidant Capacity Measurements Applicable toifferent Species, Mixtures and Solvent Media”. The authors alsoxpress their gratitude to Istanbul University Research Fund forhe support given to the Research Projects – 2724 and 5096.

eferences

[1] B. Halliwell, R. Aeschbach, J. Loliger, O.I. Auroma, Food Chem. Toxicol. 33 (1995)601–617.

[2] C. Sanchez Moreno, Food Sci. Technol. Int. 8 (2002) 121–137.[3] A. Dapkevicius, T.A. van Beek, H.A.G. Niederländer, Ae. de Groot, Anal. Chem.

71 (1999) 736–740.

[

[[

a Acta 674 (2010) 79–88

[4] A. Dapkevicius, T.A. van Beek, H.A.G. Niederländer, J. Chromatogr. A 912 (2001)73–82.

[5] A.A.H. Ogawa, H. Tanizawa, T. Miyahara, T. Toyooka, Anal. Chim. Acta 383 (1999)221–230.

[6] I.I. Koleva, H.A.G. Niederländer, T.A. van Beek, Anal. Chem. 72 (2000)2323–2328.

[7] I.I. Koleva, H.A.G. Niederländer, T.A. van Beek, Anal. Chem. 73 (2001)3373–3381.

[8] D. Bandoniene, M. Murkovic, J. Agric. Food Chem. 50 (2002) 2482–2487.[9] V. Exarchou, Y.C. Fiamegos, T. van Beek, C. Nanos, J. Vervoort, J. Chromatogr. A

1112 (2006) 293–302.10] E. Capanoglu, J. Beekwilder, D. Boyacıoglu, R. Hall, R. de Vos, J. Agric. Food Chem.

56 (2008) 964–973.11] H.A.G. Niederländer, T.A. van Beek, A. Bartasiute, I.I. Koleva, J. Chromatogr. A

1210 (2008) 121–134.12] R. Apak, K. Güclü, M. Özyürek, S.E. Karademir, J. Agric. Food Chem. 52 (2004)

7970–7981.13] R. Apak, K. Güclü, M. Özyürek, S.E. Karademir, M. Altun, Free Radic. Res. 39

(2005) 949–961.14] K. Güclü, M. Altun, M. Özyürek, S.E. Karademir, R. Apak, Int. J. Food Sci. Technol.

41 (2006) 76–85.15] H. Wang, G.J. Provan, K. Helliwell, Food Chem. 81 (2003) 307–312.16] L. Yıldız, K. Sözgen Baskan, E. Tütem, R. Apak, Talanta 77 (2008) 304–313.17] J.C. Miller, J.N. Miller, Statistics for Analytical Chemists, third ed., Ellis Hor-

wood/Prentice Hall, New York/London, 1993.18] S.E. Celik, M. Özyürek, K. Güclü, R. Apak, Talanta 81 (2010) 1300–1309.19] R. Apak, K. Güclü, M. Özyürek, S.E. Celik, Microchim. Acta 160 (2008)

413–419.20] G. Litwinienko, K.U. Ingold, J. Org. Chem. 68 (2003) 3433–3438.21] Q. Zhang, E.J.C. van der Klift, H.-G. Janssen, T.A. van Beek, J. Chromatogr. A 1216

(2009) 7268–7274.22] S.A.B.E. van Acker, D.-J. van den Berg, M.N.J.L. Tromp, D.H. Griffioen, W.P.

van Bennekom, W.J.F. van der Vijgh, A. Bast, Free Radic. Biol. Med. 20 (1996)331–342.

23] C.A. Rice-Evans, N.J. Miller, G. Paganga, Free Radic. Biol. Med. 20 (1996)933–956.

24] P.-G. Pietta, J. Nat. Prod. 63 (2000) 1035–1042.25] O. Firuzi, A. Lacanna, R. Petrucci, G. Marrosu, L. Saso, Biochim. Biophys. Acta

1721 (2005) 174–184.26] T. Oki, M. Kobayashi, T. Nakamura, A. Okuyama, M. Masuda, H. Shiratsuchi, I.

Suda, J. Food Sci. 71 (2006) C18–C22.27] R. Cardenosa, R. Mohamed, M. Pineda, M. Aguilar, J. Agric. Food Chem. 50 (2002)

3390–3395.28] A.J. Stewart, W. Mullen, A. Crozier, Mol. Nutr. Food Res. 49 (2005) 52–60.29] D. Del Rio, A.J. Stewart, W. Mullen, J. Burns, M.E.J. Lean, F. Brighenti, A. Crozier,

J. Agric. Food Chem. 52 (2004) 2807–2815.30] A. Cano, O. Alcaraz, M. Acosta, M.B. Arnao, Redox Report 7 (2002) 103–109.

(2009) 2204–2210.32] M. Kosar, D. Dorman, K. Baser, R. Hiltunen, Chromatographia 60 (2004)

635–638.33] O. Busto, M. Miracle, J. Guasch, F. Borrull, J. Chromatogr. A 757 (1997) 311–318.34] B. Özcelik, J.H. Lee, D.B. Min, J. Food Sci. 68 (2003) 487–490.