Automatic method for determination of total antioxidant capacity using 2,2-diphenyl-1-picrylhydrazyl assay

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<ul><li><p>Analytica Chimica Acta 558 (2006) 310318</p><p>Automatic method for determination of tlhale</p><p>Farmorto,</p><p>ber 2er 200</p><p>Abstract</p><p>In the pre on antotal antioxi ds pr2,2-dipheny the cantioxidant compounds monitored spectrophotometrically at 517 nm.</p><p>The influence of initial DPPH concentration and sample dilution in the present methodology was studied. It was verified that the amount ofDPPH consumed by antioxidant standards (ascorbic and caffeic acids) was independent of the initial concentration of radical except for situationswhere DPPH/antioxidant molar ratio was lower than the stoichiometric value. Furthermore, the sample dilution factor plays an important rolefor achieving results comparable to those from end-point batch method since the exhausting of scavenging ability of the sample should take placeduring the p</p><p>The propocapacity (VCcomparableabout 13 h1 2005 Else</p><p>Keywords: To</p><p>1. Introdu</p><p>There itive damagin ageingatherosclerthermore, tthat the actbeverages,tect againstective effedants specicompounds</p><p> CorresponE-mail ad</p><p>0003-2670/$doi:10.1016/jeriod of absorbance measurement.sed method was applied to several food products and the total antioxidant capacity was expressed as Vitamin C equivalent antioxidantEAC). The results obtained by the proposed method ranged from 1.1 to 318 mg of ascorbic acid/100 ml and they were statistically</p><p>to those provided by the batch method. The detection limit was 0.34 mg of ascorbic acid/100 ml and the determination frequency waswith an excellent repeatability (R.S.D. &lt; 1%, n = 10).</p><p>vier B.V. All rights reserved.</p><p>tal antioxidant capacity; DPPH; Multi-syringe flow injection; Beverages</p><p>ction</p><p>s recent evidence that free radicals induce oxida-e to biomolecules. This damage has been implicatedand in several human pathologies such as cancer,osis, rheumatoid arthritis and other diseases [1]. Fur-here is a considerable amount of studies indicatingive dietary constituents of fresh fruit, vegetables andprevent these free radical-induced diseases and pro-t foodstuff oxidative deterioration [24]. These pro-cts have been attributed, in large part, to the antioxi-es (Vitamins C and E, carotenoids and polyphenolic) which scavenge free radicals [5,6].</p><p>ding author. Tel.: +351 22 2078994; fax: +351 22 2004427.dress: msegundo@mail.ff.up.pt (M.A. Segundo).</p><p>Several methodologies, based on free radical capture orformation suppression, are used to measure the antioxidantcapacity of biological material and model compounds [7,8].The most commonly used for their ease, speed and sensitivityare those involving chromogen compounds of a radical natureto simulate radical oxygen and nitrogen species. The mostwidely used assays are based on the scavenging of radical cation2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS+assay) [9] or of the stable radical 2,2-diphenyl-1-picrylhydrazyl(DPPH assay) [10,11]. The presence of antioxidant speciesleads to the disappearance of these radical chromogens whichcan be followed by spectrophotometric methods.</p><p>Recently, the DPPH assay was implemented using automaticmethods based on flow injection analysis (FIA) [12,13], sequen-tial injection analysis (SIA) [14] or HPLC-FIA [15,16], that wereapplied for screening and evaluation of scavenging capacity ofseveral pure compounds and complex matrices such as plantextracts and beverages. In the HPLC-FIA method, the separated</p><p> see front matter 2005 Elsevier B.V. All rights reserved..aca.2005.11.013using 2,2-diphenyl-1-picryLus M. Magalhaes, Marcela A. Segundo , S</p><p>REQUIMTE, Servico de Qumica-Fsica, Faculdade deRua Anbal Cunha, 164, 4099-030 P</p><p>Received 10 October 2005; received in revised form 3 NovemAvailable online 19 Decemb</p><p>sent work, an automatic method based on multi-syringe flow injectidant capacity, measured as the cumulative capacity of the compounl-1-picrylhydrazyl (DPPH) reaction. The determination is based onotal antioxidant capacityydrazyl assaytte Reis, Jose L.F.C. Lima</p><p>acia, Universidade do Porto,Portugal005; accepted 4 November 20055</p><p>alysis (MSFIA) was developed for the determination ofesent in the sample to scavenge free radicals, using theolour disappearance due to the scavenging of DPPH by</p></li><li><p>L.M. Magalhaes et al. / Analytica Chimica Acta 558 (2006) 310318 311</p><p>analytes react postcolumn with the DPPH solution [16], and theinduced scavenging is detected as a negative peak. These meth-ods, combicapacity, pas bioassaya time conloss in activisolation an</p><p>Howevematrices dopropertiesthe synergidants implicompound[17]. For thallowed thethat enablethe matrix.</p><p>Recentlflow analysanalysis (Min order toflexibility othe objectiautomaticcapacity exanalysis.</p><p>The meantioxidantabsorbancein order toconsumptioing. Furtheand sample</p><p>2. Experim</p><p>2.1. Reage</p><p>All chemfurther purfrom Milliwere used.</p><p>2,2-Dipwere purchobtained fr(5.80 10appropriateand protect</p><p>For the fl1.45 10stock solutto 200 ml wand protectas carrier s</p><p>Ascorbimol l1) w</p><p>of the respective solid in ethanol solution 50% (v/v). Workingstandard solutions containing either ascorbic or caffeic acid in</p><p>ncen 4 1by din 50the</p><p>.25 ng stioned arderesta</p><p>trationce</p><p>100.52sam</p><p>s weintog duore imetho attnd beese ss wemple3 andete</p><p>n [21stoc/v) inm.</p><p>ppar</p><p>utionulti-</p><p>is apes inothewer</p><p>ge osigneflower vae duver-</p><p>ersoasic</p><p>numsitiondetephothroade</p><p>werening separation and evaluation of radical-scavengingresent a major advantage relatively to batch methods-guide fractionation of natural or food samples is</p><p>suming and labour-intensive process. Moreover, theity for antioxidants due to decomposition during thed purification procedures is avoided.r, the presence of antioxidant compounds in complexes not necessarily imply that the same antioxidant</p><p>are verified in the whole sample. On the other hand,stic effect that may exist between different antioxi-es that the sum of antioxidant capacities from eachisolated may not exactly reflect the overall actionis reason, automatic methods based on FIA or SIAevaluation of antioxidant capacity in an environment</p><p>d the interactions between all compounds present in</p><p>y, novel computer controlled techniques for automaticis were reported, namely multi-syringe flow injectionSFIA). This technique was proposed in 1999 [18],</p><p>combine the multi-channel operation of FIA and thef multi-commutation flow systems [19]. Therefore,</p><p>ve of the present work was the development of anflow system for the assessment of total antioxidantploiting the features introduced by MSFIA in flow</p><p>thodology was based on consumption of DPPH byspecies present in the sample by monitoring theat 517 nm. A stopped flow approach was chosen</p><p>follow the reaction development and to assess if totaln of antioxidant is attained during reaction monitor-</p><p>rmore, the influence of initial DPPH concentrationdilution are also addressed in the present work.</p><p>ental</p><p>nts and solutions</p><p>icals used were of analytical-reagent grade with noification. For the preparation of all solutions water-Q system (resistivity &gt; 18 M cm) and ethanol p.a.</p><p>henyl-1-picrylhydrazyl (DPPH) and ascorbic acidased from Sigma (St. Louis, USA). Caffeic acid wasom Aldrich (Milwaukee, USA). A stock solution4 mol l1) of DPPH was prepared by dissolving theamount in ethanol. This solution was kept at 4 C</p><p>ed from light, and it was stable during a week.ow system, the DPPH working solution containing</p><p>4 mol l1 was prepared by measuring 50 ml of theion, 50 ml of ethanol and the volume was made upith water. This working solution was prepared dailyed from light. Ethanol solution 50% (v/v) was usedolution.c and caffeic acid stock solutions (1.00 102ere prepared by dissolving the appropriate amount</p><p>the coparedsolutio</p><p>Fortion (1Workicentratdescrib</p><p>In oon theconcen</p><p>The c0.375 0.840,</p><p>Allextract90 Cbrewin</p><p>Befbatchorder twine aand thsampleThe sato 1:33</p><p>ForHansefrom a50% (vat 428</p><p>2.2. A</p><p>Solof a mFor thsyringin thevalvesexchanwas as</p><p>for thethe oththe timavoid o</p><p>A pQuickBation (the po</p><p>Asspectroa flow-heim/Bmentstration range 0.2510.00 10 mol l were pre-lution of the respective stock solution using ethanol% (v/v). These solutions were prepared daily.analysis using the batch method, a DPPH solu-104 mol l1) in ethanol (50%, v/v) was prepared.</p><p>andard solutions containing ascorbic acid in the con-range 0.252.00 104 mol l1 were prepared asbove.to evaluate the influence of DPPH concentrationblishment of calibration curves, different DPPHons were prepared in ethanol solution 50% (v/v).ntrations studied were 1.500, 0.938, 0.750 and4 mol l1 providing initial absorbance values of8, 0.426 and 0.208, respectively.ples were purchased at local markets. The green teare prepared by pouring 200 ml deionised water ata glass with tea bag (1.491.77 g of leaves) and byring 3 min [20].ntroduction into the flow system or analysis using theod, samples were first diluted 50% using ethanol inain a final concentration of 50% (v/v) in ethanol. Forer samples, the initial alcohol content was consideredamples were diluted with ethanol and water. Somere further diluted using ethanol solution 50% (v/v).dilutions used for the flow system varied from 1:2</p><p>d for the batch method varied between 1:2 and 1:200.rmination of dispersion coefficient of Ruzicka and], a bromothymol blue (BTB) solution was preparedk solution (0.50 g l1) by dilution in ethanol solutionn order to provide an absorbance value of about 0.520</p><p>atus</p><p>s were propelled through the flow network by meanssyringe burette (Crison Instruments, Allela, Spain).plication, the multi-syringe was equipped with 5 mlpositions 1 and 2 while 10 ml syringes were placedr two positions (Fig. 1). Three extra commutatione included in the module used. For all valves, theptions were classified in on/off lines. The off lined to the solution flasks and the on line was reservednetwork in the valves placed at the multi-syringe. Forlves, the positions on/off were chosen to minimisering which the valves were switched on in order toheating problems.nal computer, running lab-made software written in</p><p>4.5 (Microsoft), controlled the multi-syringe oper-ber of steps and direction of piston displacement and</p><p>of all commutation valves).ction system, a Jenway 6105 (Essex, UK) UVvistometer equipped with a thermostatic cell holder andugh cell from Hellma (80l, ref. 178.710-QS, Mull-n, Germany) was used and the absorbance measure-carried out at 517 nm. The cell holder was connected</p></li><li><p>312 L.M. Magalhaes et al. / Analytica Chimica Acta 558 (2006) 310318</p><p>Fig. 1. MSFIA manifold used for the determination of total antioxidant capacityusing DPPH assay: MS, multi-syringe; Si, syringes; Vi, commutation valves(solid and dotted lines represent the position on and off, respectively); B1 and B2,confluences; HC, holding coil (200 cm); RC, reaction coil (120 cm); D, detector;C, carrier (ethanol solution 50%, v/v); R, 2,2-diphenyl-1-picrylhydrazyl reagentprepared in Cputer; W, was</p><p>to a Tectronacquisitionat 3 Hz, usflow systemsoftware.</p><p>2.3. MSFI</p><p>The syscally in Figbridge, UKBel, Franceacrylic Y-s</p><p>The conwere 200 cThe tubingThe connethe same le</p><p>B2 was 50 cm long while the connection between the valve V7and confluence B2 was 10 cm long. The reaction coil (RC) was120 cm long.</p><p>The protocol sequence for the determination of total antiox-idant capacity using DPPH assay is listed in Table 1. Beforestarting the analytical procedure, syringe 4 was filled with car-rier solution and further propelled to detection system. Thus, theflow-through cell was filled with it and the absorbance signalwas adjusted to zero. After that, syringe 3 was filled with radicalsolution while the syringes 1 and 2 were filled with carrier solu-tion and further propelled to the detector. The absorbance valuemeasured corresponded to the absorbance of radical solution inthe absence of antioxidant compounds.</p><p>The procedure included six steps. The first step consistedof filling syringes with the respective solutions. Then, 50l ofstandard/sample was aspirated into the holding coil (HC). Aftera dummy step, applied to change the flow direction [22], thesample, carrier and reagent were sent towards the detection sys-tem. During this step the sample plug was pushed by carrier</p><p>conflyring solled uoppe0 s (s wa</p><p>selinanaltheroce</p><p>the vn posolu</p><p>medthe</p><p>ell, tby</p><p>Table 1Protocol sequ</p><p>Step De</p><p>1 Sysol</p><p>2 Sa3 Du</p><p>dir4 Sa</p><p>tow5 Flo</p><p>wi6 Ca</p><p>the</p><p>The indicated; AO, antioxidant standard solution or sample; PC, personal com-te.</p><p>S-543 (Altrincham, UK) thermostatic bath. The datawas performed through a PCL-711B interface card</p><p>ing the same software developed for controlling the. The data obtained were analysed using Origin 6.1</p><p>A manifold and procedure</p><p>tem components were arranged as shown schemati-. 1. All connections were made from Omnifit (Cam-) PTFE tubing (0.8 mm i.d.) with Gilson (Villiers-le-) end-fittings and connectors. Two laboratory-made</p><p>haped connectors were used as confluences.nections between the multi-syringe and the valve V7m long. The holding coil (HC) had the same length.</p><p>up tofrom sDPPHpropelwas sting 18cell wathe baa new</p><p>Forsame psteps,were icarrierperfor</p><p>Forflow cculatedlength between the valves V5 and V6 was 4 cm long.ction between this valve and the confluence B1 hadngth. The connection between confluences B1 and</p><p>solution inrespective58 mol1 l</p><p>ence for the determination of total antioxidant capacity using DPPH assay</p><p>scription Position of the commutation valves</p><p>1 2 3 4 5 6</p><p>ringes are filled with the respectiveutions</p><p>F F F F F F</p><p>mple is aspirated N F F F N Fmmy step to change the flowection</p><p>F F F F F F</p><p>mple, carrier and reagent are sentards detection system</p><p>N N N F F F</p><p>w stop for reaction monitoringth data acquisition</p><p>F F F F F F</p><p>rrier and reagent are sent to washsystem</p><p>N N N F F F</p><p>values for volume refer to syringe 3 (10 ml). N and F represent the positions on anduence B1, where it was diluted by carrier solutione 2. Subsequently, the diluted sample was mixed withution, after confluence B2. This mixture was furtherntil it reached the detection system. Then, the flowd and the absorbance at 517 nm was measured dur-temperature = 25 1 C). Finally, the flow-throughshed with carrier and reagent solution to re-establishe. After this last step, the flow system was ready forytical cycle.determination of intrinsic absorption of sample thedure was performed, except for steps 4 and 6. In thesealve V3 was in position off and the valves V4 and V7sition on in order to replace the DPPH solution bytion. Moreover, the absorbance measurements wereduring 30 s instead of 120 s.determination of DPPH/AO molar ratio in the</p><p>he DPPH concentration in the flow cell was cal-the ratio between absorbance value of radicalthe absence of antioxidant compounds and the</p><p>molar absorption coefficient at 517 nm ( = 11 071 cm1). The antioxidant concentration in the flow cell</p><p>Volume (l) Time (s)7</p><p>F 3000 22.50</p><p>F 100 3.00F 500 3.75</p><p>F 750 15.00</p><p>F 180.00</p><p>F 1850 37.00</p><p>off, respectively.</p></li><li><p>L.M. Magalhaes et al. / Analytica Chimica Acta 558 (2006) 310318 313</p><p>was calculated as the ratio between the concentration of the solu-tion introduced in the system and the dispersion coefficient.</p><p>2.4. End-p</p><p>The DPwas applieto a micropThe...</p></li></ul>

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