Food Chemistry 126 (2011) 679–686

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Food Chemistry

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Analytical Methods

The reduction of Cu(II)/neocuproine complexes by some polyphenols: Totalpolyphenols determination in wine samples

Gina Lee c, Maura Vincenza Rossi a, Nina Coichev b, Horacio Dorigan Moya c,⇑a Universidade Presbiteriana Mackenzie, CEP 01302-907 São Paulo, SP, Brazilb Instituto de Química, Universidade de São Paulo, CP 26.077, CEP 05599-970 São Paulo, SP, Brazilc Faculdade de Medicina da Fundação do ABC, CEPES (Centro de Estudos, Pesquisa, Prevenção e Tratamento em Saúde), CEP 09060-650 Santo André, SP, Brazil

a r t i c l e i n f o

Article history:Received 10 May 2010Received in revised form 13 October 2010Accepted 4 November 2010

Keywords:WinePolyphenolsCopperNeocuproine

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.11.020

⇑ Corresponding author. Tel.: +55 11 4993 7292; faE-mail address: hdmoya@fmabc.br (H.D. Moya).

a b s t r a c t

A new method is presented for spectrophotometric determination of total polyphenols content in wine.The procedure is a modified CUPRAC method based on the reduction of Cu(II), in hydroethanolic medium(pH 7.0) in the presence of neocuproine (2,9-dimethyl-1,10-phenanthroline), by polyphenols, yielding aCu(I) complexes with maximum absorption peak at 450 nm. The absorbance values are linear (r = 0.998,n = 6) with tannic acid concentrations from 0.4 to 3.6 lmol L�1. The limit of detection obtained was0.41 lmol L�1 and relative standard deviation 1.2% (1 lmol L�1; n = 8). Recoveries between 80% and110% (mean value of 95%) were calculated for total polyphenols determination in 14 commercials and2 synthetic wine samples (with and without sulphite). The proposed procedure is about 1.5 more sensi-tive than the official Folin–Ciocalteu method. The sensitivities of both methods were compared by theanalytical responses of several polyphenols tested in each method.

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1. Introduction wines (De Beer et al., 2004). It has pointed out a close relation of

Tannin is a word used to describe a heterogeneous mixture ofnatural polymeric phenolic compounds with a high molecularweight found in vegetables and fruits. It is the most abundant classof phenolics compounds in grapes, which is responsible for theastringent taste in wines.

The official AOAC method for tannins quantification in wines isbased on the spectrophotometry measurements after the oxidationof the polyphenolic compounds by the Folin–Ciocalteu (or FolinDenis) reagent in a very alkaline medium, using tannic acid as stan-dard solution (Horwitz, 1970). In spite of this procedure being wellestablished, this method consumes phosphomolybdic or phospho-tungstic acid resulting in waste, which is normally not recycled,and other reducing agents present in the samples such as citricand ascorbic acids and even some simple sugars may interfere.

Some authors have proposed the determination of condensedtannins in grapes and flavonoids in wines using more expensivetechniques, such as HPLC (Brossaud, Cheynier, Asselin, & Moutounet,1998), near IR spectrophotometry (Cozzolino, Cynkar, Dambergs,Mercurio, & Smith, 2008) and thermal lens spectrometry assembledwith a semiconductor diode-array laser (Cladera, Tomas, Estela,Cerda, & Ramis-Ramos, 1995).

A more recent study compared several analytical methods fordetermination of various phenolic compounds (e.g. monomeric andpolymeric phenols, flavan-3-ol, anthocyanin, tannins, etc.) in some

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the total phenol content obtained by more sophisticated techniques,like HPLC and cyclic voltammetry, when compared with the refer-ence spectrophotometric method using Folin–Ciocalteu reagent.

In fact, the spectrophotometric methods have still been attrac-tive because of the low cost, simplicity and roughness of this tech-nique since methods involving separations (chromatography orsolvent extraction) are more time consuming and generate moreamounts of waste.

The reduction of Cu(II) in the presence of neocuproine, 2,9-di-methyl-1,10-phenanthroline (NC), by a reducing agent, likehydroxylamine hydrochloride, yields a Cu(I) complex with maxi-mum absorption peak at 450 nm (e = 7.5 � 103 L cm�1 mol�1)(Tütem, Apak, & Baykut, 1991).

Table 1 shows several analytes (reducing agents and copper ion)determined based on the Cu(I)/neocuproine complexes formation.Considering only the linear range (LR) and the limit of detection(LOD) values, the analysis of reducing sugars in forage materialsand in wines samples presented the lowest limit of detection whilethe determination of copper in water shows the highest one, whichindicates that the analytical response may be related also with thecomplexity of the sample utilised.

We have found that tannic acid reduces Cu(II) to Cu(I) in the pres-ence of neocuproine at pH 7.0, kept with ammonium acetate. Theabsorbance at 450 nm, a characteristic peak of the Cu(I)/neocuproinecomplexes, can be related to the total polyphenols concentration inwines, expressed as tannic acid. The procedure is based on a modi-fied CUPRAC (cupric reducing antioxidant capacity) method. Somesamples were treated twice with gelatin solution and kaolin, so that

Table 1Selected spectrophotometric methods using the Cu(II)/NC complexes.

Sample Analyte LR (mg L�1) LOD(mg L�1)

Remarks Ref.

BSA, a-amilase, lysozyme,peroxidase, protease andtrypsin

Proteina 0.5–40 0.5 Assay temperature varied from 30–60 �C. and incubationtime was 30, 60 and 90 min.

Castro, Mendez, andSineriz (1991)

Meat, sardine and milkproducts

Proteina 8–100 1 Extraction., pptn. and redissoln procedures with analyse inalkaline medium

Sözgen, Cekic, Tütemand Apak, 2006

Pharmaceuticals a-Tocopherol 1.0–39 NA Analyte dissolved in diethyl ether/ethanol mixture; 1.0 Mammonium acetate buffer medium (pH 7.0)

Tütem, Apak, Günayd,and Sözgen (1997)

Fruit, beverage andpharmaceuticals

Ascorbic acid 0.1–40 0.040 Indirect method; complex extd. with PBITU in CHCl3 Shrivas, Agrawal, Patel,and Kumar (2005)

Fruit juice, red wine andpharmaceuticals

Ascorbic acid 1.4–14 NA Ammonium acetate buffer medium (pH 7.0) Güçlü, Sözgen, Tütem,Özyürek, and Apak(2005)

Blood serum and river water Iron and copper 0.10–8.00(Fe)

NA Simultaneous detn of iron (phen) and copper (NC) usingeither PLS and HPSAM

Safavi and Nezhad(2004)

0.12–8.75(Cu)

Different types of water Iron and copper 0.000195–0.12 (Fe)

0.000044(Fe)

Simultaneous detn of iron (TPTZ) and cooper (NC),preconc. with cation-exchange resin Sephadex sp C-25;pH 5.

Toral, Lara, Gomez, andRichter (2002)

0.000451–0.30 (Cu)

0.00014(Cu)

Synthetic soil and lemon juice DNOC (herbicide) 7.5–225 0.2 Reduction of analyte with Zn/HCl and preconc. usingoxine-impregnated-XAD-4 resin stabilized with Fe(III).

Uzer, Ercag, Parlar, Apak,and Filik (2006)

Forage materials Carbohydratesand reducingsugars

2000–8000 NA Multicommutated flow procedure with an acid hydrolysisof nonstructural carbohydrates

Tumang, Tomazzini, andReis (2003)

Wines Reducing sugarsb 1000–7000 NA FIA (50 samples/h); RSD = 1.6% (11 samples 3 g L�1) Maquieira, Castro, andValcarcel (1987)

Wines Reducing sugarsb 1200–7200 NA FIA (40 samples/h) with 3 dialysis membrane; a syntheticwines was made without polyphenols

Peris-Tortajada et al.(1991)

Wines Reducing sugars 2000–25,000(table)

1200(table)

SIA anal. varying from 14 (table) to 18 (port) samples/hour; HCl/hydrogen phthalate buffer (pH 3.4)

Araujo et al. (2000)

20,000–140,000(port)

11,200(port)

Wines Totalpolyphenolsc

0.68 – 5.1 0.22 Ammonium acetate medium buffer (pH 7.0);hydroethanolic medium.

This work

LR, linear range; LOD, limit of detection; NA, not available; PBITU, Phenylbenzimidoylthiourea; phen, 1,10 phenanthroline; TPTZ, 2,4,6-trypyridyl-1,3,5-triazine; DNOC, 4,6-dinitro-o-cresol; PLS, partial least squares; HPSAM, H-point standard addition method.

a Protein total express as bovine serum albumin (BSA).b Expressed as glucose.c Expressed as tannic acid.

680 G. Lee et al. / Food Chemistry 126 (2011) 679–686

the tannins were removed by precipitation, providing a tannin-freesample. Due to the low solubility of the Cu(II) complex in water theanalysis is better performed in a hydroethanolic medium.

For comparison purposes, the same wine samples were ana-lysed by the official AOAC method for tannins determination inwines (Horwitz, 1970), using the same standard (tannic acid). Asthe official and the present methods are based on redox reactions,other reducing agents usually present in the samples (e.g. ascorbicacid, reducing sugars and SO2) may interfere. So, the study of thepossible interference of these reagents in the proposed methodwas also investigated.

2. Materials and methods

2.1. Apparatus

All spectrophotometric measurements were made in a HPUV8453 (Agilent) spectrophotometer using 1.00 cm glass cell.

2.2. Materials

Deionised water was used to prepare the analytical-gradechemicals (except when another solvent is indicated) and also inall sample dilutions.

Neocuproine hydrochloride monohydrated, NC, (C14H12N2�HCl�H2O, Sigma Aldrich) stock solution 1.22 � 10�2 mol L�1 was pre-pared by dissolution of 0.3201 g in 100 mL of ethanol 99%.

A 2.3 mol L�1 Cu(ClO4)2 stock solution was prepared by reactionof copper(II) carbonate with a slight excess of perchloric acid. A di-luted 9.31 � 10�2 mol L�1 Cu(ClO4)2 solution was obtained by dilu-tion and the standardization was carried out by complexometrictitration with EDTA.

Ammonium acetate (C2H7NO2, Fluka A.G. Chemie) 3.0 mol L�1

stock solution used as buffer solution (pH 7.0) was prepared by dis-solution in water.

A fresh 5.0 � 10�3 mol L�1 tannic acid (C76H52O46, J.T. Baker, FW1701.2 g mol�1) stock solution was prepared by dissolving0.2127 g in 25 mL of water just before the use. A solution1.0 � 10�5 mol L�1 was obtained by dilution in water.

Luteolin (99% TLC, FW 286.2 g mol�1), quercetin dehydrate (98%HPLC, FW 338.3 g mol�1) and resveratrol (99% GC, FW 228.2g mol�1) 1.0 � 10�3 mol L�1 stock solutions, all from Sigma Aldrich,were prepared by dissolution in 50:50 v/v water:ethanol 99%.

Hesperetin (95% HPLC, FW 302.3 g mol�1), kaempferol (>90%,HPLC, FW 286.24 g mol�1), malvin (malvidin chloride 3,5-digluco-side, >90%, HPLC, FW 691.03 g mol�1), b-carotene (97%, UV, FW536.87 g mol�1) and trolox ((±)-6-hydroxy-2,5,7,8-tetramethylch-roman-2-carboxylic acid, 97%, FW 250.29 g mol�1), 1.0 �

G. Lee et al. / Food Chemistry 126 (2011) 679–686 681

10�3 mol L�1 stock solutions, from Sigma Aldrich, were preparedby dissolution in ethanol 99%.

(�)-Epigallocatechin gallate (80% HPLC, FW 458.4 g mol�1),1,2,4-benzenotriol (99%, FW 126.1 g mol�1) 1.0 � 10�3 mol L�1

stock solutions, both from Sigma Aldrich, were prepared by disso-lution in water.

Gallic acid (99%, FW 188.1 g mol�1), phloroglucinol (99%, FW126.1 g mol�1), pyrogallol (99%, FW 126.1 g mol�1), o-pyrocathecol(99%, FW 110.1 g mol�1), hydroquinone (99%, FW 110.1 g mol�1),resorcinol (99%, FW 110.1 g mol�1) and phenol (99%, FW94.11 g mol�1), all from Labsynth, 1.0 � 10�3 mol L�1 stock solu-tions, were all prepared by dissolution in water.

Acidic NaCl solution was prepared by mixing 25 mL of H2SO4

(98%, d = 1.84 g cm�3, Merck) with 375 mL of NaCl saturated solu-tion, both reagents from Merck.

Gelatin solution (0.3% m/v) was prepared by soaking 0.3 g ofcommercial gelatin from Brazilian market in saturated NaCl solu-tion for 1 h, then warming to dissolve it. After cooling it was di-luted to 100 mL with the same saturated NaCl solution.

Kaolin, Al2Si2O5(OH)4, was from Labsynth.The Folin–Ciocalteu reagent was prepared as described else-

where (Singleton, Orthofer, & Lamuela-Raventós, 1999).A modified CUPRAC reagent was used with cupric perchlorate

instead of cupric chloride. The Cu(II)/NC complexes solution wasprepared by mixing 0.75 mL of 9.31 � 10�2 mol L�1 Cu(ClO4)2,3.0 mL of 3.0 mol L�1 ammonium acetate and 15 mL of1.22 � 10�2 mol L�1 NC solutions. After that, the volume was madeup to 50 mL in a volumetric flask with ethanol 99%.

2.3. Procedures

2.3.1. Sample preparation for the total polyphenols analysisThe red and synthetic wines were diluted 500-fold and the

white wines were diluted 25-fold just before the analysis by bothCu(II)/NC complexes and Folin–Ciocalteu reagent.

2.3.2. Proposed methodThe calibration graph was obtained by adding aliquots of 200–

1000 lL from the same 1.0 � 10�5 mol L�1 standard solution oftannic acid to six volumetric flasks (5.0 mL) containing 2.5 mL offresh solution of Cu(II)/NC complexes and diluted with water to5.0 mL before the measurements.

The multiple standard addition method was used in all winesamples analysis (14 commercials and 2 synthetics). One mL ofany diluted wine sample was transferred to five volumetric flasks(5.0 mL) containing 2.5 mL of fresh solution of Cu(II)/NC com-plexes. In four out of five volumetric flasks, aliquots of 200, 300,400 or 500 lL of a 1.0 � 10�5 mol L�1 standard solution of tannicacid were added and each solution was diluted with water to5.0 mL before the measurements.

All the absorbance measurements were carried out at 450 nm. A1:1 (v/v) fresh diluted aqueous solution of Cu(II)/NC complexeswas used as reference solution (blank).

2.3.3. Reference methodThe procedure was carried out as described in the AOAC method

number 952.03 (Horwitz, 1970).The calibration graph was obtained by adding aliquots of 500–

3500 lL from the same 1.0 � 10�5 mol L�1 standard solution oftannic acid to 5.0 mL volumetric flasks where 0.25 mL of Folin–Cio-calteu reagent and 1 mL of saturated solution of Na2CO3 wereadded. Each final solution was diluted with water to 5.0 mL. After30 min the absorbance was measured at 760 nm.

The multiple standard addition method was also used in thesame wine samples. One mL of diluted wine sample was trans-ferred to a 5.0 mL volumetric flask where 0.25 mL of Folin–Ciocal-

teu reagent and 1 mL of saturated solution of Na2CO3 were added.Aliquots of 500, 1000, 1500 and 2000 lL of a 1.0 � 10�5 mol L�1

tannic acid standard solution were added and each final solutionwas diluted with water. After 30 min, the absorbance was mea-sured at 760 nm.

2.3.3.1. Sample and gelatin blank preparation and procedures forbackground corrections. This procedure was based on the treatmentdescribed before (Lau, Luk, & Huang, 1989) with some modifica-tions. For this purpose, white wines were used with no dilutionand the red ones were diluted 20-fold.

The sample was treated twice with gelatin solution and kaolin,so that the tannins were removed by precipitation, providing a tan-nin-free sample.

In the present work, this additional treatment was done onlywith some samples whose values of total polyphenols concentra-tions found by both methods showed large disagreement: threered wines (Cabernet, Pinot Noir and Pinotage) and two white wines(Chardonnay and Sauvignon Blanc).

A 10 mL volume of the wine samples (diluted or not) weretransferred into a 100 mL beaker containing 5.0 mL of the gelatinsolution (0.3% m/v), 10 mL of acidic NaCl solution followed bythe addition of 2.0 g of kaolin. The final mixture was shaken for15 min and the precipitate was allowed to settle down before fil-tering it through a sintered glass (no 4). After that, 10 mL of filtratewere transferred into another 100 mL beaker containing 6.0 mL ofwater, 3.0 mL of same gelatin solution and 6.0 mL of acidic NaClsolution, followed by the addition of 2.0 g kaolin. Then, this mix-ture was shaken again for the same period and the precipitatewas also allowed to settle down before new filtering. One mL ofthe filtrated of this solution without tannin (named as sampleblank) was analysed by both methods as described in the proce-dures. The absorbance values obtained by the proposed and refer-ence methods for these tannin-free solutions were named as Asb

(absorbance of the sample blank solution).The same procedure was carried out for the determination of

the absorbance value of a solution containing no sample but onlygelatin, kaolin and NaCl which was named as Agb (absorbance ofthe gelatin blank solution), except that 10 mL of water was used in-stead of the wine sample solution. In both analyses, for the deter-mination of Asb and Agb values, the ratio of dilution of 6.25 wasconsidered.

For the determination of total polyphenols content the same ra-tio of dilution was kept and 160 lL of diluted samples of red winesor 160 lL of white wine samples (both without any treatment withgelatine/kaolin to remove tannins) were transferred into a 5.0 mLflask. The absorbance values found by analysis of both methodswere named as Atp (absorbance of total polyphenols), correspond-ing to the absorbance value obtained with the sample which maycontain tannins and other polyphenols.

Thus, the absorbance due only to tannins, At, in the analysedsample was calculated as At = Atp � Asb � Agb. In this way, the tan-nin concentration was calculated from the tannic acid calibrationgraph (absorbance vs. tannic acid standard solution concentration)obtained with the proposed and reference procedures.

3. Results and discussion

Neocuproine (NC) forms chelate compounds CuðNCÞ2þ2 (Tütemet al., 1991) so the working solutions of CuðNCÞ2þ2 was preparedwith excess of ligand, such as the concentrations ratio ligand:metalwas about 2.5 in the final solution, i.e. NC 3.66 � 10�3 mol L�1 andCu(II) 1.40 � 10�3 mol L�1.

The calibration graph for tannic acid was linear from 0.4 to3.6 lmol L�1, described by the equation A = 0.00883 +

682 G. Lee et al. / Food Chemistry 126 (2011) 679–686

292,000 � [TA] (r = 0.998; n = 6) where A is the absorbance at450 nm and [TA] is the concentration of tannic acid in mol L�1.The limit of detection, LOD, was estimated as 0.41 lmol L�1, whichis quite lower than the required one for the determination of totalpolyphenols in the most wine samples. The relative standard devi-ation for eight measurements of 1 lmol L�1 standard solution oftannic acid was 1.2%.

3.1. Interference and recovery studies

The influence of some substances, usually present in wines, onthe reduction of CuðNCÞ2þ2 complexes was also verified. In thisstudy, solutions containing one fixed concentration of tannic acid(0.6 lmol L�1) and four solutions with this tannic acid and knownconcentrations (0.6, 6.0, 30 and 60 lmol L�1) of the potential inter-fering species were analysed by the proposed method. The absor-bance values of all solutions, after the procedure, were measuredat 450 nm and the interference was considered when the signal in-creased or decreased by 10% compared with the absorbance valueobtained for the 0.6 lmol L�1 tannic acid solution. No interferencewas observed for up to 100-fold excess (60/0.6 lmol L�1) over tan-nic acid, of malic, benzoic and tartaric acids, glucose, lactose, fruc-tose and sucrose. Sulphite interferes positively when present onlyin a 100-fold (60/0.6 lmol L�1). Citric acid has a negative interfer-ence when present in 50-fold excess (30/0.6 lmol L�1) probablybecause its complexation with Cu(II) (logb2,2 = 14) (Smith &Martell, 2004). Ascorbic acid can reduce Cu(II) in neocuproine mediumand a positive interference was observed when it was present in a10-fold excess (6.0/0.6 lmol L�1). However, the concentrations ofsulphite, citric and ascorbic acids in wine are usually much lowerthan the total polyphenols, so no significant interference of thesespecies in this method is expected.

As seen in Table 1 some authors suggested the determination ofreducing sugars in wines (Araújo, Lima, Rangel, & Segundo, 2000;Peris-Tortajada, Puchades, & Maquieira, 1991; Maquieira et al.,1987) based on the absorbance values of the CuðNCÞþ2 complexes,but the linear range, LR, and the LOD values for reducing sugarswere much larger than that for the tannic acid (present procedure).For determination of polyphenols by the present method it isnecessary a 500-fold and 25-fold dilutions for red and white wines,respectively. So, these dilution requirements avoid any possibleinterference that could come from the presence of reducing sugars.Moreover, one of the procedure cited for reducing sugar determi-nation (Peris-Tortajada et al., 1991) used synthetic wine samplesprepared without any kind of polyphenol. Therefore, the possibleinterference of these compounds on the reducing sugars determi-nation was not considered by those authors.

Recovery experiments were performed by spiking the samplewith three known amount of tannic acid standard solution (J.T. Ba-ker Chemical, USA), with the total tannic acid added being deter-mined using the calibration curve ‘‘absorbance vs. concentrationsof tannic acid standard solutions’’. The calculated mean valuesfor the recoveries present in Table 2 were (95.0 ± 7.1)% and(94.8 ± 6.6)% for the proposed and reference methods, respectively,which could reflect a good agreement between both methods.

3.2. Applications to synthetic samples

A synthetic wine sample was used to confirm the consistency ofthe proposed method and also the effect of S(IV) added in wines,present as sulphite, in both methods. The synthetic wine samplewas prepared by mixing 0.38 g lactose, 0.38 g glucose, 4.0 g glycer-ine, 1.5 g tartaric acid, 0.75 g malic acid, 0.080 citric acid, 0.45 suc-cinic acid, 0.23 g lactic acid, 0.23 g acetic acid, 1.31 g tannic acid,1.04 g sucrose and 64 g ethanol 99% and diluted in 250 mL volu-metric flask (Amarante, 2005). This solution was separated in

two portions of 125.0 mL, transferred to two 250.0 mL volumetricflasks and completed with water. In one of them it was previouslyadded 0.0411 g Na2S2O5 in order to make a synthetic wine samplewith sulphite containing 180 mg L�1 S(IV). In both synthetic sam-ples the total polyphenol content was 2.62 g L�1 as tannic acid.

As can be seen in Table 2 the recoveries found with the pro-posed method for the synthetic wine samples with and withoutsulphite were close to 102%, which are higher when compared tothe values obtained by the reference method (about 89%). Tables2 and 3 show that the presence of sulphite had a major effect onthe results obtained by the reference method, with an overesti-mated value of total polyphenol concentration, (4.5 ± 0.9) g L�1,in the presence of sulphite (Table 3) and lower values of recoveries(Table 2). However, these divergences cannot be explained by thiscomparative study. As it was pointed out by the literature, otherreducing agents, like reducing sugars and ascorbic acid, are alsosignificant in the reference method (Celeste, Tomás, Cladera, Estela,& Cerdà, 1992).

3.3. Matrix effect and antioxidant capacity studies

Table 3 also shows that the results of the total polyphenols con-centration, expressed as tannic acid, obtained by the reference andthe proposed methods are not in agreement for some samples. Atwo-way ANOVA test was performed for statistical comparison ofthe results presented in Table 3. There were no differences be-tween the two methods (p < 0.05) for the samples Tannat, Merlot,Rhône, Malbec, Carmenère, Vitige and the both synthetic wineswith and without sulphite.

In order to study the matrix effect in the present method, thecurves obtained by the multiple standard addition method forthe samples (see experimental part) were compared with the cal-ibration graph. Depending of the nature of the wine, parallelstraight lines were obtained with angular coefficient varying from0.7% to 13%. These results indicate that the matrix effects can benegligible. However, to better investigate this matrix effect in theboth methods calibration curves with several polyphenols solu-tions were obtained (Table 4). Comparing the slope of the all cali-bration curves, as it was made in a previous work dealing only withFolin–Ciocalteu reagent (Singleton et al., 1999), it is possible to ver-ify that for both methods the sensitivity tends to increase with thenumber of free phenolic hydroxyl groups (FPHG).

It can be noted that for phenol, a non oxidant compound(FPHG = 1) and b-carotene, a polyunsaturated hydrocarbon,(FPHG = 0), the Folin–Ciocalteu method is respectively about 1.6and 1.2 more sensitive than the CuðNCÞþ2 method. Alternatively,for resveratrol (FPHG = 3), (�)-epigallocatechin gallate (FPHG = 8)and tannic acid (FPHG = 12) the proposed method is 1.8 more sen-sitive than the reference one. For gallic acid (FPHG = 3), this ratioincreases to 2.8.

Taking into consideration only the benzenediols isomers(1,2 = o-pyrocatechol; 1,3 = resorcinol and 1,4 = hydroquinone) itwas found that the present method is barely 1.5 times more sensi-tive than the reference one for o-pyrocatechol (1,2-) and hydroqui-none (1,4-), but almost three times for resorcinol (1,3-). It wasfound o-pyrocatechol (1,2-) is 2.4 more sensitive than hydroqui-none (1,4-) with the Folin–Ciocalteu reagent. In contrast, resorcinol(1,3-) is 2.8 more sensitive than hydroquinone (1,4-) when theCuðNCÞþ2 is used. Considering only the FPHG, the order of sensitiv-ity using the Folin–Ciocalteu method is: hydroquinone < resor-cinol < o-pyrocatechol (1,2-) but for the CuðNCÞþ2 it ishydroquinone < o-pyrocatechol < resorcinol.

For the benzenetriols isomers (1,2,3 = pyrogallol; 1,3,5 = phloro-glucinol and 1,2,4-benzenetriol) it was found that pyrogallol (1,2,3-) is 1.6 and 2.1 more sensitive than phloroglucinol (1,3,5-) and1,2,4-benzenetriol, respectively, when the Folin–Ciocalteu method

Table 2Recoveries from wine samples obtained with three different amounts of tannic acid.

Sample Tannic acid concentration (lmol L�1)

Proposed method Reference method (Horwitz, 1970)

Diluted sample Diluted sample + added Found % Recovery Diluted sample Diluted sample + added Found % Recovery

Red wine (Tannat) 1.17 1.73 2.88 99.4 1.05 2.60 2.92 80.01.91 3.14 102 3.11 3.44 82.82.29 3.56 103 4.14 5.24 101

Red wine (Syrah) 1.15 1.71 2.75 96.1 0.831 2.38 2.96 92.31.90 2.92 95.7 2.89 3.48 93.52.27 3.32 97.1 3.92 4.73 99.6

Red wine (Bonarda) 0.931 1.55 2.30 92.8 1.33 2.87 3.77 89.71.75 2.38 88.9 3.39 4.25 90.01.96 2.68 92.8 4.42 5.32 92.6

Red wine (Merlot) 0.694 1.25 1.94 99.9 0.584 2.13 2.21 81.31.44 2.13 100 2.64 2.97 92.11.81 2.58 103 3.67 4.25 100

Red wine (Rhône) 1.05 1.66 2.64 97.7 0.891 2.94 4.21 1101.87 2.65 91.0 3.97 5.15 1062.07 2.47 79.1 5.00 6.30 107

Red wine (Malbec) 0.892 1.51 2.18 90.6 0.832 2.88 3.71 1001.71 2.43 93.4 3.91 4.06 85.61.92 2.29 81.6 4.94 6.06 105

Red wine (Cabernet Sauvignon) 0.469 0.87 1.32 98.5 0.818 2.82 3.57 98.01.27 1.74 100.2 3.82 4.55 98.21.47 1.99 102.6 4.82 5.52 97.9

Red wine (Pinot Noir) 0.339 0.74 1.05 97.1 0.748 1.75 2.48 99.21.14 1.48 100.3 2.75 3.53 100.81.34 1.76 104.7 4.75 5.38 97.8

Red wine (Pinotage) 0.511 0.91 1.26 89.0 0.906 2.91 3.77 98.81.31 1.52 83.7 3.91 4.61 95.81.51 1.81 89.4 4.91 5.73 98.6

Red wine (Carmenère) 0.629 1.23 1.77 95.4 0.758 2.76 3.44 97.71.43 1.95 94.9 3.76 4.34 96.11.63 2.05 90.6 4.76 5.22 94.6

Red wine (Vitige) 0.481 0.88 1.07 78.3 0.556 2.56 3.11 99.81.28 1.38 78.1 3.56 4.04 98.21.48 1.56 79.4 4.56 4.98 97.3

White wine (Riesling) 1.00 1.59 2.57 99.1 1.59 3.13 4.11 87.01.73 2.65 97.0 3.65 4.60 87.82.10 3.08 99.5 4.68 5.34 85.1

White wine (Chardonnay) 0.864 1.26 2.11 99.3 1.411 3.41 4.64 96.21.46 2.24 96.3 4.41 5.59 96.11.86 2.53 92.7 5.41 6.40 93.8

White wine (Sauvignon Blanc) 0.718 1.32 1.90 93.2 1.422 2.42 3.71 96.71.52 2.05 91.8 3.42 4.76 98.31.72 2.26 92.9 4.42 5.72 97.9

Synthetic winea 0.850 1.49 2.41 103 0.931 2.52 2.99 86.61.70 2.55 100 3.05 3.57 89.71.91 2.84 103 4.11 4.67 92.6

Synthetic wineb 0.867 1.50 2.37 100 1.09 2.68 3.29 87.31.71 2.60 101 3.21 3.81 88.61.93 2.88 103 4.27 4.74 88.5

a Synthetic wine without sulphite.b Synthetic wine with sulphite.

G. Lee et al. / Food Chemistry 126 (2011) 679–686 683

is used. In contrast, for the CuðNCÞþ2 method the (b) values of pyro-gallol (1,2,3-) were 2.3 and almost four times higher than 1,2,4-benzenetriol and phloroglucinol (1,3,5-), respectively. Moreover,pyrogallol (1,2,3-) and 1,2,4-benzenetriol were about three timesmore sensitive with present method when compared with the ref-erence one. Taking into account only the FPHG, the order ofsensitivity using the Folin–Ciocalteu is 1,2,4-benzenetriol < phlor-oglucinol < pyrogallol but for the CuðNCÞþ2 it is phloroglu-cinol < 1,2,4-benzenetriol < pyrogallol.

From the same Table 4 it is possible to compare the sensitivityof some flavonoids used as a pattern of their groups. No consider-

able difference on the (b) values, obtained by both methods, wasobserved for the flavone luteolin (2-phenylchromen-4-one) andthe flavanone hesperetin (2,3-dihydro-2-phenylchromen-4-one).However for the both flavonols (3-hydroxyflavone) isomers quer-cetin and kaempferol a 1.7 and 2.0 higher values of (b) were ob-tained by the present method, respectively.

For malvin, an anthocyanin pigment (FPHG = 10), the CuðNCÞþ2method presented a small increase in the sensitiveness (1.2 times)when compared to the Folin–Ciocalteu method.

As suggested by Apak, Güçlü, Özyürek, & Karademir (2004), Ta-ble 5 shows the values of the antioxidant capacity of the same

Table 3Comparison of total polyphenols determination by the proposed and the official AOACmethods expressed as tannic acid.

Samples of wine Polyphenols concentration (g L�1)

Proposedmethod

Reference method (Horwitz,1970)

Red (Tannat) 5.0 ± 0.1 4.6 ± 0.5Red (Syrah) 4.9 ± 0.3 3.5 ± 0.6Red (Bonarda) 4.0 ± 0.3 5.6 ± 0.4Red (Merlot) 3.0 ± 0.1 2.5 ± 0.3Red (Rhône) 4.5 ± 0.3 3.8 ± 0.5Red (Malbec) 3.8 ± 0.3 3.5 ± 0.7Red (Cabernet) 2.0 ± 0.1 3.5 ± 0.2

1.2a 1.3a

Red (Pinot Noir) 1.4 ± 0.2 3.2 ± 0.40.98a 1.3a

Red (Pinotage) 2.2 ± 0.4 3.8 ± 0.30.95a 1.6a

Red (Carmenère) 2.7 ± 0.4 3.2 ± 0.5Red (Vitige) 2.0 ± 0.1 2.4 ± 0.3White (Riesling) 0.23 ± 0.01 0.42 ± 0.01White (Chardonnay) 0.20 ± 0.01 0.30 ± 0.02

0.09a NDa

White (SauvignonBlanc)

0.2 ± 0.01 0.3 ± 0.02

0.1a NDa

Synthetic wineb 3.6 ± 0.2 4.0 ± 0.4Synthetic winec 3.7 ± 0.2 4.5 ± 0.9

ND = not detectable.a Values of tannins obtained after treatment for background correction.b Without sulphite.c With sulphite.

Table 5Antioxidant capacity of some phenolic compounds as trolox equivalents (TEACvalues) in the Folin–Ciocalteu and CUPRAC methods.

Compound FPHG TEACFolin TEACCUPRAC TEACCUPRAC/TEACFolin

Tannic acid 12 5.7 10 1.7Malvin 10 4.1 4.8 1.2(�)-Epigallocatechin

gallate8 1.4 2.46 1.7

Quercetin 4 1.7 2.8 1.6Luteolin 4 0.73 0.91 1.3Kaempferol 4 0.80 1.5 1.9Hesperetin 3 1.0 1.0 0.97Resveratrol 3 0.89 1.6 1.8Gallic acid 3 0.54 1.5 2.7Phloroglucinol 3 0.42 0.52 1.2Pyrogallol 3 0.69 2.0 2.91,2,4-Benzenotriol 3 0.33 0.87 2.7o-Pyrocatechol 2 0.95 1.3 1.3Hydroquinone 2 0.39 0.62 1.6Resorcinol 2 0.63 1.8 2.8Phenol 1 0.53 0.32 0.60b-Carotene 0 0.11 0.089 0.81

FPHG = free phenolic hydroxyl groups.

684 G. Lee et al. / Food Chemistry 126 (2011) 679–686

compounds presented in Table 4 calculated as trolox equivalent inboth CUPRAC (TEACCUPRAC) and Folin–Ciocalteu (TEACFolin) meth-ods. These TEAC values were obtained by the quotient betweenthe (b) value of the calibration curve of a compound and the (b) va-lue of the calibration curve of trolox, in both methods. Plotting theTEACCUPRAC vs. TEACFolin values for all compounds a straight line,represented by the equation TEACCUPRAC = 0.102 + (1.53 ±0.11) � TEACFolin with r = 0.962, is obtained revealing that the pro-posed method is about 1.5 more sensitive than the reference one.In fact, the TEACCUPRAC/TEACFolin ratio values for the compoundsinvestigated (except b-Carotene and phenol) varied from 0.97 to

Table 4Parameters of the linear regression of the calibration curves for some standard compound

Compound FPHG PPG FW Reference method (Horw

LR � 106 n a � 103

Tannic acid 12 – 1701.2 1–6 6 20.0Malvin 10 – 691.0 1–6 6 �37.0(�)-Epigallocatechin gallate 8 – 458.4 1–6 6 �1.25Quercetin 4 – 338.3 1–6 6 12.0Kaempferol 4 – 286.2 1–6 6 35.4Luteolin 4 – 286.2 1–6 6 8.98Hesperetin 3 – 302.3 1–6 6 30.9Resveratrol 3 – 228.2 1–6 5 45.2Gallic acid 3 – 188.1 1–6 4 58.4Phloroglucinol 3 1,3,5 126.1 1–6 4 4.35Pyrogallol 3 1,2,3 126.1 1–6 6 57.91,2,4-Benzenotriol 3 1,2,4 126.1 1–6 6 �1.31o-Pyrocatechol 2 1,2 110.1 1–6 5 �15.8Hydroquinone 2 1,4 110.1 1–6 6 �0.177Resorcinol 2 1,3 110.1 1–6 6 1.04Phenol 1 – 94.11 1–6 6 �1.37b-Carotene 0 – 536.9 1–5 4 1.1Trolox 2 250.3 1–6 6 �4.15

FPHG = free phenolic hydroxyl groups; PPG = position of phenolic group of the isomer; FLOD (limit of detection) in lmol L�1; n = number of points. Stock solutions (1 mM) preparhesperetin, b-caroten, kaempferol, malvin chloride and trolox (ethanol 99%).

2.9 (with a mean value 1.8) showing that the CuðNCÞþ2 method ismore responsive than the Folin–Ciocalteu. The antioxidant potencyof the compounds is related to the number and position of the hy-droxyl groups as well as the degree of conjugation of the wholemolecule (Apak et al., 2004). It was found that the sensitivity forboth methods tends to increase with the FPHG (Table 4) and theTEAC values (Table 5).

As reported before by our previous work (Marino, Sabino,Armando, Ruggiero, & Moya, 2009), this study confirms that themean analytical response for both methods depends on the antiox-idant activity of the phenolic compounds, which may explain thedifferent results obtained with some wine samples analysed. Thestandard potential of Cu(II)/Cu(I)/NC system is about 0.6 V (vs.NHE), and therefore can be a more selective oxidant compared tothe Folin–Ciocalteu reagent of unknown potential.

Several alternative methods reported in the literature alsopointed out results in disagreement on the determination of poly-phenols content in various samples like tea and beer (Lau et al.,

s (y = a + b � [C]).

itz, 1970) Proposed method

b/103 r LOD LR � 106 n a � 103 b/103 r LOD

162 0.998 0.36 1–6 6 8.83 292 0.998 0.41117 0.998 0.48 1–6 7 �16.2 142 0.998 0.5439,4 0.998 0.35 1–6 6 44.3 69,2 0.995 0.6048,5 0.997 0.47 1–6 6 �19.2 82,4 0.999 0.2522,6 0.991 0.78 1–6 6 29.3 44,6 0.993 0.7720,7 0.993 0.67 1–6 5 8.28 26,6 0.994 0.7229,5 0.999 0.17 1–6 6 5.97 29,7 0.997 0.4525,3 0.996 0.62 1–6 6 �45.5 46,3 0.998 0.3715,2 0.998 0.30 1–6 4 11.4 43,1 0.999 0.2112,0 0.998 0.37 1–6 4 �20.2 15,1 0.997 0.5919,6 0.997 0.48 1–6 4 �48.2 58,9 0.990 1.359,3 0.997 0.44 1–6 5 �0.120 25,5 0.995 0.7026,8 0.992 0.72 1–6 5 8.58 37,3 0.990 0.9411,0 0.995 0.62 1–6 5 �5.94 18,3 0.998 0.4117,8 0.994 0.64 1–6 4 �40.5 51,8 0.991 0.9014,9 0.994 0.68 1–6 5 2.12 9,4 0.993 0.863,16 0.998 0.47 1–5 5 �5.35 2,6 0.993 6.1528,3 0.995 0.82 1–6 6 �43.7 29,3 0.994 0.61

W (formula weight) in g mol�1; LR (linear range) in mol L�1.ed in water except luteonin, quercetin and resveratrol (50:50 v/v water:ethanol) and

G. Lee et al. / Food Chemistry 126 (2011) 679–686 685

1989), wine and tea (Moya, Dantoni, Rocha, & Coichev, 2008;Yebra, Gallego, & Valcarcel, 1995) and aqueous extracts of medicinalplants (Marino et al., 2009).

Some authors suggested the precipitation of tannins with bo-vine serum albumin, gelatin or even casein, to guarantee its com-plete removal from vegetables tanning baths samples (Araújoet al., 2000; Lau et al., 1989; Molinari, Buonomenna, Cassano, &Drioli, 2001; Yebra et al., 1995).

In the present work, the additional treatment with gelatin andkaolin (see Procedure) was done with some wine samples whichshowed large disagreements between the values of total polyphe-nols found by both methods (Table 3). Three red wines were trea-ted (Cabernet, Pinot Noir and Pinotage). After this treatment, theresults for tannins concentrations, obtained by both methods, forCarbernet and Pinot Noir wines (but not for Pinotage) were inagreement. On the other hand, for the white wine treated samples(Chardonnay and Sauvignon Blanc) the tannins concentrations ob-tained using the proposed method were quite low and not detect-able using the reference method. Our results may indicate that thepresent method is also sensitive for high molecular weight poly-phenols, as is better explained by the data from Tables 4 and 5.The discrepancies found in Table 4 are probably related to the dif-ferent antioxidant activity of the phenolic compounds. In fact, thestudies from De Beer et al., 2004 with 26 kinds of wines also re-vealed that in the five white wine samples analysed (after the sam-ple treatment with protein) it was not possible to detect anytannin. Experiments using HPLC techniques for the comparison ofthe results would contribute to better understanding these effects.

The determination of tannin in tea and wine samples, using amulticommuted flow-injection system, based on the reduction ofCu(II) in the presence of 4,40-dicarboxy-2,20-biquinoline (BCA), aspecific chelator for Cu(I), was already studied by our group (Moyaet al., 2008). The absorbance value at 558 nm, characteristic of theCu(I)/BCA complexes, is also proportional to tannic acid concentra-tion up to 5.00 lmol L�1. The limit of detection and coefficient ofvariation were estimated as 10 nmol L�1 (99.7% confidence level)and 1.0% (1.78 lmol L�1 tannic acid, n = 6), respectively. The molarabsorptivity values of the Cu(I)/BCA complexes (e558m = 7.7 �103 L cm�1 mol�1) (Gershuns & Koval, 1970) and of the Cu(I)/NCcomplexes (e450nm = 7.5 � 103 L cm�1 mol�1) (Tütem et al., 1991)have about the same value. Thus, these both spectrophotometricmethods might achieve similar sensitivity. However, the presentmethod has advantages over the Cu(I)/BCA method (Moya et al.,2008), since the BCA precipitates with Cu(II) while no precipitationwas observed with neocuproine. The main purpose of the presentwork is to propose better reagents for an alternative analyticalmethod but a simple mechanized flow procedure could also im-prove sample throughout and precision, diminishing reagent con-sumption and waste generation.

4. Conclusions

The modified CUPRAC procedure presented here is a sensitivespectrophotometric method to estimate the total polyphenols inwine, expressed as tannic acid concentration, with a low limit ofdetection (LOD = 0.41 lmol L�1), comparable to the limits of detec-tion achieved by other spectrophotometric procedures (LOD =0.01–0.6 lmol L�1) (Dressler, Machado, & Martins, 1995; Horwitz,1970; Moya et al., 2008). In addition, it is free from interferencesof the common species present in wines and does not requireexpensive analytical instrumentation.

Acknowledgements

The authors acknowledge the financial support from Brazilianagencies: FAPESP (Fundação de Amparo à Pesquisa do Estado

de São Paulo), CNPq (Conselho Nacional de DesenvolvimentoCientífico e Tecnológico) and NEPAS (Núcleo de Ensino, Pesquisae Assessoria a Saúde) from FMABC.

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