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Research Article Four Derivative Spectrophotometric Methods for the Simultaneous Determination of Carmoisine and Ponceau 4R in Drinks and Comparison with High Performance Liquid Chromatography Fatma Turak, 1 Mithat Dinç, 2 Öznur Dülger, 1 and Mahmure Ustun Özgür 1 1 Department of Chemistry, Faculty of Science and Art, Yıldız Technical University, 34220 Istanbul, Turkey 2 Tekirdag City Food Control Laboratory, 59000 Tekirdag, Turkey Correspondence should be addressed to Fatma Turak; ſt[email protected] Received 6 October 2013; Revised 10 December 2013; Accepted 10 December 2013; Published 12 February 2014 Academic Editor: Hian Kee Lee Copyright © 2014 Fatma Turak et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Four simple, rapid, and accurate spectrophotometric methods were developed for the simultaneous determination of two food colorants, Carmoisine (E122) and Ponceau 4R (E124), in their binary mixtures and soſt drinks. e first method is based on recording the first derivative curves and determining each component using the zero-crossing technique. e second method uses the first derivative of ratio spectra. e ratio spectra are obtained by dividing the absorption spectra of the binary mixture by that of one of the components. e third method, derivative differential procedure, is based on the measurement of difference absorptivities derivatized in first order of solution of drink samples in 0,1N NaOH relative to that of an equimolar solution in 0,1N HCl at wavelengths of 366 and 451 nm for Carmoisine and Ponceau 4R, respectively. e last method, based on the compensation method is presented for derivative spectrophotometric determination of E122 and E124 mixtures with overlapping spectra. By using ratios of the derivative maxima, the exact compensation of either component in the mixture can be achieved, followed by its determination. ese proposed methods have been successfully applied to the binary mixtures and soſt drinks and the results were statistically compared with the reference HPLC method (NMKL 130). 1. Introduction Color is a vital constituent of food and probably the first characteristic perceived by the human senses. Probably, for economic reasons, brightly colored and stable synthetic colorants have been widely used as food additives. ese colorants are used to supplement and enhance natural colors destroyed during processing or storage and substantially increase the appeal and acceptability of food stuff. e term color additive can be applied to any dye, pigment, or other substances artificially made or obtained from a vegetable, animal, mineral, or another natural source [1]. Some synthetic colorants and their metabolic products are consumed in large amounts maybe toxic and cause cancer, deformation, and so forth [2]. Carmoisine (E122) and Ponceau 4R (E124) are synthetic dyes that contain azo and aromatic ring structures and are oſten used in dyeing food, drink, medicine, and cosmetics (Scheme 1). e development of chemical and instrumental methods for the separation, identification, and quantitative analysis of synthetic food colorants has become extremely important for the food and beverages industry and academic and governmental institutions to assess the quality and safety of food products [3]. Although the amounts of synthetic colors that are added to food and drinks are strictly controlled, they may exceed the authorized levels. us monitoring of the levels of colorants in high consumption products such as beverages becomes of great importance. e number of works Hindawi Publishing Corporation International Journal of Analytical Chemistry Volume 2014, Article ID 650465, 11 pages http://dx.doi.org/10.1155/2014/650465

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Page 1: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

Research ArticleFour Derivative Spectrophotometric Methods forthe Simultaneous Determination of Carmoisine and Ponceau4R in Drinks and Comparison with High PerformanceLiquid Chromatography

Fatma Turak1 Mithat Dinccedil2 Oumlznur Duumllger1 and Mahmure Ustun Oumlzguumlr1

1 Department of Chemistry Faculty of Science and Art Yıldız Technical University 34220 Istanbul Turkey2 Tekirdag City Food Control Laboratory 59000 Tekirdag Turkey

Correspondence should be addressed to Fatma Turak fturakyildizedutr

Received 6 October 2013 Revised 10 December 2013 Accepted 10 December 2013 Published 12 February 2014

Academic Editor Hian Kee Lee

Copyright copy 2014 Fatma Turak et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Four simple rapid and accurate spectrophotometric methods were developed for the simultaneous determination of two foodcolorants Carmoisine (E122) andPonceau 4R (E124) in their binarymixtures and softdrinksThefirstmethod is based on recordingthe first derivative curves and determining each component using the zero-crossing technique The second method uses the firstderivative of ratio spectra The ratio spectra are obtained by dividing the absorption spectra of the binary mixture by that of oneof the components The third method derivative differential procedure is based on the measurement of difference absorptivitiesderivatized in first order of solution of drink samples in 01 N NaOH relative to that of an equimolar solution in 01 N HCl atwavelengths of 366 and 451 nm for Carmoisine and Ponceau 4R respectivelyThe last method based on the compensation methodis presented for derivative spectrophotometric determination of E122 and E124mixtures with overlapping spectra By using ratios ofthe derivative maxima the exact compensation of either component in the mixture can be achieved followed by its determinationThese proposed methods have been successfully applied to the binary mixtures and soft drinks and the results were statisticallycompared with the reference HPLC method (NMKL 130)

1 Introduction

Color is a vital constituent of food and probably the firstcharacteristic perceived by the human senses Probablyfor economic reasons brightly colored and stable syntheticcolorants have been widely used as food additives Thesecolorants are used to supplement and enhance natural colorsdestroyed during processing or storage and substantiallyincrease the appeal and acceptability of food stuff The termcolor additive can be applied to any dye pigment or othersubstances artificially made or obtained from a vegetableanimalmineral or another natural source [1] Some syntheticcolorants and their metabolic products are consumed in largeamounts maybe toxic and cause cancer deformation and soforth [2]

Carmoisine (E122) and Ponceau 4R (E124) are syntheticdyes that contain azo and aromatic ring structures and areoften used in dyeing food drink medicine and cosmetics(Scheme 1)

The development of chemical and instrumental methodsfor the separation identification and quantitative analysisof synthetic food colorants has become extremely importantfor the food and beverages industry and academic andgovernmental institutions to assess the quality and safety offood products [3] Although the amounts of synthetic colorsthat are added to food and drinks are strictly controlledthey may exceed the authorized levels Thus monitoring ofthe levels of colorants in high consumption products such asbeverages becomes of great importanceThenumber ofworks

Hindawi Publishing CorporationInternational Journal of Analytical ChemistryVolume 2014 Article ID 650465 11 pageshttpdxdoiorg1011552014650465

2 International Journal of Analytical Chemistry

N N

N N

OH

HO

NaO3S

NaO3S

NaO3S

NaO3S

SO3Na

Carmoisine (Azorubine E122) CI 14720 Ponceau 4R (Cochineal Red A E124) CI 16255

Scheme 1 Chemical structures common names European community numbers (E) and color index (CI) numbers of synthetic foodcolorants studied

published during the last years demonstrates the importanceof this problemand the need for developing fast accurate andselective techniques for synthetic colorants analysis [4]

Various techniques have been introduced for the deter-mination of mixtures of colorants with three or more com-ponent combination in food samples

These include capillary electrophoresis [5] differentialpulse polarography [4] high performance liquid chromatog-raphy [6ndash9] electrospray mass spectrometry [10] spec-trophotometry [11ndash13] partial least squares [14] and spec-trofluorimetry [15] Chromatographic methods have beenused for colorant analysis in foods and they are recom-mended when the mixture contains many different colorants[6 7 16 17] However there are some potential drawbackssuch as usage of toxic solvents the need for complex samplepretreatments and the resulting waste product Capillaryelectrophoresis is also very suitable when the sample containsseveral colorants but foods beverages and pharmaceuticalscontain only one two or rarely three food dyes and thesemethods need special equipment that may not be availablein certain quality laboratories All multivariate calibrationmethods including Partial Least Square (PLS) require thedata processing with powerful software as well as the manip-ulations of the abstract vector space and its application tothe regression analysis and various chemometric methodsare also often used for more complex mixtures Thus ana-lytical methods from alternative technique are always usefulespecially if the methods are simple cheap and compara-tively fast Spectrophotometry as alternative methodologyis suitable for routine laboratories especially for developingcountries and sometimes serious analytical problems canbe resolved by this common technique Among computer-controlled instruments derivative techniques (ratio deriva-tive difference derivative and compensation method) areplaying a very important role in the binary analysis ofmixtures by UV-VIS molecular absorption spectrophotom-etry All approaches are useful in the resolution of bandoverlapping in quantitative analysis

In this present work selective sensitive simple andlow-cost procedures were developed for the simultaneous

determination of Carmoisine (C) and Ponceau 4R (P) insyntheticmixture and drink samples by using derivative spec-trophotometry (DSM) ratio derivative spectrophotometry(RDM) derivative differential spectrophotometry (DDM)and compensation method (CM) These methods have beensuccessfully applied to synthetic mixtures and drink samplesand the results obtained were compared with those obtainedby HPLC method according to NMKL 130 [18]

No extraction or evaporation step no complexationagent andnoharmful chemicals are involved in the suggestedmethods in the suggestedmethods so can be used for routineanalysis of both colorants in quality control and routinelaboratories

2 Experimental

21 Apparatus All spectral measurements and treatment ofdata were carried out using a Shimadzu UV-2450 doublebeam UV-Vis spectrophotometer with two matched 1-cmquartz cells connected to a Hewlett Packard compatiblepersonal computer and a HP 1102 Laser Jet Printer BundledUV-PC personal spectroscopy software of version 221 wasused to process the absorption and the derivative spectraThe spectral band width was 1 nm with wavelength scanningspeed ofmediumHPLCmeasurements were performedwithan Agilent 1100 series HPLC-DAD system with a vacuumdegasser quaternary pump auto sampler and injector with a100 120583L loop volume Separation was achieved using a WatersSpherisorb C18 column (46 times 250mm 5120583m) and variablewavelength UV-VIS DAD detector measuring at 520 nm forCarmoisine and 512 nm for Ponceau 4R

22 Chromatographic Conditions Separation was achievedusing aWaters Spherisorb C

18column (46times 250mm 5 120583m)

The solvent system consisted of a mixture of an aqueoussolution containing 0005M tetrabutylammonium hydro-gen sulphate (TBA) and a phosphate buffer solution (pH65) methanol (50 50 vv) It was pumped isocratically at07mLminminus1 for the separation The eluents were monitoredat 512 and 520 nm for P and C respectively

International Journal of Analytical Chemistry 3

Table 1 Experimental parameters calculated for the simultaneous determination of Carmoisine and Ponceau 4R in binary mixture bycompensation method

Colorants Concentration range (120583gmLminus1) Ratio Meana RSD ()Carmoisine 2ndash10 1D472

1D569 0600 plusmn 0002 0333Ponceau 4R 2ndash10 1D470

1D554 0690 plusmn 0009 1304aMean of five separate determinations

Table 2 Statistical parameters of the simultaneous determination of Carmoisine and Ponceau 4R by DSM RDM and DDM

Methods AnalyteSelected

wavelength(nm)

Concentrationrange

(120583gmLminus1)Regression equationsa

Correlationcoefficient

(119903)

Detection limit(120583gmLminus1)

Quantificationlimit

(120583gmLminus1)

DSM C 1D331 2ndash10 156 sdot 10minus4119862+190 sdot 10

minus5 09995 0086 0286P 1D517 2ndash10 232 sdot 10

minus4119862minus320 sdot 10

minus5 09996 0091 0304

RDM C 1D323 2ndash10 413 sdot 10minus3119862minus323 sdot 10

minus4 09994 0079 0263P 1D344 2ndash10 195 sdot 10

minus3119862minus343 sdot 10

minus3 09996 0082 0273

DDM C 1D366 2ndash10 325 sdot 10minus4119862+127 sdot 10

minus4 09996 0071 0236P 1D451 2ndash10 439 sdot 10

minus4119862minus156 sdot 10

minus4 09998 0081 0270aFive separate determinations were performed and mean was calculated119862lowast the concentration of the analyte (120583gmLminus1)

23 Chemicals andMaterials Distilledwaterwas used to pre-pare the solutions and for mobile phase All chemicals usedwere of analytical reagent grade and were purchased fromMerck (Darmstadt Germany) Ponceau 4R (E124 CochinealRed A molecular weight 60447 CI 16255 CAS number 2611-82-7) and Carmoisine (E122 Azorubine molecular weight50244 CI 14720CAS number 53026-69-9) were from Sigma-Aldrich (Steinheim Germany) Soft drinks were purchasedfrom commercial markets

24 Standard Solutions and Calibration Graphs Stock solu-tions of Carmoisine and Ponceau 4R (100 120583gmLminus1) wereprepared by dissolving appropriate weights in distilled waterVarious aliquots of each stock solution within the concen-tration range stated in Table 2 were transferred into five setsof 10mL volumetric flasks and the volume was completedwith distilled water The stability of the working solutions ofCarmoisine and Ponceau 4R was studied by recording thetime-dependent absorption spectra no changes in the spectrawere observed for at least one month when the solutions arestored at room temperature in the dark

For DDMmethod accurate volume aliquots of the stocksolution were transferred into two sets of 10mL volumetricflasks and the volume was made up with 01 N HCI and 01 NNaOH to give a series of equimolar solutions containing 20ndash100 120583gmLminus1 of both Carmoisine and Ponceau 4R Calibra-tion solutions (119899 = 5) were used to construct the calibrationcurves in the standardization of the cited methods

For HPLC method stock solutions of (25mg100mL)Carmoisine and Ponceau 4R were prepared in a 1 1 vvmixture of methanolmdashTBA buffer solution (0005M TBA +0005M phosphate buffer pH 65) The standard solutions

were prepared containing 5ndash20120583gmLminus1 of Carmoisine andPonceau 4R in the same solvent system All prepared solu-tions were filtered through 045120583m membrane filter beforeinjection

Commercial drink samples due to turbid are treatmentwith the Carrez I and II solutions

Carrez solution I it contains potassium hexacyano-ferrate (II) trihydrate (K

4[Fe (CN)

6] times 3H

2O) 100mL

concentration 15 g times 100mLminus1

Carrez solution II it contains zinc sulfate heptahy-drate (ZnSO

4times 7H2O) 100mL concentration 30 g times

100mLminus1

25 Spectrophotometric Measurements

(i) Final solutions for measurements were prepared overthe concentration ranges given in Table 2

(ii) The derivative spectra were recorded over the wave-length range of 300ndash700 nm The amplitude of thederivative curve was measured at the selected wave-lengths for each method (Table 1)

(iii) For the compensation method the absorbance differ-ence spectra (sample versus reference) were recordedover the wavelength range of 300ndash700 nm usingdifferent concentrations of the reference solutionprepared from the stock solution

(iv) For the DDmethod the Carmoisine and Ponceau 4Rsolutions in 01 N HCI difference derivative spectrawere recorded against corresponding 01 N NaOHsolutions of the colorants as a blank The amplitudeof the first derivative spectra (Table 2) was measuredat the chosen wavelengths

4 International Journal of Analytical Chemistry

26 Procedures

261 Derivative Spectrophotometric Method (DSM) Ali-quots of Carmoisine working solutions equivalent to 20ndash100 120583gmLminus1 were placed into 10mL calibration flasks andthe volume was made up with distilled water The absorptionspectra were recorded versus distilled water Next the zero-order spectra were transformed into first derivative Theamplitude from the baseline to peak at 331 nm (zero-crossingpoint for Ponceau 4R) was related to the actual contents ofCarmoisine

Appropriate aliquots of aqueous working solutions ofPonceau 4R were placed into 10mL calibration flasks andthe volume was made up with distilled water The spectra ofstandard solutions in the concentration range 20ndash10120583gmLminus1were recorded and subjected to the mathematical treatment(first derivative) The values of 1D

517measured at 517 nm

(zero-crossing point for Carmoisine) were used for construc-tion of calibration graph

262 Ratio Derivative Spectrophotometric Method (RDM)Accurately aliquots of 02 04 06 08 and 10mLof colorantsstock solution were transferred into 10mL volumetric flasksand the volume wasmade up with distilled waterThe spectraof the prepared standard solutions were scanned from 300 to700 nm and stored in the computer For the determination ofCarmoisine the stored spectra of Carmoisine were dividedseparately by the spectrumof 6120583gmLminus1 Ponceau 4R to obtainthe ratio spectra The first derivative corresponding to eachratio spectrum (1DD) was recorded A calibration graphrelating the peak amplitudes (1DD

323) to the corresponding

concentrations in 120583gmLminus1 of Carmoisine was constructedIn order to determine Ponceau 4R stored spectra of Pon-

ceau 4R standard solutions were divided by a standard spec-trum of Carmoisine of 6120583gmLminus1 The first ratio derivativespectra were calculatedThe concentration of the Ponceau 4Rwas proportional to the amplitude of themaximumat 344 nm(1D344

)

263 Derivative Differential Spectrophotometric Method(DDM) The difference spectra between the acidic (01 NHCI) solution and equimolar basic (01 N NaOH) solutionof pure colorants and drink samples were recorded from3000 to 7000 nm by placing the acidic solution in thesample compartment and the basic solution in the referencecompartment A first derivative spectrum of each of thedifferential curves was subsequently recordedThe derivativeabsorbance values were measured at 366 and 451 nm forCarmoisine and Ponceau 4R respectively

264 Compensation Method (CM)

(1) Determination of Standard Ratios The first derivativespectra for each set of standard solutions were recorded usingthe appropriate blank solution The ratios of the first deriva-tive maxima and minima ( 1D

1205821

1D1205822

) where appropriate at

the specified wavelengths (1205821and 120582

2) as indicated in Table 1

were calculated

(2) Compensation Spectrophotometric Measurements Thesolution of the mixture (containing compounds Carmoisineand Ponceau 4R) was placed in the sample cell A series ofsolutions containing various concentrations (2ndash10120583gmLminus1)of Carmoisine above and below that present in the mixturesolution were prepared and placed in succession in thereference cell The first derivative spectra (1D) were recordedand the corresponding ratios (Table 1) in each instancewere calculated and followed the calculated ratio for purecolorant Ponceau 4R The exact balance point (the ratio ofthe mixture is equal to that of pure compound Ponceau 4R)was determined at which the concentration of Carmoisine insample solution is equal to that in the reference solution

By analogy the same steps were followed by using solu-tions of pure Ponceau 4R in the reference cell to determine itsconcentration in the mixture at the balance point

The same principle was also applied for the analysis ofdrink samples

(3) Graphical Method To avoid the excessive preparation ofseveral standard solutions of either compoundCarmoisine orin order to locate the exact balance point a graphical methodis recommended [19 20]

The same steps as for the compensation method werefollowed and the derivative ratio of themixture (in the samplecell) was calculated at each time and plotted against the con-centration of pure compound Carmoisine (in the referencecell) A line with slight curvature is usually obtained Theconcentration of Carmosine can easily be interpolated fromthe graph by substituting the ratio of pure Ponceau 4R Atthe point at which the ratio of the mixture is equal to thatof pure Ponceau 4R the concentration of Carmoisine in thereference cell is equal to that in themixture in the sample cellBy analogy the concentration of compound Ponceau 4R canbe obtained

265 HPLC Method (Reference Method NMKL 130) Thisreference method is described in detail in Nordic Committeeon Food Analysis [18] About 20 120583L of each sample wasinjected and the concentrations were calculated on the basisof the ratios of peak areaswith those of the standard solutionsThe chromatograms of two compounds and drink sampleswere plotted and stored in the computer Separation wascarried out in room temperature The mobile phase wasprepared daily filtered through a 045120583m membrane filterAll chromatograms were run with the use of a (50 50 vv)mixture of methanol and TBA buffer Other experimentalparameters were flow rate 07mL minminus1 retention times554min for Ponceau 4R and 2162min for Carmoisine

266 Application of the Proposed Methods for the Deter-mination of Two Colorants in Drink Sample Commercialsamples weremixed with Carrez (I) and Carrez (II) solutionsOnce precipitated the samples were centrifuged 50mL ofthe sample that is clarified by Carrez precipitation was

International Journal of Analytical Chemistry 5

0175

0375

0575

0775

300

350

400

450

500

550

600

650

700

Abso

rban

ce

Wavelength (nm)

C

Mix

P

minus0025

(a)

00002

00022

300

320

340

Firs

t der

ivat

ive

Wavelength (nm)

P

C

00002

00022

475

500

525

550

575

Firs

t der

ivat

ive

Wavelength (nm)

C

P

minus00098

minus00078

minus00058

minus00038

minus00018

minus00098

minus00078

minus00058

minus00038

minus00018

(b)

Figure 1 Zero-order spectra (a) of 10 120583gmLminus1 Carmoisine 10120583gmLminus1 Ponceau 4R and their binary mixture first derivative spectra (b) ofcolorants (Carmoisine(mdash) 2ndash10120583gmLminus1 Ponceau 4R (- -) 2ndash10120583gmLminus1)

transferred to 100mL volumetric flask and the volume wasmade up with distilled water A series of dilution wasprepared quantitatively with water from this solution toobtain standard solutions to reach the concentration ranges ofcalibration curves graphed for each of the proposed methods(DSM RDM and CM) For the DDM method the sameprocedure was used but in this case the final volume wasmade up with 01 N HCI and 01 N NaOH separately forthe determination of Carmoisine and Ponceau 4R HPLCmethod the same sample preparation procedure describedabove was followed using a mixture 1 1 vv of methanolmdashTBA buffer solution After dissolution process preparedsolutions were filtered using 045 120583m disposable membranefilters (Sartrious Minisart 045120583m) by using a syringe Thefinal solutionwas diluted to theworking concentration range

27 Method Validation The validity and suitability of theproposed methods were assessed by accuracy (reported aspercentage recovered) precision (reported as RSD) lin-earity (evaluated by regression equation) limit of detection(LOD) and limit of quantification (LOQ) [21]

LOD and LOQ were based on the standard deviation ofresponse and the slope of the corresponding curve using thefollowing equations

LOD = 3 119904119898 LOQ = 10 119904

119898 (1)

where 119904 the noise estimate is the standard deviation of thederivative amplitude of the blank and 119898 is the slope of therelated calibration graphs [22]

The linearity of the methods was established in theconcentration ranges of 2ndash10120583gmLminus1 for Carmoisine and

Ponceau 4R respectively In this study five concentrationswere analyzed in five replicates The linearity was evaluatedby the least square regression method

In order to demonstrate the validity and suitability ofthe proposed methods intra- and interday variability studieswere performed at three different concentrations (2 6 and10 120583gmLminus1) in the presence of the certain concentration (26 and 10 120583gmLminus1) of the other colorants three times in a dayand also three different days Method efficiency was tested interms of RSDrsquos for both intraday and interday precisions

The precision was ascertained by carrying out five repli-cate determinations of synthetic mixtures at different con-centration ratios of Carmoisine and Ponceau 4R Recoverytest also confirmed the accuracy and applicability of theproposed methods by analyzing several synthetic mixturesof Carmoisine and Ponceau 4R which reproduced differentcomposition ratios within the linearity rangeThese ratios arealso the ratios in drinksThemean percentage recoveries andRSD values were calculated

Recovery of the colorants of interest from a given matrixcan be used as a measure of the accuracy or the bias of themethod The same ranges of concentrations as employed inthe linearity studies were used

In our study the applicability of the derivative spec-trophotometric methods was also validated by comparisonwith the HPLC technique

3 Result and Discussion

Figure 1(a) shows the absorption spectra of CarmoisinePonceau 4R and themixture of them in the wavelength rangeof 300ndash700 nm Zero-order absorption spectra of Carmoisine

6 International Journal of Analytical Chemistry

Table 3 Method validation for the simultaneous determination of Carmoisine and Ponceau 4R in laboratory prepared mixtures by theproposed methods

Method Accuracy (meanlowastplusmn RSD ) Precision repeatabilitya(mean plusmn RSD )

Intermediate precisionb

(mean plusmn RSD )

DSM C (1D331) 1008 plusmn 087 1001 plusmn 096 9991 plusmn 102

P (1D517) 991 plusmn 102 1005 plusmn 105 1001 plusmn 092

RDM C (1D323) 1010 plusmn 095 9998 plusmn 099 1006 plusmn 105

P (1D344) 9825 plusmn 093 9983 plusmn 098 1004 plusmn 096

DDM C (1D366) 1004 plusmn 107 9895 plusmn 105 9992 plusmn 098

P (1D451) 9955 plusmn 095 1005 plusmn 095 1007 plusmn 096

CM C (1D4721D569) 9975 plusmn 102 998 plusmn 101 1001 plusmn 097

P (1D4701D554) 985 plusmn 106 9975 plusmn 103 1003 plusmn 106

aThe intraday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times within the daybThe interday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times in three successive dayslowastThe values of recovery are an average of five replicates of each of five synthetic mixtures at different concentration ratios of C and P (2ndash10 120583gmLminus1)RSD relative standard deviation

and Ponceau 4R in distilled water gave rise to maximumpeaks at 517 nm and 508 nm respectively As can be seenthe spectra of the colorants were strongly overlapped in the400ndash600 nm wavelength region Hence the direct absorp-tion measurement for assaying binary mixture seems to beimpossible On the other hand the absorption spectra of thetwo components within a wavelength range of 300ndash380 nmwere less overlapped than the region 400ndash600 nm So in thiswork the region of 300ndash380 nm was chosen for the analysisof Carmoisine In this spectral range DSM RDM and DDMwere used for the determination of the studied colorants inlaboratory prepared mixtures and soft drink samples Thesemethods are also applied to analyze Ponceau 4R in the region320ndash520 nm

31 Derivative Spectrophotometric Method (DSM) Undercomputer-controlled instrumentation derivative spectro-photometry played a very important role in the resolutionof band overlapping in quantitative analysis [23] Figure 1(b)shows the first derivative spectra of Carmoisine and Ponceau4R As can be seen in Figure 1(b) in the region of 300ndash520 nm the first derivative spectra allowed the simultaneousdetermination of Carmoisine and Ponceau 4R At this regionthe 1D spectra (Figure 1(b)) show a characteristic peak at331 nm for Carmoisine while Ponceau 4R shows no responseTherefore the absolute values of the 1D peak amplitudes at331 nm can be used to quantify Carmoisine in this mixtureOn the other hand Ponceau 4R can be assayed in the presenceof Carmoisine by measuring its 1D response at 517 nm (zero-crossing point of Carmoisine)

Under the experimental conditions described the cal-ibration graphs obtained by plotting the derivative valuesversus concentrations for Carmoisine and Ponceau 4R inthe concentration range stated in Table 2 exhibited linearrelationships Statistical analysis of the data showed thatlinearity of the calibration graphs and compliance withBeerrsquos law were validated and the small values of the inter-cepts as illustrated by the excellent values of correlation

coefficients of the regression equations To prove the valid-ity and applicability of the proposed method laboratory-prepared mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R (Table 3) The obtainedresults for the analysis of these mixtures in five replicates bythe described method showed high accuracy and precisionof the proposed method (Table 3)

32 Ratio Derivative Method (RDM) While the main dis-advantages of the zero-crossing method in derivative spec-trophotometry for resolving a mixture of components withoverlapped spectra are the risk of small drifts of the workingwavelengths and the circumstance that the working wave-lengths generally do not fall in correspondence of peaks ofthe derivative spectrum this may be particularly dangerouswhen the slope of the spectrum is very high with consequentloss of accuracy and precision and the working wavelengthis in proximity of the base of the spectrum which causespoor sensitivity The main advantage of the RDM is thechance of doing easy measurements in correspondence ofpeaks so it permits the use of the wavelength of highestvalue of analytical signals (a maximum or a minimum) andmoreover the presence of a lot of maxima and minima isanother advantage by the fact that these wavelengths give anopportunity for the determination of active compounds in thepresence of other compounds and ingredients which possiblyinterfere in the assay [24ndash27]

This method is based on the use of the first derivativeof the ratio spectra The ratio spectra were obtained bydividing the absorption spectrum of the mixture by thatof one of the components As we depicted above directabsorption measurements are not possible due to spectraloverlap This spectral overlapping was sufficiently enough todemonstrate the resolving power of the proposed methodFigure 2(a) shows the first ratio derivative spectra of differ-ent Carmoisine standard solutions (spectra divided by thespectrum of 6 120583gmLminus1 Ponceau 4R solution) The ratio firstderivative amplitudes at 323 nm (1DD

323) corresponding to

International Journal of Analytical Chemistry 7

001

003

308 328 348 368

Firs

t der

ivat

ive

Carm

oisin

ePo

ncea

u 4R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

Firs

t der

ivat

ive

Ponc

eau

4RC

arm

oisin

e

Wavelength (nm)

fg

hi

j

minus002678

minus001678

minus000678

(b)

Figure 2 First derivative ratio spectra of Carmoisine (a) and Ponceau 4R (b) for different concentrations (Carmoisine (6 120583gmLminus1 Ponceau4R was used as a divisor) a 2 120583gmLminus1 b 4 120583gmLminus1 c 6120583gmLminus1 d 8 120583gmLminus1 e 10120583gmLminus1 Ponceau 4R (6120583gmLminus1 Carmoisine was usedas a divisor) f 2120583gmLminus1 g 4 120583gmLminus1 h 6 120583gmLminus1 i 8120583gmLminus1 j 10120583gmLminus1)

Table 4 Assay results for the determination of Carmoisine and Ponceau 4R in soft drinks using the proposed methods and the referenceHPLC method (NMKL 130)

Methods Analyte Selected wavelengths(nm)

Assay results mean plusmn SDlowast (119905calculated 119865calculated)lowastlowast

Soft drink I (120583g 100mLminus1) Soft drink II (120583g 100mLminus1)

DSM C 1D331 930 plusmn 022 (068 145) 628 plusmn 029 (061 144)

P 1D517 220 plusmn 011 (118 118) 139 plusmn 013 (040 179)

RDM C 1D323 926 plusmn 023 (037 145) 636 plusmn 021 (128 120)

P 1D344 235 plusmn 011 (044 125) 143 plusmn 013 (013 169)

DDM C 1D366 931 plusmn 024 (073 169) 641 plusmn 024 (153 164)

P 1D451 249 plusmn 017 (189 302) 144 plusmn 016 (023 268)

CM C 1D4721D569 930 plusmn 026 (063 188) 636 plusmn 028 (107 211)

P 1D4701D554 230 plusmn 016 (023 256) 143 plusmn 019 (010 368)

HPLC C 520 921 plusmn 018 620 plusmn 019

P 512 232 plusmn 010 142 plusmn 010

lowastResults obtained are the average of five experiments for each method SD standard deviationlowastlowastThe corresponding theoretical value for 119905 and 119865 at 119875 005 (119905theoretical 231 119865theoretical 639)

a maximum wavelength are proportional to the Carmoisineconcentration (Figure 2(a)) To determine the other com-ponent Ponceau 4R an analogues procedure was followedFor determination of Ponceau 4R the stored spectra ofthe mixtures are divided by a standard spectrum of 6mgLminus1 Carmoisine (Figure 2(b)) Standard solutions of Ponceau4R and the content of Ponceau 4R were determined bymeasuring the signals at 344 nm (1DD

344) Figure 2(b)

Under the described experimental conditions the cal-ibration graphs obtained by plotting the derivative valuesof each colorant versus concentration in the concentration

range stated in Table 2 showed linear relationships Con-formity with Beerrsquos law was evident in the concentrationrange of the final dilution cited in Table 2 For this methodthe characteristic parameters of regression equations andcorrelation coefficients are given in Table 2 The correlationcoefficients were 09996 and 09994 indicating good linearityLOD and LOQ were calculated for each colorant and areshown in the same table The detection limits were foundto be 0079120583gmLminus1 for Carmoisine and 0082 120583gmLminus1 forPonceau 4R while the quantification limits were estimatedto be 0263 120583gmLminus1 for Carmoisine and 0273120583gmLminus1 for

8 International Journal of Analytical Chemistry

000988

008988

016988

024988

032988

300 400 500 600 700

Abso

rban

ce

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

Diff

eren

ce fi

rst d

eriv

ativ

e

Wavelength (nm)

C

P

minus00068

minus00028

(b)

Figure 3 Difference absorption spectra of 10 120583gmLminus1 Carmoisine 10 120583gmLminus1 Ponceau 4R and their binary mixture (a) Difference firstderivative spectra (b) of colorants (Carmoisine 2ndash10120583gmLminus1 Ponceau 4R 2ndash10 120583gmLminus1)

Ponceau 4R The proposed method was also applied to thedrink samples (Table 4) The accuracy of RDM was checkedby analyzing the laboratory preparedmixtures of Carmoisineand Ponceau 4R at various concentration ratios ranging from2 to 10 120583gmLminus1 (Table 3) All data represent the average offive determinations Low values of relative standard deviationindicate very good precision Satisfactory recoveries withsmall standard deviations were obtained which indicated thehigh repeatability and accuracy of the method

33 Derivative DifferentialMethod (DDM) Differential spec-trophotometry (ΔD

1) based on pH changes has also been

reported to be useful in the determination of binary mix-tures This method that depends on utilization of differenceabsorption spectra corresponding to the same compoundobtained at two different pHrsquos has been investigated for theanalysis of binary mixtures The procedure is comprised ofthe measurement of ΔD

1food colorantsrsquo in acidic solutions

against their alkaline solutions as blanks The ΔD1has

been successfully used to eliminate interferences from drinksamples There are few reports on utilization of the abovetwo combined techniques (difference and derivative) for theestimation of individual drug substances and for combinedpreparations [28ndash30]

The difference absorption spectra of Carmoisine Pon-ceau 4R and their binary mixture in 01 N NaOH and in01 N HCI are shown in Figure 3(a) Figure 3(b) shows thefirst derivative difference spectra of colorants in the concen-tration range 2ndash10 120583gmLminus1 The spectra of both colorantsin Figure 3(b) offer an advantage for their simultaneousdetermination by having zero-crossing points In particularthe first derivative amplitudes at 366 nm for Carmoisine and

451 nm for Ponceau 4R in the Carmoisine and Ponceau 4Rmixture were considered as the optimum working wave-lengths for their determination By measuring the values ofthe ΔD

1amplitudes at these wavelengths the concentration

of each colorant can be directly calculated since the dif-ferential first derivative measurement cancels the irrelevantabsorbance due to the drinks matrix at these wavelengths

Under the experimental conditions described the cali-bration graphs obtained by plotting the derivative values ofeach colorant in the mixture versus concentration in therange concentration stated in Table 2 show linear relation-ships The correlation coefficients were 09996 and 09998indicating good linearity The LOD were 0071 120583gmLminus1 forCarmoisine and 0081 120583gmLminus1 for Ponceau 4R while theLOQ were 0236 120583gmLminus1 for Carmoisine and 0270 120583gmLminus1for Ponceau 4R The proposed procedure was success-fully applied for the determination of these colorants inlaboratory-prepared mixtures and drink samples The resultsare presented in Tables 3 and 4The values of relative standarddeviation indicate very good reproducibility In additionapplication of the DDMwas found to be correct for the drinkmatrix interference and to enhance the sensitivity

34 CompensationMethod (CM) Thecompensationmethodis a nonmathematical method for the detection and elimi-nation of unwanted absorption during spectrophotometricanalysis [19 20 31 32] The accuracy of the method dependson the evaluation of the balance point Figure 1(b) showsthe first derivative spectra of Carmoisine and Ponceau 4Rin the concentration range 2ndash10 120583gmLminus1 The first derivativespectra were recorded for each reference solution of thecomponents and the ratios of the 1D maximum and 1D

International Journal of Analytical Chemistry 9

000011000211000411000611

401 451 501 551 601Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

Firs

t der

ivat

ive

(a)

000080002800048

400 440 480 520 560 600

Firs

t der

ivat

ive

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)

Figure 4 (a) First derivative spectra between the mixture solution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in the sample cell anddifferent concentrations (2ndash10120583gmLminus1) of Ponceau 4R solutions in the reference cell First derivative spectra between the mixture solution(6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and 6120583gmLminus1 Ponceau 4R in the reference cell at balance points andcomparison of these spectra with 6 120583gmLminus1 concentration of Carmoisine (- - -) spectrum (b) First derivative spectra between the mixturesolution (6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and different concentrations (2ndash10120583gmLminus1) of Carmoisinesolutions in the reference cell Derivative spectra between the mixture solution (6120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in thesample cell and 6 120583gmLminus1 Carmoisine in the reference cell at balance points and comparison of these spectra with 6120583gmLminus1 concentrationof Ponceau 4R (- - -) spectrum

minimum were calculated Table 1 shows the mean valuesof the ratios calculated for five different determinations foreach solutionThe ratios are constant and characteristic of thepure substance independent of concentration and presenceof another component For the determination of Carmoisineconcentrations in Carmoisine-Ponceau 4R binary mixturesand drink samples the sample cell was filled with themixturesolution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R)and the reference cell was filled in succession with aseries of reference Ponceau 4R with solutions in differentconcentrations Ponceau 4R as shown in Figure 4(a) Theratios of the mixture calculated from the recorded 1D spectrawere compared with those of Carmoisine At the exactbalance point the ratio of the mixture corresponds to thatof Carmoisine where the concentration of Carmoisine in themixtures in the sample cell is equal to that of the referencesolution in the reference cell For determining the othercomponent the same steps were followed using solutions ofpure compoundCarmoisine in the reference cell to determineits concentration in the mixture (Figure 4(b)) Conformitywith Beerrsquos law was evident in the concentration range from 2to 10 120583gmLminus1 of Carmoisine and Ponceau 4R In thismethodsynthetic mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R and the recoveries ofthe method were found to be in the range of 9850 plusmn 105ndash9975 plusmn 110 The presented results in Table 3 are in goodagreement with the other proposed methods

35 Application of the Developed Methods to the Soft DrinksThe developed and validated methods were successfullyapplied to the determination of Carmoisine and Ponceau 4Rcontent in the commercial soft drinks The obtained resultsare presented in Table 4 Good agreement was observed forthe assay results of the drinks by application of the fourmethods in this paper (Table 4) The values of standarddeviation indicate very good reproducibility The results ofthe proposed spectrophotometric methods were statisticallycompared with the results of the HPLC method given in

the literature [18] at the 95 confidence level with studentrsquos 119905-test and by the variance ratio 119865-test (Table 4) The calculated119905- and119865-values never exceed the theoretical 119905- and119865-values at005 level of significant difference There was no interferenceof ingredients in the products examined so no additiveextraction or separation procedures were required duringtheir assay The results of all methods were very close toeach other This suggests that the four methods are equallyapplicable

36 Validation of the Proposed Methods

361 Linearity The linearity of the proposed methods wasevaluated under different concentrations of Carmoisine andPonceau 4R within the concentration range stated in Table 2The good linearity of the calibration graphs and negligiblescatter of the experimental points is clearly evident by thevalues of the correlation coefficients and variances around theslope (Table 2)

362 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD is expressed as the analyte concentration cor-responding to the sample blank value plus three standarddeviations and LOQ is the analyte concentration correspond-ing to the sample blank value ten standard deviations [21]All the proposed methods data was presented in Table 2The LOD and LOQ values found were between 0071 and0086 and 0236 and 0286 120583gmLminus1 for Carmoisine and 0081and 0091 and 0270 and 0304 120583gmLminus1 for Ponceau 4Rrespectively The data shows that the methods are sensitivefor the determination of Carmoisine and Ponceau 4R

363 Precision (Repeatability) and Accuracy (Recovery )The precision of the developed methods was expressedas a percentage of relative standard deviation (RSD) forrepeatability (intraday precision) and intermediate precision(interday precision) The data obtained were less than 106(Table 3) indicating reasonable repeatability of the proposed

10 International Journal of Analytical Chemistry

methods The intraday and interday accuracy ranges werefrom 9895 to 1006 for Carmoisine and from 9975 to1007 for Ponceau 4R respectively The results obtainedfrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria To confirm the accuracy of the proposed methodssyntheticmixtures of different concentration ratios consistingof each food colorant were prepared The resulting mixtureswere assayed according to above proposed procedures infive replicates and the average results were calculated asthe percentage of analyte recovered The percentages meanaccuracy for Carmoisine were 1008 plusmn 087 for method DSM1010 plusmn 095 for RDM 10040 plusmn 107 for DDM and 9975 plusmn102 for CM respectively And those of P were 9910 plusmn 102for method DSM 9825 plusmn 093 for RDM 9955 plusmn 095 forDDM and 9850 plusmn 106 for CM The good recovery percent(RSD) assured the high accuracy of the proposed methods(Table 3) The relative standard deviations were found tobe less than 107 and 106 for Carmoisine and Ponceau4R respectively indicating reasonable repeatability of theproposed methods

4 Conclusion

Four derivative spectrophotometric methods for the simul-taneous analysis of two common food colorants have beensuccessfully researched developed applied and compared byreference HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination ofCarmosine and Ponceau 4R in synthetic mixtures and softdrinks without any interference from ingredients presentThe developed methods are simple (as there is no needfor pretreatment) rapid (as they require measurements of998779D1 and D1 values at a single wavelength) and direct (asthey estimate each colorant independently of the other) UVmethods offer a cost effective and time saving alternative toother methods for example colorimetric complexometricand chromatographic analyses The methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetricmethods Also themethodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities of the proposed meth-ods are almost comparable and can be used for the determina-tion of the two colorants in commercial drinks in the absenceof official method

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research has been supported by Scientific ResearchProject Coordination Center of Yildiz Technical University(Project no 2012-01-02-KAP07)

References

[1] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 1989

[2] J Vries Food Safety and Toxicity CRC London UK 1996[3] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination of

food dyes in soft drinks containing natural pigments by liquidchromatography with minimal clean-uprdquo Food Control vol 16no 3 pp 293ndash297 2005

[4] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination of carmoisine allura red and Ponceau4R in sweets and soft drinks by differential pulse polarographyrdquoJournal of Food Composition andAnalysis vol 18 no 6 pp 503ndash515 2005

[5] N Dossi R Toniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem forthe rapid in-channel amperometric detection of synthetic dyesin foodrdquo Journal of Electroanalytical Chemistry vol 601 no 1-2pp 1ndash7 2007

[6] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination of eight synthetic permitted and five commonlyencountered nonpermitted food colors in various foodmatrixesby high-performance liquid chromatographyrdquo Journal of AOACInternational vol 93 no 5 pp 1503ndash1514 2010

[7] S Dixit S K Khanna and M Das ldquoA simple method forsimultaneous determination of basic dyes encountered in foodpreparations by reversed-phase HPLCrdquo Journal of AOAC Inter-national vol 94 no 6 pp 1874ndash1881 2011

[8] N Yoshioka and K Ichihashi ldquoDetermination of 40 syntheticfood colors in drinks and candies by high-performance liquidchromatography using a short column with photodiode arraydetectionrdquo Talanta vol 74 no 5 pp 1408ndash1413 2008

[9] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination of synthetic dyes in selectedfoodstuffs by high performance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 107 no 1 pp 489ndash496 2008

[10] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination of water-soluble and fat-soluble synthetic colorantsin foodstuffbyhigh-performance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal ofChromatography A vol 1103 no 1 pp 170ndash176 2006

[11] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination of food dyes by firstderivative spectrophotometry with sorption onto polyurethanefoamrdquo Analytical Sciences vol 21 no 2 pp 149ndash153 2005

[12] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilfroush ldquoSimultaneous determination of carmoisine andPonceau 4Rrdquo Food Analytical Methods vol 1 no 3 pp 214ndash2192008

[13] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination of carmoisine andbrilliant blue FCF in food samplesrdquo Talanta vol 84 no 1 pp240ndash243 2011

[14] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan andB Asci ldquoAbilities of partial least-squares (PLS-2)multivariate calibration in the analysis of quaternary mixture offood colors (E-110 E-122 E-124 E-131)rdquo Analytical Letters vol40 no 10 pp 1893ndash1903 2007

[15] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectrofluorimetric determination of rhodamine dyein cosmeticsrdquo Talanta vol 75 no 1 pp 135ndash140 2008

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

Submit your manuscripts athttpwwwhindawicom

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CatalystsJournal of

Page 2: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

2 International Journal of Analytical Chemistry

N N

N N

OH

HO

NaO3S

NaO3S

NaO3S

NaO3S

SO3Na

Carmoisine (Azorubine E122) CI 14720 Ponceau 4R (Cochineal Red A E124) CI 16255

Scheme 1 Chemical structures common names European community numbers (E) and color index (CI) numbers of synthetic foodcolorants studied

published during the last years demonstrates the importanceof this problemand the need for developing fast accurate andselective techniques for synthetic colorants analysis [4]

Various techniques have been introduced for the deter-mination of mixtures of colorants with three or more com-ponent combination in food samples

These include capillary electrophoresis [5] differentialpulse polarography [4] high performance liquid chromatog-raphy [6ndash9] electrospray mass spectrometry [10] spec-trophotometry [11ndash13] partial least squares [14] and spec-trofluorimetry [15] Chromatographic methods have beenused for colorant analysis in foods and they are recom-mended when the mixture contains many different colorants[6 7 16 17] However there are some potential drawbackssuch as usage of toxic solvents the need for complex samplepretreatments and the resulting waste product Capillaryelectrophoresis is also very suitable when the sample containsseveral colorants but foods beverages and pharmaceuticalscontain only one two or rarely three food dyes and thesemethods need special equipment that may not be availablein certain quality laboratories All multivariate calibrationmethods including Partial Least Square (PLS) require thedata processing with powerful software as well as the manip-ulations of the abstract vector space and its application tothe regression analysis and various chemometric methodsare also often used for more complex mixtures Thus ana-lytical methods from alternative technique are always usefulespecially if the methods are simple cheap and compara-tively fast Spectrophotometry as alternative methodologyis suitable for routine laboratories especially for developingcountries and sometimes serious analytical problems canbe resolved by this common technique Among computer-controlled instruments derivative techniques (ratio deriva-tive difference derivative and compensation method) areplaying a very important role in the binary analysis ofmixtures by UV-VIS molecular absorption spectrophotom-etry All approaches are useful in the resolution of bandoverlapping in quantitative analysis

In this present work selective sensitive simple andlow-cost procedures were developed for the simultaneous

determination of Carmoisine (C) and Ponceau 4R (P) insyntheticmixture and drink samples by using derivative spec-trophotometry (DSM) ratio derivative spectrophotometry(RDM) derivative differential spectrophotometry (DDM)and compensation method (CM) These methods have beensuccessfully applied to synthetic mixtures and drink samplesand the results obtained were compared with those obtainedby HPLC method according to NMKL 130 [18]

No extraction or evaporation step no complexationagent andnoharmful chemicals are involved in the suggestedmethods in the suggestedmethods so can be used for routineanalysis of both colorants in quality control and routinelaboratories

2 Experimental

21 Apparatus All spectral measurements and treatment ofdata were carried out using a Shimadzu UV-2450 doublebeam UV-Vis spectrophotometer with two matched 1-cmquartz cells connected to a Hewlett Packard compatiblepersonal computer and a HP 1102 Laser Jet Printer BundledUV-PC personal spectroscopy software of version 221 wasused to process the absorption and the derivative spectraThe spectral band width was 1 nm with wavelength scanningspeed ofmediumHPLCmeasurements were performedwithan Agilent 1100 series HPLC-DAD system with a vacuumdegasser quaternary pump auto sampler and injector with a100 120583L loop volume Separation was achieved using a WatersSpherisorb C18 column (46 times 250mm 5120583m) and variablewavelength UV-VIS DAD detector measuring at 520 nm forCarmoisine and 512 nm for Ponceau 4R

22 Chromatographic Conditions Separation was achievedusing aWaters Spherisorb C

18column (46times 250mm 5 120583m)

The solvent system consisted of a mixture of an aqueoussolution containing 0005M tetrabutylammonium hydro-gen sulphate (TBA) and a phosphate buffer solution (pH65) methanol (50 50 vv) It was pumped isocratically at07mLminminus1 for the separation The eluents were monitoredat 512 and 520 nm for P and C respectively

International Journal of Analytical Chemistry 3

Table 1 Experimental parameters calculated for the simultaneous determination of Carmoisine and Ponceau 4R in binary mixture bycompensation method

Colorants Concentration range (120583gmLminus1) Ratio Meana RSD ()Carmoisine 2ndash10 1D472

1D569 0600 plusmn 0002 0333Ponceau 4R 2ndash10 1D470

1D554 0690 plusmn 0009 1304aMean of five separate determinations

Table 2 Statistical parameters of the simultaneous determination of Carmoisine and Ponceau 4R by DSM RDM and DDM

Methods AnalyteSelected

wavelength(nm)

Concentrationrange

(120583gmLminus1)Regression equationsa

Correlationcoefficient

(119903)

Detection limit(120583gmLminus1)

Quantificationlimit

(120583gmLminus1)

DSM C 1D331 2ndash10 156 sdot 10minus4119862+190 sdot 10

minus5 09995 0086 0286P 1D517 2ndash10 232 sdot 10

minus4119862minus320 sdot 10

minus5 09996 0091 0304

RDM C 1D323 2ndash10 413 sdot 10minus3119862minus323 sdot 10

minus4 09994 0079 0263P 1D344 2ndash10 195 sdot 10

minus3119862minus343 sdot 10

minus3 09996 0082 0273

DDM C 1D366 2ndash10 325 sdot 10minus4119862+127 sdot 10

minus4 09996 0071 0236P 1D451 2ndash10 439 sdot 10

minus4119862minus156 sdot 10

minus4 09998 0081 0270aFive separate determinations were performed and mean was calculated119862lowast the concentration of the analyte (120583gmLminus1)

23 Chemicals andMaterials Distilledwaterwas used to pre-pare the solutions and for mobile phase All chemicals usedwere of analytical reagent grade and were purchased fromMerck (Darmstadt Germany) Ponceau 4R (E124 CochinealRed A molecular weight 60447 CI 16255 CAS number 2611-82-7) and Carmoisine (E122 Azorubine molecular weight50244 CI 14720CAS number 53026-69-9) were from Sigma-Aldrich (Steinheim Germany) Soft drinks were purchasedfrom commercial markets

24 Standard Solutions and Calibration Graphs Stock solu-tions of Carmoisine and Ponceau 4R (100 120583gmLminus1) wereprepared by dissolving appropriate weights in distilled waterVarious aliquots of each stock solution within the concen-tration range stated in Table 2 were transferred into five setsof 10mL volumetric flasks and the volume was completedwith distilled water The stability of the working solutions ofCarmoisine and Ponceau 4R was studied by recording thetime-dependent absorption spectra no changes in the spectrawere observed for at least one month when the solutions arestored at room temperature in the dark

For DDMmethod accurate volume aliquots of the stocksolution were transferred into two sets of 10mL volumetricflasks and the volume was made up with 01 N HCI and 01 NNaOH to give a series of equimolar solutions containing 20ndash100 120583gmLminus1 of both Carmoisine and Ponceau 4R Calibra-tion solutions (119899 = 5) were used to construct the calibrationcurves in the standardization of the cited methods

For HPLC method stock solutions of (25mg100mL)Carmoisine and Ponceau 4R were prepared in a 1 1 vvmixture of methanolmdashTBA buffer solution (0005M TBA +0005M phosphate buffer pH 65) The standard solutions

were prepared containing 5ndash20120583gmLminus1 of Carmoisine andPonceau 4R in the same solvent system All prepared solu-tions were filtered through 045120583m membrane filter beforeinjection

Commercial drink samples due to turbid are treatmentwith the Carrez I and II solutions

Carrez solution I it contains potassium hexacyano-ferrate (II) trihydrate (K

4[Fe (CN)

6] times 3H

2O) 100mL

concentration 15 g times 100mLminus1

Carrez solution II it contains zinc sulfate heptahy-drate (ZnSO

4times 7H2O) 100mL concentration 30 g times

100mLminus1

25 Spectrophotometric Measurements

(i) Final solutions for measurements were prepared overthe concentration ranges given in Table 2

(ii) The derivative spectra were recorded over the wave-length range of 300ndash700 nm The amplitude of thederivative curve was measured at the selected wave-lengths for each method (Table 1)

(iii) For the compensation method the absorbance differ-ence spectra (sample versus reference) were recordedover the wavelength range of 300ndash700 nm usingdifferent concentrations of the reference solutionprepared from the stock solution

(iv) For the DDmethod the Carmoisine and Ponceau 4Rsolutions in 01 N HCI difference derivative spectrawere recorded against corresponding 01 N NaOHsolutions of the colorants as a blank The amplitudeof the first derivative spectra (Table 2) was measuredat the chosen wavelengths

4 International Journal of Analytical Chemistry

26 Procedures

261 Derivative Spectrophotometric Method (DSM) Ali-quots of Carmoisine working solutions equivalent to 20ndash100 120583gmLminus1 were placed into 10mL calibration flasks andthe volume was made up with distilled water The absorptionspectra were recorded versus distilled water Next the zero-order spectra were transformed into first derivative Theamplitude from the baseline to peak at 331 nm (zero-crossingpoint for Ponceau 4R) was related to the actual contents ofCarmoisine

Appropriate aliquots of aqueous working solutions ofPonceau 4R were placed into 10mL calibration flasks andthe volume was made up with distilled water The spectra ofstandard solutions in the concentration range 20ndash10120583gmLminus1were recorded and subjected to the mathematical treatment(first derivative) The values of 1D

517measured at 517 nm

(zero-crossing point for Carmoisine) were used for construc-tion of calibration graph

262 Ratio Derivative Spectrophotometric Method (RDM)Accurately aliquots of 02 04 06 08 and 10mLof colorantsstock solution were transferred into 10mL volumetric flasksand the volume wasmade up with distilled waterThe spectraof the prepared standard solutions were scanned from 300 to700 nm and stored in the computer For the determination ofCarmoisine the stored spectra of Carmoisine were dividedseparately by the spectrumof 6120583gmLminus1 Ponceau 4R to obtainthe ratio spectra The first derivative corresponding to eachratio spectrum (1DD) was recorded A calibration graphrelating the peak amplitudes (1DD

323) to the corresponding

concentrations in 120583gmLminus1 of Carmoisine was constructedIn order to determine Ponceau 4R stored spectra of Pon-

ceau 4R standard solutions were divided by a standard spec-trum of Carmoisine of 6120583gmLminus1 The first ratio derivativespectra were calculatedThe concentration of the Ponceau 4Rwas proportional to the amplitude of themaximumat 344 nm(1D344

)

263 Derivative Differential Spectrophotometric Method(DDM) The difference spectra between the acidic (01 NHCI) solution and equimolar basic (01 N NaOH) solutionof pure colorants and drink samples were recorded from3000 to 7000 nm by placing the acidic solution in thesample compartment and the basic solution in the referencecompartment A first derivative spectrum of each of thedifferential curves was subsequently recordedThe derivativeabsorbance values were measured at 366 and 451 nm forCarmoisine and Ponceau 4R respectively

264 Compensation Method (CM)

(1) Determination of Standard Ratios The first derivativespectra for each set of standard solutions were recorded usingthe appropriate blank solution The ratios of the first deriva-tive maxima and minima ( 1D

1205821

1D1205822

) where appropriate at

the specified wavelengths (1205821and 120582

2) as indicated in Table 1

were calculated

(2) Compensation Spectrophotometric Measurements Thesolution of the mixture (containing compounds Carmoisineand Ponceau 4R) was placed in the sample cell A series ofsolutions containing various concentrations (2ndash10120583gmLminus1)of Carmoisine above and below that present in the mixturesolution were prepared and placed in succession in thereference cell The first derivative spectra (1D) were recordedand the corresponding ratios (Table 1) in each instancewere calculated and followed the calculated ratio for purecolorant Ponceau 4R The exact balance point (the ratio ofthe mixture is equal to that of pure compound Ponceau 4R)was determined at which the concentration of Carmoisine insample solution is equal to that in the reference solution

By analogy the same steps were followed by using solu-tions of pure Ponceau 4R in the reference cell to determine itsconcentration in the mixture at the balance point

The same principle was also applied for the analysis ofdrink samples

(3) Graphical Method To avoid the excessive preparation ofseveral standard solutions of either compoundCarmoisine orin order to locate the exact balance point a graphical methodis recommended [19 20]

The same steps as for the compensation method werefollowed and the derivative ratio of themixture (in the samplecell) was calculated at each time and plotted against the con-centration of pure compound Carmoisine (in the referencecell) A line with slight curvature is usually obtained Theconcentration of Carmosine can easily be interpolated fromthe graph by substituting the ratio of pure Ponceau 4R Atthe point at which the ratio of the mixture is equal to thatof pure Ponceau 4R the concentration of Carmoisine in thereference cell is equal to that in themixture in the sample cellBy analogy the concentration of compound Ponceau 4R canbe obtained

265 HPLC Method (Reference Method NMKL 130) Thisreference method is described in detail in Nordic Committeeon Food Analysis [18] About 20 120583L of each sample wasinjected and the concentrations were calculated on the basisof the ratios of peak areaswith those of the standard solutionsThe chromatograms of two compounds and drink sampleswere plotted and stored in the computer Separation wascarried out in room temperature The mobile phase wasprepared daily filtered through a 045120583m membrane filterAll chromatograms were run with the use of a (50 50 vv)mixture of methanol and TBA buffer Other experimentalparameters were flow rate 07mL minminus1 retention times554min for Ponceau 4R and 2162min for Carmoisine

266 Application of the Proposed Methods for the Deter-mination of Two Colorants in Drink Sample Commercialsamples weremixed with Carrez (I) and Carrez (II) solutionsOnce precipitated the samples were centrifuged 50mL ofthe sample that is clarified by Carrez precipitation was

International Journal of Analytical Chemistry 5

0175

0375

0575

0775

300

350

400

450

500

550

600

650

700

Abso

rban

ce

Wavelength (nm)

C

Mix

P

minus0025

(a)

00002

00022

300

320

340

Firs

t der

ivat

ive

Wavelength (nm)

P

C

00002

00022

475

500

525

550

575

Firs

t der

ivat

ive

Wavelength (nm)

C

P

minus00098

minus00078

minus00058

minus00038

minus00018

minus00098

minus00078

minus00058

minus00038

minus00018

(b)

Figure 1 Zero-order spectra (a) of 10 120583gmLminus1 Carmoisine 10120583gmLminus1 Ponceau 4R and their binary mixture first derivative spectra (b) ofcolorants (Carmoisine(mdash) 2ndash10120583gmLminus1 Ponceau 4R (- -) 2ndash10120583gmLminus1)

transferred to 100mL volumetric flask and the volume wasmade up with distilled water A series of dilution wasprepared quantitatively with water from this solution toobtain standard solutions to reach the concentration ranges ofcalibration curves graphed for each of the proposed methods(DSM RDM and CM) For the DDM method the sameprocedure was used but in this case the final volume wasmade up with 01 N HCI and 01 N NaOH separately forthe determination of Carmoisine and Ponceau 4R HPLCmethod the same sample preparation procedure describedabove was followed using a mixture 1 1 vv of methanolmdashTBA buffer solution After dissolution process preparedsolutions were filtered using 045 120583m disposable membranefilters (Sartrious Minisart 045120583m) by using a syringe Thefinal solutionwas diluted to theworking concentration range

27 Method Validation The validity and suitability of theproposed methods were assessed by accuracy (reported aspercentage recovered) precision (reported as RSD) lin-earity (evaluated by regression equation) limit of detection(LOD) and limit of quantification (LOQ) [21]

LOD and LOQ were based on the standard deviation ofresponse and the slope of the corresponding curve using thefollowing equations

LOD = 3 119904119898 LOQ = 10 119904

119898 (1)

where 119904 the noise estimate is the standard deviation of thederivative amplitude of the blank and 119898 is the slope of therelated calibration graphs [22]

The linearity of the methods was established in theconcentration ranges of 2ndash10120583gmLminus1 for Carmoisine and

Ponceau 4R respectively In this study five concentrationswere analyzed in five replicates The linearity was evaluatedby the least square regression method

In order to demonstrate the validity and suitability ofthe proposed methods intra- and interday variability studieswere performed at three different concentrations (2 6 and10 120583gmLminus1) in the presence of the certain concentration (26 and 10 120583gmLminus1) of the other colorants three times in a dayand also three different days Method efficiency was tested interms of RSDrsquos for both intraday and interday precisions

The precision was ascertained by carrying out five repli-cate determinations of synthetic mixtures at different con-centration ratios of Carmoisine and Ponceau 4R Recoverytest also confirmed the accuracy and applicability of theproposed methods by analyzing several synthetic mixturesof Carmoisine and Ponceau 4R which reproduced differentcomposition ratios within the linearity rangeThese ratios arealso the ratios in drinksThemean percentage recoveries andRSD values were calculated

Recovery of the colorants of interest from a given matrixcan be used as a measure of the accuracy or the bias of themethod The same ranges of concentrations as employed inthe linearity studies were used

In our study the applicability of the derivative spec-trophotometric methods was also validated by comparisonwith the HPLC technique

3 Result and Discussion

Figure 1(a) shows the absorption spectra of CarmoisinePonceau 4R and themixture of them in the wavelength rangeof 300ndash700 nm Zero-order absorption spectra of Carmoisine

6 International Journal of Analytical Chemistry

Table 3 Method validation for the simultaneous determination of Carmoisine and Ponceau 4R in laboratory prepared mixtures by theproposed methods

Method Accuracy (meanlowastplusmn RSD ) Precision repeatabilitya(mean plusmn RSD )

Intermediate precisionb

(mean plusmn RSD )

DSM C (1D331) 1008 plusmn 087 1001 plusmn 096 9991 plusmn 102

P (1D517) 991 plusmn 102 1005 plusmn 105 1001 plusmn 092

RDM C (1D323) 1010 plusmn 095 9998 plusmn 099 1006 plusmn 105

P (1D344) 9825 plusmn 093 9983 plusmn 098 1004 plusmn 096

DDM C (1D366) 1004 plusmn 107 9895 plusmn 105 9992 plusmn 098

P (1D451) 9955 plusmn 095 1005 plusmn 095 1007 plusmn 096

CM C (1D4721D569) 9975 plusmn 102 998 plusmn 101 1001 plusmn 097

P (1D4701D554) 985 plusmn 106 9975 plusmn 103 1003 plusmn 106

aThe intraday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times within the daybThe interday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times in three successive dayslowastThe values of recovery are an average of five replicates of each of five synthetic mixtures at different concentration ratios of C and P (2ndash10 120583gmLminus1)RSD relative standard deviation

and Ponceau 4R in distilled water gave rise to maximumpeaks at 517 nm and 508 nm respectively As can be seenthe spectra of the colorants were strongly overlapped in the400ndash600 nm wavelength region Hence the direct absorp-tion measurement for assaying binary mixture seems to beimpossible On the other hand the absorption spectra of thetwo components within a wavelength range of 300ndash380 nmwere less overlapped than the region 400ndash600 nm So in thiswork the region of 300ndash380 nm was chosen for the analysisof Carmoisine In this spectral range DSM RDM and DDMwere used for the determination of the studied colorants inlaboratory prepared mixtures and soft drink samples Thesemethods are also applied to analyze Ponceau 4R in the region320ndash520 nm

31 Derivative Spectrophotometric Method (DSM) Undercomputer-controlled instrumentation derivative spectro-photometry played a very important role in the resolutionof band overlapping in quantitative analysis [23] Figure 1(b)shows the first derivative spectra of Carmoisine and Ponceau4R As can be seen in Figure 1(b) in the region of 300ndash520 nm the first derivative spectra allowed the simultaneousdetermination of Carmoisine and Ponceau 4R At this regionthe 1D spectra (Figure 1(b)) show a characteristic peak at331 nm for Carmoisine while Ponceau 4R shows no responseTherefore the absolute values of the 1D peak amplitudes at331 nm can be used to quantify Carmoisine in this mixtureOn the other hand Ponceau 4R can be assayed in the presenceof Carmoisine by measuring its 1D response at 517 nm (zero-crossing point of Carmoisine)

Under the experimental conditions described the cal-ibration graphs obtained by plotting the derivative valuesversus concentrations for Carmoisine and Ponceau 4R inthe concentration range stated in Table 2 exhibited linearrelationships Statistical analysis of the data showed thatlinearity of the calibration graphs and compliance withBeerrsquos law were validated and the small values of the inter-cepts as illustrated by the excellent values of correlation

coefficients of the regression equations To prove the valid-ity and applicability of the proposed method laboratory-prepared mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R (Table 3) The obtainedresults for the analysis of these mixtures in five replicates bythe described method showed high accuracy and precisionof the proposed method (Table 3)

32 Ratio Derivative Method (RDM) While the main dis-advantages of the zero-crossing method in derivative spec-trophotometry for resolving a mixture of components withoverlapped spectra are the risk of small drifts of the workingwavelengths and the circumstance that the working wave-lengths generally do not fall in correspondence of peaks ofthe derivative spectrum this may be particularly dangerouswhen the slope of the spectrum is very high with consequentloss of accuracy and precision and the working wavelengthis in proximity of the base of the spectrum which causespoor sensitivity The main advantage of the RDM is thechance of doing easy measurements in correspondence ofpeaks so it permits the use of the wavelength of highestvalue of analytical signals (a maximum or a minimum) andmoreover the presence of a lot of maxima and minima isanother advantage by the fact that these wavelengths give anopportunity for the determination of active compounds in thepresence of other compounds and ingredients which possiblyinterfere in the assay [24ndash27]

This method is based on the use of the first derivativeof the ratio spectra The ratio spectra were obtained bydividing the absorption spectrum of the mixture by thatof one of the components As we depicted above directabsorption measurements are not possible due to spectraloverlap This spectral overlapping was sufficiently enough todemonstrate the resolving power of the proposed methodFigure 2(a) shows the first ratio derivative spectra of differ-ent Carmoisine standard solutions (spectra divided by thespectrum of 6 120583gmLminus1 Ponceau 4R solution) The ratio firstderivative amplitudes at 323 nm (1DD

323) corresponding to

International Journal of Analytical Chemistry 7

001

003

308 328 348 368

Firs

t der

ivat

ive

Carm

oisin

ePo

ncea

u 4R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

Firs

t der

ivat

ive

Ponc

eau

4RC

arm

oisin

e

Wavelength (nm)

fg

hi

j

minus002678

minus001678

minus000678

(b)

Figure 2 First derivative ratio spectra of Carmoisine (a) and Ponceau 4R (b) for different concentrations (Carmoisine (6 120583gmLminus1 Ponceau4R was used as a divisor) a 2 120583gmLminus1 b 4 120583gmLminus1 c 6120583gmLminus1 d 8 120583gmLminus1 e 10120583gmLminus1 Ponceau 4R (6120583gmLminus1 Carmoisine was usedas a divisor) f 2120583gmLminus1 g 4 120583gmLminus1 h 6 120583gmLminus1 i 8120583gmLminus1 j 10120583gmLminus1)

Table 4 Assay results for the determination of Carmoisine and Ponceau 4R in soft drinks using the proposed methods and the referenceHPLC method (NMKL 130)

Methods Analyte Selected wavelengths(nm)

Assay results mean plusmn SDlowast (119905calculated 119865calculated)lowastlowast

Soft drink I (120583g 100mLminus1) Soft drink II (120583g 100mLminus1)

DSM C 1D331 930 plusmn 022 (068 145) 628 plusmn 029 (061 144)

P 1D517 220 plusmn 011 (118 118) 139 plusmn 013 (040 179)

RDM C 1D323 926 plusmn 023 (037 145) 636 plusmn 021 (128 120)

P 1D344 235 plusmn 011 (044 125) 143 plusmn 013 (013 169)

DDM C 1D366 931 plusmn 024 (073 169) 641 plusmn 024 (153 164)

P 1D451 249 plusmn 017 (189 302) 144 plusmn 016 (023 268)

CM C 1D4721D569 930 plusmn 026 (063 188) 636 plusmn 028 (107 211)

P 1D4701D554 230 plusmn 016 (023 256) 143 plusmn 019 (010 368)

HPLC C 520 921 plusmn 018 620 plusmn 019

P 512 232 plusmn 010 142 plusmn 010

lowastResults obtained are the average of five experiments for each method SD standard deviationlowastlowastThe corresponding theoretical value for 119905 and 119865 at 119875 005 (119905theoretical 231 119865theoretical 639)

a maximum wavelength are proportional to the Carmoisineconcentration (Figure 2(a)) To determine the other com-ponent Ponceau 4R an analogues procedure was followedFor determination of Ponceau 4R the stored spectra ofthe mixtures are divided by a standard spectrum of 6mgLminus1 Carmoisine (Figure 2(b)) Standard solutions of Ponceau4R and the content of Ponceau 4R were determined bymeasuring the signals at 344 nm (1DD

344) Figure 2(b)

Under the described experimental conditions the cal-ibration graphs obtained by plotting the derivative valuesof each colorant versus concentration in the concentration

range stated in Table 2 showed linear relationships Con-formity with Beerrsquos law was evident in the concentrationrange of the final dilution cited in Table 2 For this methodthe characteristic parameters of regression equations andcorrelation coefficients are given in Table 2 The correlationcoefficients were 09996 and 09994 indicating good linearityLOD and LOQ were calculated for each colorant and areshown in the same table The detection limits were foundto be 0079120583gmLminus1 for Carmoisine and 0082 120583gmLminus1 forPonceau 4R while the quantification limits were estimatedto be 0263 120583gmLminus1 for Carmoisine and 0273120583gmLminus1 for

8 International Journal of Analytical Chemistry

000988

008988

016988

024988

032988

300 400 500 600 700

Abso

rban

ce

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

Diff

eren

ce fi

rst d

eriv

ativ

e

Wavelength (nm)

C

P

minus00068

minus00028

(b)

Figure 3 Difference absorption spectra of 10 120583gmLminus1 Carmoisine 10 120583gmLminus1 Ponceau 4R and their binary mixture (a) Difference firstderivative spectra (b) of colorants (Carmoisine 2ndash10120583gmLminus1 Ponceau 4R 2ndash10 120583gmLminus1)

Ponceau 4R The proposed method was also applied to thedrink samples (Table 4) The accuracy of RDM was checkedby analyzing the laboratory preparedmixtures of Carmoisineand Ponceau 4R at various concentration ratios ranging from2 to 10 120583gmLminus1 (Table 3) All data represent the average offive determinations Low values of relative standard deviationindicate very good precision Satisfactory recoveries withsmall standard deviations were obtained which indicated thehigh repeatability and accuracy of the method

33 Derivative DifferentialMethod (DDM) Differential spec-trophotometry (ΔD

1) based on pH changes has also been

reported to be useful in the determination of binary mix-tures This method that depends on utilization of differenceabsorption spectra corresponding to the same compoundobtained at two different pHrsquos has been investigated for theanalysis of binary mixtures The procedure is comprised ofthe measurement of ΔD

1food colorantsrsquo in acidic solutions

against their alkaline solutions as blanks The ΔD1has

been successfully used to eliminate interferences from drinksamples There are few reports on utilization of the abovetwo combined techniques (difference and derivative) for theestimation of individual drug substances and for combinedpreparations [28ndash30]

The difference absorption spectra of Carmoisine Pon-ceau 4R and their binary mixture in 01 N NaOH and in01 N HCI are shown in Figure 3(a) Figure 3(b) shows thefirst derivative difference spectra of colorants in the concen-tration range 2ndash10 120583gmLminus1 The spectra of both colorantsin Figure 3(b) offer an advantage for their simultaneousdetermination by having zero-crossing points In particularthe first derivative amplitudes at 366 nm for Carmoisine and

451 nm for Ponceau 4R in the Carmoisine and Ponceau 4Rmixture were considered as the optimum working wave-lengths for their determination By measuring the values ofthe ΔD

1amplitudes at these wavelengths the concentration

of each colorant can be directly calculated since the dif-ferential first derivative measurement cancels the irrelevantabsorbance due to the drinks matrix at these wavelengths

Under the experimental conditions described the cali-bration graphs obtained by plotting the derivative values ofeach colorant in the mixture versus concentration in therange concentration stated in Table 2 show linear relation-ships The correlation coefficients were 09996 and 09998indicating good linearity The LOD were 0071 120583gmLminus1 forCarmoisine and 0081 120583gmLminus1 for Ponceau 4R while theLOQ were 0236 120583gmLminus1 for Carmoisine and 0270 120583gmLminus1for Ponceau 4R The proposed procedure was success-fully applied for the determination of these colorants inlaboratory-prepared mixtures and drink samples The resultsare presented in Tables 3 and 4The values of relative standarddeviation indicate very good reproducibility In additionapplication of the DDMwas found to be correct for the drinkmatrix interference and to enhance the sensitivity

34 CompensationMethod (CM) Thecompensationmethodis a nonmathematical method for the detection and elimi-nation of unwanted absorption during spectrophotometricanalysis [19 20 31 32] The accuracy of the method dependson the evaluation of the balance point Figure 1(b) showsthe first derivative spectra of Carmoisine and Ponceau 4Rin the concentration range 2ndash10 120583gmLminus1 The first derivativespectra were recorded for each reference solution of thecomponents and the ratios of the 1D maximum and 1D

International Journal of Analytical Chemistry 9

000011000211000411000611

401 451 501 551 601Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

Firs

t der

ivat

ive

(a)

000080002800048

400 440 480 520 560 600

Firs

t der

ivat

ive

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)

Figure 4 (a) First derivative spectra between the mixture solution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in the sample cell anddifferent concentrations (2ndash10120583gmLminus1) of Ponceau 4R solutions in the reference cell First derivative spectra between the mixture solution(6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and 6120583gmLminus1 Ponceau 4R in the reference cell at balance points andcomparison of these spectra with 6 120583gmLminus1 concentration of Carmoisine (- - -) spectrum (b) First derivative spectra between the mixturesolution (6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and different concentrations (2ndash10120583gmLminus1) of Carmoisinesolutions in the reference cell Derivative spectra between the mixture solution (6120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in thesample cell and 6 120583gmLminus1 Carmoisine in the reference cell at balance points and comparison of these spectra with 6120583gmLminus1 concentrationof Ponceau 4R (- - -) spectrum

minimum were calculated Table 1 shows the mean valuesof the ratios calculated for five different determinations foreach solutionThe ratios are constant and characteristic of thepure substance independent of concentration and presenceof another component For the determination of Carmoisineconcentrations in Carmoisine-Ponceau 4R binary mixturesand drink samples the sample cell was filled with themixturesolution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R)and the reference cell was filled in succession with aseries of reference Ponceau 4R with solutions in differentconcentrations Ponceau 4R as shown in Figure 4(a) Theratios of the mixture calculated from the recorded 1D spectrawere compared with those of Carmoisine At the exactbalance point the ratio of the mixture corresponds to thatof Carmoisine where the concentration of Carmoisine in themixtures in the sample cell is equal to that of the referencesolution in the reference cell For determining the othercomponent the same steps were followed using solutions ofpure compoundCarmoisine in the reference cell to determineits concentration in the mixture (Figure 4(b)) Conformitywith Beerrsquos law was evident in the concentration range from 2to 10 120583gmLminus1 of Carmoisine and Ponceau 4R In thismethodsynthetic mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R and the recoveries ofthe method were found to be in the range of 9850 plusmn 105ndash9975 plusmn 110 The presented results in Table 3 are in goodagreement with the other proposed methods

35 Application of the Developed Methods to the Soft DrinksThe developed and validated methods were successfullyapplied to the determination of Carmoisine and Ponceau 4Rcontent in the commercial soft drinks The obtained resultsare presented in Table 4 Good agreement was observed forthe assay results of the drinks by application of the fourmethods in this paper (Table 4) The values of standarddeviation indicate very good reproducibility The results ofthe proposed spectrophotometric methods were statisticallycompared with the results of the HPLC method given in

the literature [18] at the 95 confidence level with studentrsquos 119905-test and by the variance ratio 119865-test (Table 4) The calculated119905- and119865-values never exceed the theoretical 119905- and119865-values at005 level of significant difference There was no interferenceof ingredients in the products examined so no additiveextraction or separation procedures were required duringtheir assay The results of all methods were very close toeach other This suggests that the four methods are equallyapplicable

36 Validation of the Proposed Methods

361 Linearity The linearity of the proposed methods wasevaluated under different concentrations of Carmoisine andPonceau 4R within the concentration range stated in Table 2The good linearity of the calibration graphs and negligiblescatter of the experimental points is clearly evident by thevalues of the correlation coefficients and variances around theslope (Table 2)

362 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD is expressed as the analyte concentration cor-responding to the sample blank value plus three standarddeviations and LOQ is the analyte concentration correspond-ing to the sample blank value ten standard deviations [21]All the proposed methods data was presented in Table 2The LOD and LOQ values found were between 0071 and0086 and 0236 and 0286 120583gmLminus1 for Carmoisine and 0081and 0091 and 0270 and 0304 120583gmLminus1 for Ponceau 4Rrespectively The data shows that the methods are sensitivefor the determination of Carmoisine and Ponceau 4R

363 Precision (Repeatability) and Accuracy (Recovery )The precision of the developed methods was expressedas a percentage of relative standard deviation (RSD) forrepeatability (intraday precision) and intermediate precision(interday precision) The data obtained were less than 106(Table 3) indicating reasonable repeatability of the proposed

10 International Journal of Analytical Chemistry

methods The intraday and interday accuracy ranges werefrom 9895 to 1006 for Carmoisine and from 9975 to1007 for Ponceau 4R respectively The results obtainedfrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria To confirm the accuracy of the proposed methodssyntheticmixtures of different concentration ratios consistingof each food colorant were prepared The resulting mixtureswere assayed according to above proposed procedures infive replicates and the average results were calculated asthe percentage of analyte recovered The percentages meanaccuracy for Carmoisine were 1008 plusmn 087 for method DSM1010 plusmn 095 for RDM 10040 plusmn 107 for DDM and 9975 plusmn102 for CM respectively And those of P were 9910 plusmn 102for method DSM 9825 plusmn 093 for RDM 9955 plusmn 095 forDDM and 9850 plusmn 106 for CM The good recovery percent(RSD) assured the high accuracy of the proposed methods(Table 3) The relative standard deviations were found tobe less than 107 and 106 for Carmoisine and Ponceau4R respectively indicating reasonable repeatability of theproposed methods

4 Conclusion

Four derivative spectrophotometric methods for the simul-taneous analysis of two common food colorants have beensuccessfully researched developed applied and compared byreference HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination ofCarmosine and Ponceau 4R in synthetic mixtures and softdrinks without any interference from ingredients presentThe developed methods are simple (as there is no needfor pretreatment) rapid (as they require measurements of998779D1 and D1 values at a single wavelength) and direct (asthey estimate each colorant independently of the other) UVmethods offer a cost effective and time saving alternative toother methods for example colorimetric complexometricand chromatographic analyses The methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetricmethods Also themethodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities of the proposed meth-ods are almost comparable and can be used for the determina-tion of the two colorants in commercial drinks in the absenceof official method

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research has been supported by Scientific ResearchProject Coordination Center of Yildiz Technical University(Project no 2012-01-02-KAP07)

References

[1] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 1989

[2] J Vries Food Safety and Toxicity CRC London UK 1996[3] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination of

food dyes in soft drinks containing natural pigments by liquidchromatography with minimal clean-uprdquo Food Control vol 16no 3 pp 293ndash297 2005

[4] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination of carmoisine allura red and Ponceau4R in sweets and soft drinks by differential pulse polarographyrdquoJournal of Food Composition andAnalysis vol 18 no 6 pp 503ndash515 2005

[5] N Dossi R Toniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem forthe rapid in-channel amperometric detection of synthetic dyesin foodrdquo Journal of Electroanalytical Chemistry vol 601 no 1-2pp 1ndash7 2007

[6] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination of eight synthetic permitted and five commonlyencountered nonpermitted food colors in various foodmatrixesby high-performance liquid chromatographyrdquo Journal of AOACInternational vol 93 no 5 pp 1503ndash1514 2010

[7] S Dixit S K Khanna and M Das ldquoA simple method forsimultaneous determination of basic dyes encountered in foodpreparations by reversed-phase HPLCrdquo Journal of AOAC Inter-national vol 94 no 6 pp 1874ndash1881 2011

[8] N Yoshioka and K Ichihashi ldquoDetermination of 40 syntheticfood colors in drinks and candies by high-performance liquidchromatography using a short column with photodiode arraydetectionrdquo Talanta vol 74 no 5 pp 1408ndash1413 2008

[9] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination of synthetic dyes in selectedfoodstuffs by high performance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 107 no 1 pp 489ndash496 2008

[10] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination of water-soluble and fat-soluble synthetic colorantsin foodstuffbyhigh-performance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal ofChromatography A vol 1103 no 1 pp 170ndash176 2006

[11] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination of food dyes by firstderivative spectrophotometry with sorption onto polyurethanefoamrdquo Analytical Sciences vol 21 no 2 pp 149ndash153 2005

[12] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilfroush ldquoSimultaneous determination of carmoisine andPonceau 4Rrdquo Food Analytical Methods vol 1 no 3 pp 214ndash2192008

[13] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination of carmoisine andbrilliant blue FCF in food samplesrdquo Talanta vol 84 no 1 pp240ndash243 2011

[14] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan andB Asci ldquoAbilities of partial least-squares (PLS-2)multivariate calibration in the analysis of quaternary mixture offood colors (E-110 E-122 E-124 E-131)rdquo Analytical Letters vol40 no 10 pp 1893ndash1903 2007

[15] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectrofluorimetric determination of rhodamine dyein cosmeticsrdquo Talanta vol 75 no 1 pp 135ndash140 2008

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

Submit your manuscripts athttpwwwhindawicom

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Page 3: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

International Journal of Analytical Chemistry 3

Table 1 Experimental parameters calculated for the simultaneous determination of Carmoisine and Ponceau 4R in binary mixture bycompensation method

Colorants Concentration range (120583gmLminus1) Ratio Meana RSD ()Carmoisine 2ndash10 1D472

1D569 0600 plusmn 0002 0333Ponceau 4R 2ndash10 1D470

1D554 0690 plusmn 0009 1304aMean of five separate determinations

Table 2 Statistical parameters of the simultaneous determination of Carmoisine and Ponceau 4R by DSM RDM and DDM

Methods AnalyteSelected

wavelength(nm)

Concentrationrange

(120583gmLminus1)Regression equationsa

Correlationcoefficient

(119903)

Detection limit(120583gmLminus1)

Quantificationlimit

(120583gmLminus1)

DSM C 1D331 2ndash10 156 sdot 10minus4119862+190 sdot 10

minus5 09995 0086 0286P 1D517 2ndash10 232 sdot 10

minus4119862minus320 sdot 10

minus5 09996 0091 0304

RDM C 1D323 2ndash10 413 sdot 10minus3119862minus323 sdot 10

minus4 09994 0079 0263P 1D344 2ndash10 195 sdot 10

minus3119862minus343 sdot 10

minus3 09996 0082 0273

DDM C 1D366 2ndash10 325 sdot 10minus4119862+127 sdot 10

minus4 09996 0071 0236P 1D451 2ndash10 439 sdot 10

minus4119862minus156 sdot 10

minus4 09998 0081 0270aFive separate determinations were performed and mean was calculated119862lowast the concentration of the analyte (120583gmLminus1)

23 Chemicals andMaterials Distilledwaterwas used to pre-pare the solutions and for mobile phase All chemicals usedwere of analytical reagent grade and were purchased fromMerck (Darmstadt Germany) Ponceau 4R (E124 CochinealRed A molecular weight 60447 CI 16255 CAS number 2611-82-7) and Carmoisine (E122 Azorubine molecular weight50244 CI 14720CAS number 53026-69-9) were from Sigma-Aldrich (Steinheim Germany) Soft drinks were purchasedfrom commercial markets

24 Standard Solutions and Calibration Graphs Stock solu-tions of Carmoisine and Ponceau 4R (100 120583gmLminus1) wereprepared by dissolving appropriate weights in distilled waterVarious aliquots of each stock solution within the concen-tration range stated in Table 2 were transferred into five setsof 10mL volumetric flasks and the volume was completedwith distilled water The stability of the working solutions ofCarmoisine and Ponceau 4R was studied by recording thetime-dependent absorption spectra no changes in the spectrawere observed for at least one month when the solutions arestored at room temperature in the dark

For DDMmethod accurate volume aliquots of the stocksolution were transferred into two sets of 10mL volumetricflasks and the volume was made up with 01 N HCI and 01 NNaOH to give a series of equimolar solutions containing 20ndash100 120583gmLminus1 of both Carmoisine and Ponceau 4R Calibra-tion solutions (119899 = 5) were used to construct the calibrationcurves in the standardization of the cited methods

For HPLC method stock solutions of (25mg100mL)Carmoisine and Ponceau 4R were prepared in a 1 1 vvmixture of methanolmdashTBA buffer solution (0005M TBA +0005M phosphate buffer pH 65) The standard solutions

were prepared containing 5ndash20120583gmLminus1 of Carmoisine andPonceau 4R in the same solvent system All prepared solu-tions were filtered through 045120583m membrane filter beforeinjection

Commercial drink samples due to turbid are treatmentwith the Carrez I and II solutions

Carrez solution I it contains potassium hexacyano-ferrate (II) trihydrate (K

4[Fe (CN)

6] times 3H

2O) 100mL

concentration 15 g times 100mLminus1

Carrez solution II it contains zinc sulfate heptahy-drate (ZnSO

4times 7H2O) 100mL concentration 30 g times

100mLminus1

25 Spectrophotometric Measurements

(i) Final solutions for measurements were prepared overthe concentration ranges given in Table 2

(ii) The derivative spectra were recorded over the wave-length range of 300ndash700 nm The amplitude of thederivative curve was measured at the selected wave-lengths for each method (Table 1)

(iii) For the compensation method the absorbance differ-ence spectra (sample versus reference) were recordedover the wavelength range of 300ndash700 nm usingdifferent concentrations of the reference solutionprepared from the stock solution

(iv) For the DDmethod the Carmoisine and Ponceau 4Rsolutions in 01 N HCI difference derivative spectrawere recorded against corresponding 01 N NaOHsolutions of the colorants as a blank The amplitudeof the first derivative spectra (Table 2) was measuredat the chosen wavelengths

4 International Journal of Analytical Chemistry

26 Procedures

261 Derivative Spectrophotometric Method (DSM) Ali-quots of Carmoisine working solutions equivalent to 20ndash100 120583gmLminus1 were placed into 10mL calibration flasks andthe volume was made up with distilled water The absorptionspectra were recorded versus distilled water Next the zero-order spectra were transformed into first derivative Theamplitude from the baseline to peak at 331 nm (zero-crossingpoint for Ponceau 4R) was related to the actual contents ofCarmoisine

Appropriate aliquots of aqueous working solutions ofPonceau 4R were placed into 10mL calibration flasks andthe volume was made up with distilled water The spectra ofstandard solutions in the concentration range 20ndash10120583gmLminus1were recorded and subjected to the mathematical treatment(first derivative) The values of 1D

517measured at 517 nm

(zero-crossing point for Carmoisine) were used for construc-tion of calibration graph

262 Ratio Derivative Spectrophotometric Method (RDM)Accurately aliquots of 02 04 06 08 and 10mLof colorantsstock solution were transferred into 10mL volumetric flasksand the volume wasmade up with distilled waterThe spectraof the prepared standard solutions were scanned from 300 to700 nm and stored in the computer For the determination ofCarmoisine the stored spectra of Carmoisine were dividedseparately by the spectrumof 6120583gmLminus1 Ponceau 4R to obtainthe ratio spectra The first derivative corresponding to eachratio spectrum (1DD) was recorded A calibration graphrelating the peak amplitudes (1DD

323) to the corresponding

concentrations in 120583gmLminus1 of Carmoisine was constructedIn order to determine Ponceau 4R stored spectra of Pon-

ceau 4R standard solutions were divided by a standard spec-trum of Carmoisine of 6120583gmLminus1 The first ratio derivativespectra were calculatedThe concentration of the Ponceau 4Rwas proportional to the amplitude of themaximumat 344 nm(1D344

)

263 Derivative Differential Spectrophotometric Method(DDM) The difference spectra between the acidic (01 NHCI) solution and equimolar basic (01 N NaOH) solutionof pure colorants and drink samples were recorded from3000 to 7000 nm by placing the acidic solution in thesample compartment and the basic solution in the referencecompartment A first derivative spectrum of each of thedifferential curves was subsequently recordedThe derivativeabsorbance values were measured at 366 and 451 nm forCarmoisine and Ponceau 4R respectively

264 Compensation Method (CM)

(1) Determination of Standard Ratios The first derivativespectra for each set of standard solutions were recorded usingthe appropriate blank solution The ratios of the first deriva-tive maxima and minima ( 1D

1205821

1D1205822

) where appropriate at

the specified wavelengths (1205821and 120582

2) as indicated in Table 1

were calculated

(2) Compensation Spectrophotometric Measurements Thesolution of the mixture (containing compounds Carmoisineand Ponceau 4R) was placed in the sample cell A series ofsolutions containing various concentrations (2ndash10120583gmLminus1)of Carmoisine above and below that present in the mixturesolution were prepared and placed in succession in thereference cell The first derivative spectra (1D) were recordedand the corresponding ratios (Table 1) in each instancewere calculated and followed the calculated ratio for purecolorant Ponceau 4R The exact balance point (the ratio ofthe mixture is equal to that of pure compound Ponceau 4R)was determined at which the concentration of Carmoisine insample solution is equal to that in the reference solution

By analogy the same steps were followed by using solu-tions of pure Ponceau 4R in the reference cell to determine itsconcentration in the mixture at the balance point

The same principle was also applied for the analysis ofdrink samples

(3) Graphical Method To avoid the excessive preparation ofseveral standard solutions of either compoundCarmoisine orin order to locate the exact balance point a graphical methodis recommended [19 20]

The same steps as for the compensation method werefollowed and the derivative ratio of themixture (in the samplecell) was calculated at each time and plotted against the con-centration of pure compound Carmoisine (in the referencecell) A line with slight curvature is usually obtained Theconcentration of Carmosine can easily be interpolated fromthe graph by substituting the ratio of pure Ponceau 4R Atthe point at which the ratio of the mixture is equal to thatof pure Ponceau 4R the concentration of Carmoisine in thereference cell is equal to that in themixture in the sample cellBy analogy the concentration of compound Ponceau 4R canbe obtained

265 HPLC Method (Reference Method NMKL 130) Thisreference method is described in detail in Nordic Committeeon Food Analysis [18] About 20 120583L of each sample wasinjected and the concentrations were calculated on the basisof the ratios of peak areaswith those of the standard solutionsThe chromatograms of two compounds and drink sampleswere plotted and stored in the computer Separation wascarried out in room temperature The mobile phase wasprepared daily filtered through a 045120583m membrane filterAll chromatograms were run with the use of a (50 50 vv)mixture of methanol and TBA buffer Other experimentalparameters were flow rate 07mL minminus1 retention times554min for Ponceau 4R and 2162min for Carmoisine

266 Application of the Proposed Methods for the Deter-mination of Two Colorants in Drink Sample Commercialsamples weremixed with Carrez (I) and Carrez (II) solutionsOnce precipitated the samples were centrifuged 50mL ofthe sample that is clarified by Carrez precipitation was

International Journal of Analytical Chemistry 5

0175

0375

0575

0775

300

350

400

450

500

550

600

650

700

Abso

rban

ce

Wavelength (nm)

C

Mix

P

minus0025

(a)

00002

00022

300

320

340

Firs

t der

ivat

ive

Wavelength (nm)

P

C

00002

00022

475

500

525

550

575

Firs

t der

ivat

ive

Wavelength (nm)

C

P

minus00098

minus00078

minus00058

minus00038

minus00018

minus00098

minus00078

minus00058

minus00038

minus00018

(b)

Figure 1 Zero-order spectra (a) of 10 120583gmLminus1 Carmoisine 10120583gmLminus1 Ponceau 4R and their binary mixture first derivative spectra (b) ofcolorants (Carmoisine(mdash) 2ndash10120583gmLminus1 Ponceau 4R (- -) 2ndash10120583gmLminus1)

transferred to 100mL volumetric flask and the volume wasmade up with distilled water A series of dilution wasprepared quantitatively with water from this solution toobtain standard solutions to reach the concentration ranges ofcalibration curves graphed for each of the proposed methods(DSM RDM and CM) For the DDM method the sameprocedure was used but in this case the final volume wasmade up with 01 N HCI and 01 N NaOH separately forthe determination of Carmoisine and Ponceau 4R HPLCmethod the same sample preparation procedure describedabove was followed using a mixture 1 1 vv of methanolmdashTBA buffer solution After dissolution process preparedsolutions were filtered using 045 120583m disposable membranefilters (Sartrious Minisart 045120583m) by using a syringe Thefinal solutionwas diluted to theworking concentration range

27 Method Validation The validity and suitability of theproposed methods were assessed by accuracy (reported aspercentage recovered) precision (reported as RSD) lin-earity (evaluated by regression equation) limit of detection(LOD) and limit of quantification (LOQ) [21]

LOD and LOQ were based on the standard deviation ofresponse and the slope of the corresponding curve using thefollowing equations

LOD = 3 119904119898 LOQ = 10 119904

119898 (1)

where 119904 the noise estimate is the standard deviation of thederivative amplitude of the blank and 119898 is the slope of therelated calibration graphs [22]

The linearity of the methods was established in theconcentration ranges of 2ndash10120583gmLminus1 for Carmoisine and

Ponceau 4R respectively In this study five concentrationswere analyzed in five replicates The linearity was evaluatedby the least square regression method

In order to demonstrate the validity and suitability ofthe proposed methods intra- and interday variability studieswere performed at three different concentrations (2 6 and10 120583gmLminus1) in the presence of the certain concentration (26 and 10 120583gmLminus1) of the other colorants three times in a dayand also three different days Method efficiency was tested interms of RSDrsquos for both intraday and interday precisions

The precision was ascertained by carrying out five repli-cate determinations of synthetic mixtures at different con-centration ratios of Carmoisine and Ponceau 4R Recoverytest also confirmed the accuracy and applicability of theproposed methods by analyzing several synthetic mixturesof Carmoisine and Ponceau 4R which reproduced differentcomposition ratios within the linearity rangeThese ratios arealso the ratios in drinksThemean percentage recoveries andRSD values were calculated

Recovery of the colorants of interest from a given matrixcan be used as a measure of the accuracy or the bias of themethod The same ranges of concentrations as employed inthe linearity studies were used

In our study the applicability of the derivative spec-trophotometric methods was also validated by comparisonwith the HPLC technique

3 Result and Discussion

Figure 1(a) shows the absorption spectra of CarmoisinePonceau 4R and themixture of them in the wavelength rangeof 300ndash700 nm Zero-order absorption spectra of Carmoisine

6 International Journal of Analytical Chemistry

Table 3 Method validation for the simultaneous determination of Carmoisine and Ponceau 4R in laboratory prepared mixtures by theproposed methods

Method Accuracy (meanlowastplusmn RSD ) Precision repeatabilitya(mean plusmn RSD )

Intermediate precisionb

(mean plusmn RSD )

DSM C (1D331) 1008 plusmn 087 1001 plusmn 096 9991 plusmn 102

P (1D517) 991 plusmn 102 1005 plusmn 105 1001 plusmn 092

RDM C (1D323) 1010 plusmn 095 9998 plusmn 099 1006 plusmn 105

P (1D344) 9825 plusmn 093 9983 plusmn 098 1004 plusmn 096

DDM C (1D366) 1004 plusmn 107 9895 plusmn 105 9992 plusmn 098

P (1D451) 9955 plusmn 095 1005 plusmn 095 1007 plusmn 096

CM C (1D4721D569) 9975 plusmn 102 998 plusmn 101 1001 plusmn 097

P (1D4701D554) 985 plusmn 106 9975 plusmn 103 1003 plusmn 106

aThe intraday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times within the daybThe interday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times in three successive dayslowastThe values of recovery are an average of five replicates of each of five synthetic mixtures at different concentration ratios of C and P (2ndash10 120583gmLminus1)RSD relative standard deviation

and Ponceau 4R in distilled water gave rise to maximumpeaks at 517 nm and 508 nm respectively As can be seenthe spectra of the colorants were strongly overlapped in the400ndash600 nm wavelength region Hence the direct absorp-tion measurement for assaying binary mixture seems to beimpossible On the other hand the absorption spectra of thetwo components within a wavelength range of 300ndash380 nmwere less overlapped than the region 400ndash600 nm So in thiswork the region of 300ndash380 nm was chosen for the analysisof Carmoisine In this spectral range DSM RDM and DDMwere used for the determination of the studied colorants inlaboratory prepared mixtures and soft drink samples Thesemethods are also applied to analyze Ponceau 4R in the region320ndash520 nm

31 Derivative Spectrophotometric Method (DSM) Undercomputer-controlled instrumentation derivative spectro-photometry played a very important role in the resolutionof band overlapping in quantitative analysis [23] Figure 1(b)shows the first derivative spectra of Carmoisine and Ponceau4R As can be seen in Figure 1(b) in the region of 300ndash520 nm the first derivative spectra allowed the simultaneousdetermination of Carmoisine and Ponceau 4R At this regionthe 1D spectra (Figure 1(b)) show a characteristic peak at331 nm for Carmoisine while Ponceau 4R shows no responseTherefore the absolute values of the 1D peak amplitudes at331 nm can be used to quantify Carmoisine in this mixtureOn the other hand Ponceau 4R can be assayed in the presenceof Carmoisine by measuring its 1D response at 517 nm (zero-crossing point of Carmoisine)

Under the experimental conditions described the cal-ibration graphs obtained by plotting the derivative valuesversus concentrations for Carmoisine and Ponceau 4R inthe concentration range stated in Table 2 exhibited linearrelationships Statistical analysis of the data showed thatlinearity of the calibration graphs and compliance withBeerrsquos law were validated and the small values of the inter-cepts as illustrated by the excellent values of correlation

coefficients of the regression equations To prove the valid-ity and applicability of the proposed method laboratory-prepared mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R (Table 3) The obtainedresults for the analysis of these mixtures in five replicates bythe described method showed high accuracy and precisionof the proposed method (Table 3)

32 Ratio Derivative Method (RDM) While the main dis-advantages of the zero-crossing method in derivative spec-trophotometry for resolving a mixture of components withoverlapped spectra are the risk of small drifts of the workingwavelengths and the circumstance that the working wave-lengths generally do not fall in correspondence of peaks ofthe derivative spectrum this may be particularly dangerouswhen the slope of the spectrum is very high with consequentloss of accuracy and precision and the working wavelengthis in proximity of the base of the spectrum which causespoor sensitivity The main advantage of the RDM is thechance of doing easy measurements in correspondence ofpeaks so it permits the use of the wavelength of highestvalue of analytical signals (a maximum or a minimum) andmoreover the presence of a lot of maxima and minima isanother advantage by the fact that these wavelengths give anopportunity for the determination of active compounds in thepresence of other compounds and ingredients which possiblyinterfere in the assay [24ndash27]

This method is based on the use of the first derivativeof the ratio spectra The ratio spectra were obtained bydividing the absorption spectrum of the mixture by thatof one of the components As we depicted above directabsorption measurements are not possible due to spectraloverlap This spectral overlapping was sufficiently enough todemonstrate the resolving power of the proposed methodFigure 2(a) shows the first ratio derivative spectra of differ-ent Carmoisine standard solutions (spectra divided by thespectrum of 6 120583gmLminus1 Ponceau 4R solution) The ratio firstderivative amplitudes at 323 nm (1DD

323) corresponding to

International Journal of Analytical Chemistry 7

001

003

308 328 348 368

Firs

t der

ivat

ive

Carm

oisin

ePo

ncea

u 4R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

Firs

t der

ivat

ive

Ponc

eau

4RC

arm

oisin

e

Wavelength (nm)

fg

hi

j

minus002678

minus001678

minus000678

(b)

Figure 2 First derivative ratio spectra of Carmoisine (a) and Ponceau 4R (b) for different concentrations (Carmoisine (6 120583gmLminus1 Ponceau4R was used as a divisor) a 2 120583gmLminus1 b 4 120583gmLminus1 c 6120583gmLminus1 d 8 120583gmLminus1 e 10120583gmLminus1 Ponceau 4R (6120583gmLminus1 Carmoisine was usedas a divisor) f 2120583gmLminus1 g 4 120583gmLminus1 h 6 120583gmLminus1 i 8120583gmLminus1 j 10120583gmLminus1)

Table 4 Assay results for the determination of Carmoisine and Ponceau 4R in soft drinks using the proposed methods and the referenceHPLC method (NMKL 130)

Methods Analyte Selected wavelengths(nm)

Assay results mean plusmn SDlowast (119905calculated 119865calculated)lowastlowast

Soft drink I (120583g 100mLminus1) Soft drink II (120583g 100mLminus1)

DSM C 1D331 930 plusmn 022 (068 145) 628 plusmn 029 (061 144)

P 1D517 220 plusmn 011 (118 118) 139 plusmn 013 (040 179)

RDM C 1D323 926 plusmn 023 (037 145) 636 plusmn 021 (128 120)

P 1D344 235 plusmn 011 (044 125) 143 plusmn 013 (013 169)

DDM C 1D366 931 plusmn 024 (073 169) 641 plusmn 024 (153 164)

P 1D451 249 plusmn 017 (189 302) 144 plusmn 016 (023 268)

CM C 1D4721D569 930 plusmn 026 (063 188) 636 plusmn 028 (107 211)

P 1D4701D554 230 plusmn 016 (023 256) 143 plusmn 019 (010 368)

HPLC C 520 921 plusmn 018 620 plusmn 019

P 512 232 plusmn 010 142 plusmn 010

lowastResults obtained are the average of five experiments for each method SD standard deviationlowastlowastThe corresponding theoretical value for 119905 and 119865 at 119875 005 (119905theoretical 231 119865theoretical 639)

a maximum wavelength are proportional to the Carmoisineconcentration (Figure 2(a)) To determine the other com-ponent Ponceau 4R an analogues procedure was followedFor determination of Ponceau 4R the stored spectra ofthe mixtures are divided by a standard spectrum of 6mgLminus1 Carmoisine (Figure 2(b)) Standard solutions of Ponceau4R and the content of Ponceau 4R were determined bymeasuring the signals at 344 nm (1DD

344) Figure 2(b)

Under the described experimental conditions the cal-ibration graphs obtained by plotting the derivative valuesof each colorant versus concentration in the concentration

range stated in Table 2 showed linear relationships Con-formity with Beerrsquos law was evident in the concentrationrange of the final dilution cited in Table 2 For this methodthe characteristic parameters of regression equations andcorrelation coefficients are given in Table 2 The correlationcoefficients were 09996 and 09994 indicating good linearityLOD and LOQ were calculated for each colorant and areshown in the same table The detection limits were foundto be 0079120583gmLminus1 for Carmoisine and 0082 120583gmLminus1 forPonceau 4R while the quantification limits were estimatedto be 0263 120583gmLminus1 for Carmoisine and 0273120583gmLminus1 for

8 International Journal of Analytical Chemistry

000988

008988

016988

024988

032988

300 400 500 600 700

Abso

rban

ce

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

Diff

eren

ce fi

rst d

eriv

ativ

e

Wavelength (nm)

C

P

minus00068

minus00028

(b)

Figure 3 Difference absorption spectra of 10 120583gmLminus1 Carmoisine 10 120583gmLminus1 Ponceau 4R and their binary mixture (a) Difference firstderivative spectra (b) of colorants (Carmoisine 2ndash10120583gmLminus1 Ponceau 4R 2ndash10 120583gmLminus1)

Ponceau 4R The proposed method was also applied to thedrink samples (Table 4) The accuracy of RDM was checkedby analyzing the laboratory preparedmixtures of Carmoisineand Ponceau 4R at various concentration ratios ranging from2 to 10 120583gmLminus1 (Table 3) All data represent the average offive determinations Low values of relative standard deviationindicate very good precision Satisfactory recoveries withsmall standard deviations were obtained which indicated thehigh repeatability and accuracy of the method

33 Derivative DifferentialMethod (DDM) Differential spec-trophotometry (ΔD

1) based on pH changes has also been

reported to be useful in the determination of binary mix-tures This method that depends on utilization of differenceabsorption spectra corresponding to the same compoundobtained at two different pHrsquos has been investigated for theanalysis of binary mixtures The procedure is comprised ofthe measurement of ΔD

1food colorantsrsquo in acidic solutions

against their alkaline solutions as blanks The ΔD1has

been successfully used to eliminate interferences from drinksamples There are few reports on utilization of the abovetwo combined techniques (difference and derivative) for theestimation of individual drug substances and for combinedpreparations [28ndash30]

The difference absorption spectra of Carmoisine Pon-ceau 4R and their binary mixture in 01 N NaOH and in01 N HCI are shown in Figure 3(a) Figure 3(b) shows thefirst derivative difference spectra of colorants in the concen-tration range 2ndash10 120583gmLminus1 The spectra of both colorantsin Figure 3(b) offer an advantage for their simultaneousdetermination by having zero-crossing points In particularthe first derivative amplitudes at 366 nm for Carmoisine and

451 nm for Ponceau 4R in the Carmoisine and Ponceau 4Rmixture were considered as the optimum working wave-lengths for their determination By measuring the values ofthe ΔD

1amplitudes at these wavelengths the concentration

of each colorant can be directly calculated since the dif-ferential first derivative measurement cancels the irrelevantabsorbance due to the drinks matrix at these wavelengths

Under the experimental conditions described the cali-bration graphs obtained by plotting the derivative values ofeach colorant in the mixture versus concentration in therange concentration stated in Table 2 show linear relation-ships The correlation coefficients were 09996 and 09998indicating good linearity The LOD were 0071 120583gmLminus1 forCarmoisine and 0081 120583gmLminus1 for Ponceau 4R while theLOQ were 0236 120583gmLminus1 for Carmoisine and 0270 120583gmLminus1for Ponceau 4R The proposed procedure was success-fully applied for the determination of these colorants inlaboratory-prepared mixtures and drink samples The resultsare presented in Tables 3 and 4The values of relative standarddeviation indicate very good reproducibility In additionapplication of the DDMwas found to be correct for the drinkmatrix interference and to enhance the sensitivity

34 CompensationMethod (CM) Thecompensationmethodis a nonmathematical method for the detection and elimi-nation of unwanted absorption during spectrophotometricanalysis [19 20 31 32] The accuracy of the method dependson the evaluation of the balance point Figure 1(b) showsthe first derivative spectra of Carmoisine and Ponceau 4Rin the concentration range 2ndash10 120583gmLminus1 The first derivativespectra were recorded for each reference solution of thecomponents and the ratios of the 1D maximum and 1D

International Journal of Analytical Chemistry 9

000011000211000411000611

401 451 501 551 601Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

Firs

t der

ivat

ive

(a)

000080002800048

400 440 480 520 560 600

Firs

t der

ivat

ive

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)

Figure 4 (a) First derivative spectra between the mixture solution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in the sample cell anddifferent concentrations (2ndash10120583gmLminus1) of Ponceau 4R solutions in the reference cell First derivative spectra between the mixture solution(6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and 6120583gmLminus1 Ponceau 4R in the reference cell at balance points andcomparison of these spectra with 6 120583gmLminus1 concentration of Carmoisine (- - -) spectrum (b) First derivative spectra between the mixturesolution (6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and different concentrations (2ndash10120583gmLminus1) of Carmoisinesolutions in the reference cell Derivative spectra between the mixture solution (6120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in thesample cell and 6 120583gmLminus1 Carmoisine in the reference cell at balance points and comparison of these spectra with 6120583gmLminus1 concentrationof Ponceau 4R (- - -) spectrum

minimum were calculated Table 1 shows the mean valuesof the ratios calculated for five different determinations foreach solutionThe ratios are constant and characteristic of thepure substance independent of concentration and presenceof another component For the determination of Carmoisineconcentrations in Carmoisine-Ponceau 4R binary mixturesand drink samples the sample cell was filled with themixturesolution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R)and the reference cell was filled in succession with aseries of reference Ponceau 4R with solutions in differentconcentrations Ponceau 4R as shown in Figure 4(a) Theratios of the mixture calculated from the recorded 1D spectrawere compared with those of Carmoisine At the exactbalance point the ratio of the mixture corresponds to thatof Carmoisine where the concentration of Carmoisine in themixtures in the sample cell is equal to that of the referencesolution in the reference cell For determining the othercomponent the same steps were followed using solutions ofpure compoundCarmoisine in the reference cell to determineits concentration in the mixture (Figure 4(b)) Conformitywith Beerrsquos law was evident in the concentration range from 2to 10 120583gmLminus1 of Carmoisine and Ponceau 4R In thismethodsynthetic mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R and the recoveries ofthe method were found to be in the range of 9850 plusmn 105ndash9975 plusmn 110 The presented results in Table 3 are in goodagreement with the other proposed methods

35 Application of the Developed Methods to the Soft DrinksThe developed and validated methods were successfullyapplied to the determination of Carmoisine and Ponceau 4Rcontent in the commercial soft drinks The obtained resultsare presented in Table 4 Good agreement was observed forthe assay results of the drinks by application of the fourmethods in this paper (Table 4) The values of standarddeviation indicate very good reproducibility The results ofthe proposed spectrophotometric methods were statisticallycompared with the results of the HPLC method given in

the literature [18] at the 95 confidence level with studentrsquos 119905-test and by the variance ratio 119865-test (Table 4) The calculated119905- and119865-values never exceed the theoretical 119905- and119865-values at005 level of significant difference There was no interferenceof ingredients in the products examined so no additiveextraction or separation procedures were required duringtheir assay The results of all methods were very close toeach other This suggests that the four methods are equallyapplicable

36 Validation of the Proposed Methods

361 Linearity The linearity of the proposed methods wasevaluated under different concentrations of Carmoisine andPonceau 4R within the concentration range stated in Table 2The good linearity of the calibration graphs and negligiblescatter of the experimental points is clearly evident by thevalues of the correlation coefficients and variances around theslope (Table 2)

362 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD is expressed as the analyte concentration cor-responding to the sample blank value plus three standarddeviations and LOQ is the analyte concentration correspond-ing to the sample blank value ten standard deviations [21]All the proposed methods data was presented in Table 2The LOD and LOQ values found were between 0071 and0086 and 0236 and 0286 120583gmLminus1 for Carmoisine and 0081and 0091 and 0270 and 0304 120583gmLminus1 for Ponceau 4Rrespectively The data shows that the methods are sensitivefor the determination of Carmoisine and Ponceau 4R

363 Precision (Repeatability) and Accuracy (Recovery )The precision of the developed methods was expressedas a percentage of relative standard deviation (RSD) forrepeatability (intraday precision) and intermediate precision(interday precision) The data obtained were less than 106(Table 3) indicating reasonable repeatability of the proposed

10 International Journal of Analytical Chemistry

methods The intraday and interday accuracy ranges werefrom 9895 to 1006 for Carmoisine and from 9975 to1007 for Ponceau 4R respectively The results obtainedfrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria To confirm the accuracy of the proposed methodssyntheticmixtures of different concentration ratios consistingof each food colorant were prepared The resulting mixtureswere assayed according to above proposed procedures infive replicates and the average results were calculated asthe percentage of analyte recovered The percentages meanaccuracy for Carmoisine were 1008 plusmn 087 for method DSM1010 plusmn 095 for RDM 10040 plusmn 107 for DDM and 9975 plusmn102 for CM respectively And those of P were 9910 plusmn 102for method DSM 9825 plusmn 093 for RDM 9955 plusmn 095 forDDM and 9850 plusmn 106 for CM The good recovery percent(RSD) assured the high accuracy of the proposed methods(Table 3) The relative standard deviations were found tobe less than 107 and 106 for Carmoisine and Ponceau4R respectively indicating reasonable repeatability of theproposed methods

4 Conclusion

Four derivative spectrophotometric methods for the simul-taneous analysis of two common food colorants have beensuccessfully researched developed applied and compared byreference HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination ofCarmosine and Ponceau 4R in synthetic mixtures and softdrinks without any interference from ingredients presentThe developed methods are simple (as there is no needfor pretreatment) rapid (as they require measurements of998779D1 and D1 values at a single wavelength) and direct (asthey estimate each colorant independently of the other) UVmethods offer a cost effective and time saving alternative toother methods for example colorimetric complexometricand chromatographic analyses The methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetricmethods Also themethodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities of the proposed meth-ods are almost comparable and can be used for the determina-tion of the two colorants in commercial drinks in the absenceof official method

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research has been supported by Scientific ResearchProject Coordination Center of Yildiz Technical University(Project no 2012-01-02-KAP07)

References

[1] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 1989

[2] J Vries Food Safety and Toxicity CRC London UK 1996[3] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination of

food dyes in soft drinks containing natural pigments by liquidchromatography with minimal clean-uprdquo Food Control vol 16no 3 pp 293ndash297 2005

[4] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination of carmoisine allura red and Ponceau4R in sweets and soft drinks by differential pulse polarographyrdquoJournal of Food Composition andAnalysis vol 18 no 6 pp 503ndash515 2005

[5] N Dossi R Toniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem forthe rapid in-channel amperometric detection of synthetic dyesin foodrdquo Journal of Electroanalytical Chemistry vol 601 no 1-2pp 1ndash7 2007

[6] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination of eight synthetic permitted and five commonlyencountered nonpermitted food colors in various foodmatrixesby high-performance liquid chromatographyrdquo Journal of AOACInternational vol 93 no 5 pp 1503ndash1514 2010

[7] S Dixit S K Khanna and M Das ldquoA simple method forsimultaneous determination of basic dyes encountered in foodpreparations by reversed-phase HPLCrdquo Journal of AOAC Inter-national vol 94 no 6 pp 1874ndash1881 2011

[8] N Yoshioka and K Ichihashi ldquoDetermination of 40 syntheticfood colors in drinks and candies by high-performance liquidchromatography using a short column with photodiode arraydetectionrdquo Talanta vol 74 no 5 pp 1408ndash1413 2008

[9] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination of synthetic dyes in selectedfoodstuffs by high performance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 107 no 1 pp 489ndash496 2008

[10] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination of water-soluble and fat-soluble synthetic colorantsin foodstuffbyhigh-performance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal ofChromatography A vol 1103 no 1 pp 170ndash176 2006

[11] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination of food dyes by firstderivative spectrophotometry with sorption onto polyurethanefoamrdquo Analytical Sciences vol 21 no 2 pp 149ndash153 2005

[12] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilfroush ldquoSimultaneous determination of carmoisine andPonceau 4Rrdquo Food Analytical Methods vol 1 no 3 pp 214ndash2192008

[13] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination of carmoisine andbrilliant blue FCF in food samplesrdquo Talanta vol 84 no 1 pp240ndash243 2011

[14] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan andB Asci ldquoAbilities of partial least-squares (PLS-2)multivariate calibration in the analysis of quaternary mixture offood colors (E-110 E-122 E-124 E-131)rdquo Analytical Letters vol40 no 10 pp 1893ndash1903 2007

[15] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectrofluorimetric determination of rhodamine dyein cosmeticsrdquo Talanta vol 75 no 1 pp 135ndash140 2008

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

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Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Quantum Chemistry

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CatalystsJournal of

Page 4: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

4 International Journal of Analytical Chemistry

26 Procedures

261 Derivative Spectrophotometric Method (DSM) Ali-quots of Carmoisine working solutions equivalent to 20ndash100 120583gmLminus1 were placed into 10mL calibration flasks andthe volume was made up with distilled water The absorptionspectra were recorded versus distilled water Next the zero-order spectra were transformed into first derivative Theamplitude from the baseline to peak at 331 nm (zero-crossingpoint for Ponceau 4R) was related to the actual contents ofCarmoisine

Appropriate aliquots of aqueous working solutions ofPonceau 4R were placed into 10mL calibration flasks andthe volume was made up with distilled water The spectra ofstandard solutions in the concentration range 20ndash10120583gmLminus1were recorded and subjected to the mathematical treatment(first derivative) The values of 1D

517measured at 517 nm

(zero-crossing point for Carmoisine) were used for construc-tion of calibration graph

262 Ratio Derivative Spectrophotometric Method (RDM)Accurately aliquots of 02 04 06 08 and 10mLof colorantsstock solution were transferred into 10mL volumetric flasksand the volume wasmade up with distilled waterThe spectraof the prepared standard solutions were scanned from 300 to700 nm and stored in the computer For the determination ofCarmoisine the stored spectra of Carmoisine were dividedseparately by the spectrumof 6120583gmLminus1 Ponceau 4R to obtainthe ratio spectra The first derivative corresponding to eachratio spectrum (1DD) was recorded A calibration graphrelating the peak amplitudes (1DD

323) to the corresponding

concentrations in 120583gmLminus1 of Carmoisine was constructedIn order to determine Ponceau 4R stored spectra of Pon-

ceau 4R standard solutions were divided by a standard spec-trum of Carmoisine of 6120583gmLminus1 The first ratio derivativespectra were calculatedThe concentration of the Ponceau 4Rwas proportional to the amplitude of themaximumat 344 nm(1D344

)

263 Derivative Differential Spectrophotometric Method(DDM) The difference spectra between the acidic (01 NHCI) solution and equimolar basic (01 N NaOH) solutionof pure colorants and drink samples were recorded from3000 to 7000 nm by placing the acidic solution in thesample compartment and the basic solution in the referencecompartment A first derivative spectrum of each of thedifferential curves was subsequently recordedThe derivativeabsorbance values were measured at 366 and 451 nm forCarmoisine and Ponceau 4R respectively

264 Compensation Method (CM)

(1) Determination of Standard Ratios The first derivativespectra for each set of standard solutions were recorded usingthe appropriate blank solution The ratios of the first deriva-tive maxima and minima ( 1D

1205821

1D1205822

) where appropriate at

the specified wavelengths (1205821and 120582

2) as indicated in Table 1

were calculated

(2) Compensation Spectrophotometric Measurements Thesolution of the mixture (containing compounds Carmoisineand Ponceau 4R) was placed in the sample cell A series ofsolutions containing various concentrations (2ndash10120583gmLminus1)of Carmoisine above and below that present in the mixturesolution were prepared and placed in succession in thereference cell The first derivative spectra (1D) were recordedand the corresponding ratios (Table 1) in each instancewere calculated and followed the calculated ratio for purecolorant Ponceau 4R The exact balance point (the ratio ofthe mixture is equal to that of pure compound Ponceau 4R)was determined at which the concentration of Carmoisine insample solution is equal to that in the reference solution

By analogy the same steps were followed by using solu-tions of pure Ponceau 4R in the reference cell to determine itsconcentration in the mixture at the balance point

The same principle was also applied for the analysis ofdrink samples

(3) Graphical Method To avoid the excessive preparation ofseveral standard solutions of either compoundCarmoisine orin order to locate the exact balance point a graphical methodis recommended [19 20]

The same steps as for the compensation method werefollowed and the derivative ratio of themixture (in the samplecell) was calculated at each time and plotted against the con-centration of pure compound Carmoisine (in the referencecell) A line with slight curvature is usually obtained Theconcentration of Carmosine can easily be interpolated fromthe graph by substituting the ratio of pure Ponceau 4R Atthe point at which the ratio of the mixture is equal to thatof pure Ponceau 4R the concentration of Carmoisine in thereference cell is equal to that in themixture in the sample cellBy analogy the concentration of compound Ponceau 4R canbe obtained

265 HPLC Method (Reference Method NMKL 130) Thisreference method is described in detail in Nordic Committeeon Food Analysis [18] About 20 120583L of each sample wasinjected and the concentrations were calculated on the basisof the ratios of peak areaswith those of the standard solutionsThe chromatograms of two compounds and drink sampleswere plotted and stored in the computer Separation wascarried out in room temperature The mobile phase wasprepared daily filtered through a 045120583m membrane filterAll chromatograms were run with the use of a (50 50 vv)mixture of methanol and TBA buffer Other experimentalparameters were flow rate 07mL minminus1 retention times554min for Ponceau 4R and 2162min for Carmoisine

266 Application of the Proposed Methods for the Deter-mination of Two Colorants in Drink Sample Commercialsamples weremixed with Carrez (I) and Carrez (II) solutionsOnce precipitated the samples were centrifuged 50mL ofthe sample that is clarified by Carrez precipitation was

International Journal of Analytical Chemistry 5

0175

0375

0575

0775

300

350

400

450

500

550

600

650

700

Abso

rban

ce

Wavelength (nm)

C

Mix

P

minus0025

(a)

00002

00022

300

320

340

Firs

t der

ivat

ive

Wavelength (nm)

P

C

00002

00022

475

500

525

550

575

Firs

t der

ivat

ive

Wavelength (nm)

C

P

minus00098

minus00078

minus00058

minus00038

minus00018

minus00098

minus00078

minus00058

minus00038

minus00018

(b)

Figure 1 Zero-order spectra (a) of 10 120583gmLminus1 Carmoisine 10120583gmLminus1 Ponceau 4R and their binary mixture first derivative spectra (b) ofcolorants (Carmoisine(mdash) 2ndash10120583gmLminus1 Ponceau 4R (- -) 2ndash10120583gmLminus1)

transferred to 100mL volumetric flask and the volume wasmade up with distilled water A series of dilution wasprepared quantitatively with water from this solution toobtain standard solutions to reach the concentration ranges ofcalibration curves graphed for each of the proposed methods(DSM RDM and CM) For the DDM method the sameprocedure was used but in this case the final volume wasmade up with 01 N HCI and 01 N NaOH separately forthe determination of Carmoisine and Ponceau 4R HPLCmethod the same sample preparation procedure describedabove was followed using a mixture 1 1 vv of methanolmdashTBA buffer solution After dissolution process preparedsolutions were filtered using 045 120583m disposable membranefilters (Sartrious Minisart 045120583m) by using a syringe Thefinal solutionwas diluted to theworking concentration range

27 Method Validation The validity and suitability of theproposed methods were assessed by accuracy (reported aspercentage recovered) precision (reported as RSD) lin-earity (evaluated by regression equation) limit of detection(LOD) and limit of quantification (LOQ) [21]

LOD and LOQ were based on the standard deviation ofresponse and the slope of the corresponding curve using thefollowing equations

LOD = 3 119904119898 LOQ = 10 119904

119898 (1)

where 119904 the noise estimate is the standard deviation of thederivative amplitude of the blank and 119898 is the slope of therelated calibration graphs [22]

The linearity of the methods was established in theconcentration ranges of 2ndash10120583gmLminus1 for Carmoisine and

Ponceau 4R respectively In this study five concentrationswere analyzed in five replicates The linearity was evaluatedby the least square regression method

In order to demonstrate the validity and suitability ofthe proposed methods intra- and interday variability studieswere performed at three different concentrations (2 6 and10 120583gmLminus1) in the presence of the certain concentration (26 and 10 120583gmLminus1) of the other colorants three times in a dayand also three different days Method efficiency was tested interms of RSDrsquos for both intraday and interday precisions

The precision was ascertained by carrying out five repli-cate determinations of synthetic mixtures at different con-centration ratios of Carmoisine and Ponceau 4R Recoverytest also confirmed the accuracy and applicability of theproposed methods by analyzing several synthetic mixturesof Carmoisine and Ponceau 4R which reproduced differentcomposition ratios within the linearity rangeThese ratios arealso the ratios in drinksThemean percentage recoveries andRSD values were calculated

Recovery of the colorants of interest from a given matrixcan be used as a measure of the accuracy or the bias of themethod The same ranges of concentrations as employed inthe linearity studies were used

In our study the applicability of the derivative spec-trophotometric methods was also validated by comparisonwith the HPLC technique

3 Result and Discussion

Figure 1(a) shows the absorption spectra of CarmoisinePonceau 4R and themixture of them in the wavelength rangeof 300ndash700 nm Zero-order absorption spectra of Carmoisine

6 International Journal of Analytical Chemistry

Table 3 Method validation for the simultaneous determination of Carmoisine and Ponceau 4R in laboratory prepared mixtures by theproposed methods

Method Accuracy (meanlowastplusmn RSD ) Precision repeatabilitya(mean plusmn RSD )

Intermediate precisionb

(mean plusmn RSD )

DSM C (1D331) 1008 plusmn 087 1001 plusmn 096 9991 plusmn 102

P (1D517) 991 plusmn 102 1005 plusmn 105 1001 plusmn 092

RDM C (1D323) 1010 plusmn 095 9998 plusmn 099 1006 plusmn 105

P (1D344) 9825 plusmn 093 9983 plusmn 098 1004 plusmn 096

DDM C (1D366) 1004 plusmn 107 9895 plusmn 105 9992 plusmn 098

P (1D451) 9955 plusmn 095 1005 plusmn 095 1007 plusmn 096

CM C (1D4721D569) 9975 plusmn 102 998 plusmn 101 1001 plusmn 097

P (1D4701D554) 985 plusmn 106 9975 plusmn 103 1003 plusmn 106

aThe intraday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times within the daybThe interday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times in three successive dayslowastThe values of recovery are an average of five replicates of each of five synthetic mixtures at different concentration ratios of C and P (2ndash10 120583gmLminus1)RSD relative standard deviation

and Ponceau 4R in distilled water gave rise to maximumpeaks at 517 nm and 508 nm respectively As can be seenthe spectra of the colorants were strongly overlapped in the400ndash600 nm wavelength region Hence the direct absorp-tion measurement for assaying binary mixture seems to beimpossible On the other hand the absorption spectra of thetwo components within a wavelength range of 300ndash380 nmwere less overlapped than the region 400ndash600 nm So in thiswork the region of 300ndash380 nm was chosen for the analysisof Carmoisine In this spectral range DSM RDM and DDMwere used for the determination of the studied colorants inlaboratory prepared mixtures and soft drink samples Thesemethods are also applied to analyze Ponceau 4R in the region320ndash520 nm

31 Derivative Spectrophotometric Method (DSM) Undercomputer-controlled instrumentation derivative spectro-photometry played a very important role in the resolutionof band overlapping in quantitative analysis [23] Figure 1(b)shows the first derivative spectra of Carmoisine and Ponceau4R As can be seen in Figure 1(b) in the region of 300ndash520 nm the first derivative spectra allowed the simultaneousdetermination of Carmoisine and Ponceau 4R At this regionthe 1D spectra (Figure 1(b)) show a characteristic peak at331 nm for Carmoisine while Ponceau 4R shows no responseTherefore the absolute values of the 1D peak amplitudes at331 nm can be used to quantify Carmoisine in this mixtureOn the other hand Ponceau 4R can be assayed in the presenceof Carmoisine by measuring its 1D response at 517 nm (zero-crossing point of Carmoisine)

Under the experimental conditions described the cal-ibration graphs obtained by plotting the derivative valuesversus concentrations for Carmoisine and Ponceau 4R inthe concentration range stated in Table 2 exhibited linearrelationships Statistical analysis of the data showed thatlinearity of the calibration graphs and compliance withBeerrsquos law were validated and the small values of the inter-cepts as illustrated by the excellent values of correlation

coefficients of the regression equations To prove the valid-ity and applicability of the proposed method laboratory-prepared mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R (Table 3) The obtainedresults for the analysis of these mixtures in five replicates bythe described method showed high accuracy and precisionof the proposed method (Table 3)

32 Ratio Derivative Method (RDM) While the main dis-advantages of the zero-crossing method in derivative spec-trophotometry for resolving a mixture of components withoverlapped spectra are the risk of small drifts of the workingwavelengths and the circumstance that the working wave-lengths generally do not fall in correspondence of peaks ofthe derivative spectrum this may be particularly dangerouswhen the slope of the spectrum is very high with consequentloss of accuracy and precision and the working wavelengthis in proximity of the base of the spectrum which causespoor sensitivity The main advantage of the RDM is thechance of doing easy measurements in correspondence ofpeaks so it permits the use of the wavelength of highestvalue of analytical signals (a maximum or a minimum) andmoreover the presence of a lot of maxima and minima isanother advantage by the fact that these wavelengths give anopportunity for the determination of active compounds in thepresence of other compounds and ingredients which possiblyinterfere in the assay [24ndash27]

This method is based on the use of the first derivativeof the ratio spectra The ratio spectra were obtained bydividing the absorption spectrum of the mixture by thatof one of the components As we depicted above directabsorption measurements are not possible due to spectraloverlap This spectral overlapping was sufficiently enough todemonstrate the resolving power of the proposed methodFigure 2(a) shows the first ratio derivative spectra of differ-ent Carmoisine standard solutions (spectra divided by thespectrum of 6 120583gmLminus1 Ponceau 4R solution) The ratio firstderivative amplitudes at 323 nm (1DD

323) corresponding to

International Journal of Analytical Chemistry 7

001

003

308 328 348 368

Firs

t der

ivat

ive

Carm

oisin

ePo

ncea

u 4R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

Firs

t der

ivat

ive

Ponc

eau

4RC

arm

oisin

e

Wavelength (nm)

fg

hi

j

minus002678

minus001678

minus000678

(b)

Figure 2 First derivative ratio spectra of Carmoisine (a) and Ponceau 4R (b) for different concentrations (Carmoisine (6 120583gmLminus1 Ponceau4R was used as a divisor) a 2 120583gmLminus1 b 4 120583gmLminus1 c 6120583gmLminus1 d 8 120583gmLminus1 e 10120583gmLminus1 Ponceau 4R (6120583gmLminus1 Carmoisine was usedas a divisor) f 2120583gmLminus1 g 4 120583gmLminus1 h 6 120583gmLminus1 i 8120583gmLminus1 j 10120583gmLminus1)

Table 4 Assay results for the determination of Carmoisine and Ponceau 4R in soft drinks using the proposed methods and the referenceHPLC method (NMKL 130)

Methods Analyte Selected wavelengths(nm)

Assay results mean plusmn SDlowast (119905calculated 119865calculated)lowastlowast

Soft drink I (120583g 100mLminus1) Soft drink II (120583g 100mLminus1)

DSM C 1D331 930 plusmn 022 (068 145) 628 plusmn 029 (061 144)

P 1D517 220 plusmn 011 (118 118) 139 plusmn 013 (040 179)

RDM C 1D323 926 plusmn 023 (037 145) 636 plusmn 021 (128 120)

P 1D344 235 plusmn 011 (044 125) 143 plusmn 013 (013 169)

DDM C 1D366 931 plusmn 024 (073 169) 641 plusmn 024 (153 164)

P 1D451 249 plusmn 017 (189 302) 144 plusmn 016 (023 268)

CM C 1D4721D569 930 plusmn 026 (063 188) 636 plusmn 028 (107 211)

P 1D4701D554 230 plusmn 016 (023 256) 143 plusmn 019 (010 368)

HPLC C 520 921 plusmn 018 620 plusmn 019

P 512 232 plusmn 010 142 plusmn 010

lowastResults obtained are the average of five experiments for each method SD standard deviationlowastlowastThe corresponding theoretical value for 119905 and 119865 at 119875 005 (119905theoretical 231 119865theoretical 639)

a maximum wavelength are proportional to the Carmoisineconcentration (Figure 2(a)) To determine the other com-ponent Ponceau 4R an analogues procedure was followedFor determination of Ponceau 4R the stored spectra ofthe mixtures are divided by a standard spectrum of 6mgLminus1 Carmoisine (Figure 2(b)) Standard solutions of Ponceau4R and the content of Ponceau 4R were determined bymeasuring the signals at 344 nm (1DD

344) Figure 2(b)

Under the described experimental conditions the cal-ibration graphs obtained by plotting the derivative valuesof each colorant versus concentration in the concentration

range stated in Table 2 showed linear relationships Con-formity with Beerrsquos law was evident in the concentrationrange of the final dilution cited in Table 2 For this methodthe characteristic parameters of regression equations andcorrelation coefficients are given in Table 2 The correlationcoefficients were 09996 and 09994 indicating good linearityLOD and LOQ were calculated for each colorant and areshown in the same table The detection limits were foundto be 0079120583gmLminus1 for Carmoisine and 0082 120583gmLminus1 forPonceau 4R while the quantification limits were estimatedto be 0263 120583gmLminus1 for Carmoisine and 0273120583gmLminus1 for

8 International Journal of Analytical Chemistry

000988

008988

016988

024988

032988

300 400 500 600 700

Abso

rban

ce

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

Diff

eren

ce fi

rst d

eriv

ativ

e

Wavelength (nm)

C

P

minus00068

minus00028

(b)

Figure 3 Difference absorption spectra of 10 120583gmLminus1 Carmoisine 10 120583gmLminus1 Ponceau 4R and their binary mixture (a) Difference firstderivative spectra (b) of colorants (Carmoisine 2ndash10120583gmLminus1 Ponceau 4R 2ndash10 120583gmLminus1)

Ponceau 4R The proposed method was also applied to thedrink samples (Table 4) The accuracy of RDM was checkedby analyzing the laboratory preparedmixtures of Carmoisineand Ponceau 4R at various concentration ratios ranging from2 to 10 120583gmLminus1 (Table 3) All data represent the average offive determinations Low values of relative standard deviationindicate very good precision Satisfactory recoveries withsmall standard deviations were obtained which indicated thehigh repeatability and accuracy of the method

33 Derivative DifferentialMethod (DDM) Differential spec-trophotometry (ΔD

1) based on pH changes has also been

reported to be useful in the determination of binary mix-tures This method that depends on utilization of differenceabsorption spectra corresponding to the same compoundobtained at two different pHrsquos has been investigated for theanalysis of binary mixtures The procedure is comprised ofthe measurement of ΔD

1food colorantsrsquo in acidic solutions

against their alkaline solutions as blanks The ΔD1has

been successfully used to eliminate interferences from drinksamples There are few reports on utilization of the abovetwo combined techniques (difference and derivative) for theestimation of individual drug substances and for combinedpreparations [28ndash30]

The difference absorption spectra of Carmoisine Pon-ceau 4R and their binary mixture in 01 N NaOH and in01 N HCI are shown in Figure 3(a) Figure 3(b) shows thefirst derivative difference spectra of colorants in the concen-tration range 2ndash10 120583gmLminus1 The spectra of both colorantsin Figure 3(b) offer an advantage for their simultaneousdetermination by having zero-crossing points In particularthe first derivative amplitudes at 366 nm for Carmoisine and

451 nm for Ponceau 4R in the Carmoisine and Ponceau 4Rmixture were considered as the optimum working wave-lengths for their determination By measuring the values ofthe ΔD

1amplitudes at these wavelengths the concentration

of each colorant can be directly calculated since the dif-ferential first derivative measurement cancels the irrelevantabsorbance due to the drinks matrix at these wavelengths

Under the experimental conditions described the cali-bration graphs obtained by plotting the derivative values ofeach colorant in the mixture versus concentration in therange concentration stated in Table 2 show linear relation-ships The correlation coefficients were 09996 and 09998indicating good linearity The LOD were 0071 120583gmLminus1 forCarmoisine and 0081 120583gmLminus1 for Ponceau 4R while theLOQ were 0236 120583gmLminus1 for Carmoisine and 0270 120583gmLminus1for Ponceau 4R The proposed procedure was success-fully applied for the determination of these colorants inlaboratory-prepared mixtures and drink samples The resultsare presented in Tables 3 and 4The values of relative standarddeviation indicate very good reproducibility In additionapplication of the DDMwas found to be correct for the drinkmatrix interference and to enhance the sensitivity

34 CompensationMethod (CM) Thecompensationmethodis a nonmathematical method for the detection and elimi-nation of unwanted absorption during spectrophotometricanalysis [19 20 31 32] The accuracy of the method dependson the evaluation of the balance point Figure 1(b) showsthe first derivative spectra of Carmoisine and Ponceau 4Rin the concentration range 2ndash10 120583gmLminus1 The first derivativespectra were recorded for each reference solution of thecomponents and the ratios of the 1D maximum and 1D

International Journal of Analytical Chemistry 9

000011000211000411000611

401 451 501 551 601Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

Firs

t der

ivat

ive

(a)

000080002800048

400 440 480 520 560 600

Firs

t der

ivat

ive

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)

Figure 4 (a) First derivative spectra between the mixture solution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in the sample cell anddifferent concentrations (2ndash10120583gmLminus1) of Ponceau 4R solutions in the reference cell First derivative spectra between the mixture solution(6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and 6120583gmLminus1 Ponceau 4R in the reference cell at balance points andcomparison of these spectra with 6 120583gmLminus1 concentration of Carmoisine (- - -) spectrum (b) First derivative spectra between the mixturesolution (6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and different concentrations (2ndash10120583gmLminus1) of Carmoisinesolutions in the reference cell Derivative spectra between the mixture solution (6120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in thesample cell and 6 120583gmLminus1 Carmoisine in the reference cell at balance points and comparison of these spectra with 6120583gmLminus1 concentrationof Ponceau 4R (- - -) spectrum

minimum were calculated Table 1 shows the mean valuesof the ratios calculated for five different determinations foreach solutionThe ratios are constant and characteristic of thepure substance independent of concentration and presenceof another component For the determination of Carmoisineconcentrations in Carmoisine-Ponceau 4R binary mixturesand drink samples the sample cell was filled with themixturesolution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R)and the reference cell was filled in succession with aseries of reference Ponceau 4R with solutions in differentconcentrations Ponceau 4R as shown in Figure 4(a) Theratios of the mixture calculated from the recorded 1D spectrawere compared with those of Carmoisine At the exactbalance point the ratio of the mixture corresponds to thatof Carmoisine where the concentration of Carmoisine in themixtures in the sample cell is equal to that of the referencesolution in the reference cell For determining the othercomponent the same steps were followed using solutions ofpure compoundCarmoisine in the reference cell to determineits concentration in the mixture (Figure 4(b)) Conformitywith Beerrsquos law was evident in the concentration range from 2to 10 120583gmLminus1 of Carmoisine and Ponceau 4R In thismethodsynthetic mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R and the recoveries ofthe method were found to be in the range of 9850 plusmn 105ndash9975 plusmn 110 The presented results in Table 3 are in goodagreement with the other proposed methods

35 Application of the Developed Methods to the Soft DrinksThe developed and validated methods were successfullyapplied to the determination of Carmoisine and Ponceau 4Rcontent in the commercial soft drinks The obtained resultsare presented in Table 4 Good agreement was observed forthe assay results of the drinks by application of the fourmethods in this paper (Table 4) The values of standarddeviation indicate very good reproducibility The results ofthe proposed spectrophotometric methods were statisticallycompared with the results of the HPLC method given in

the literature [18] at the 95 confidence level with studentrsquos 119905-test and by the variance ratio 119865-test (Table 4) The calculated119905- and119865-values never exceed the theoretical 119905- and119865-values at005 level of significant difference There was no interferenceof ingredients in the products examined so no additiveextraction or separation procedures were required duringtheir assay The results of all methods were very close toeach other This suggests that the four methods are equallyapplicable

36 Validation of the Proposed Methods

361 Linearity The linearity of the proposed methods wasevaluated under different concentrations of Carmoisine andPonceau 4R within the concentration range stated in Table 2The good linearity of the calibration graphs and negligiblescatter of the experimental points is clearly evident by thevalues of the correlation coefficients and variances around theslope (Table 2)

362 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD is expressed as the analyte concentration cor-responding to the sample blank value plus three standarddeviations and LOQ is the analyte concentration correspond-ing to the sample blank value ten standard deviations [21]All the proposed methods data was presented in Table 2The LOD and LOQ values found were between 0071 and0086 and 0236 and 0286 120583gmLminus1 for Carmoisine and 0081and 0091 and 0270 and 0304 120583gmLminus1 for Ponceau 4Rrespectively The data shows that the methods are sensitivefor the determination of Carmoisine and Ponceau 4R

363 Precision (Repeatability) and Accuracy (Recovery )The precision of the developed methods was expressedas a percentage of relative standard deviation (RSD) forrepeatability (intraday precision) and intermediate precision(interday precision) The data obtained were less than 106(Table 3) indicating reasonable repeatability of the proposed

10 International Journal of Analytical Chemistry

methods The intraday and interday accuracy ranges werefrom 9895 to 1006 for Carmoisine and from 9975 to1007 for Ponceau 4R respectively The results obtainedfrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria To confirm the accuracy of the proposed methodssyntheticmixtures of different concentration ratios consistingof each food colorant were prepared The resulting mixtureswere assayed according to above proposed procedures infive replicates and the average results were calculated asthe percentage of analyte recovered The percentages meanaccuracy for Carmoisine were 1008 plusmn 087 for method DSM1010 plusmn 095 for RDM 10040 plusmn 107 for DDM and 9975 plusmn102 for CM respectively And those of P were 9910 plusmn 102for method DSM 9825 plusmn 093 for RDM 9955 plusmn 095 forDDM and 9850 plusmn 106 for CM The good recovery percent(RSD) assured the high accuracy of the proposed methods(Table 3) The relative standard deviations were found tobe less than 107 and 106 for Carmoisine and Ponceau4R respectively indicating reasonable repeatability of theproposed methods

4 Conclusion

Four derivative spectrophotometric methods for the simul-taneous analysis of two common food colorants have beensuccessfully researched developed applied and compared byreference HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination ofCarmosine and Ponceau 4R in synthetic mixtures and softdrinks without any interference from ingredients presentThe developed methods are simple (as there is no needfor pretreatment) rapid (as they require measurements of998779D1 and D1 values at a single wavelength) and direct (asthey estimate each colorant independently of the other) UVmethods offer a cost effective and time saving alternative toother methods for example colorimetric complexometricand chromatographic analyses The methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetricmethods Also themethodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities of the proposed meth-ods are almost comparable and can be used for the determina-tion of the two colorants in commercial drinks in the absenceof official method

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research has been supported by Scientific ResearchProject Coordination Center of Yildiz Technical University(Project no 2012-01-02-KAP07)

References

[1] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 1989

[2] J Vries Food Safety and Toxicity CRC London UK 1996[3] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination of

food dyes in soft drinks containing natural pigments by liquidchromatography with minimal clean-uprdquo Food Control vol 16no 3 pp 293ndash297 2005

[4] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination of carmoisine allura red and Ponceau4R in sweets and soft drinks by differential pulse polarographyrdquoJournal of Food Composition andAnalysis vol 18 no 6 pp 503ndash515 2005

[5] N Dossi R Toniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem forthe rapid in-channel amperometric detection of synthetic dyesin foodrdquo Journal of Electroanalytical Chemistry vol 601 no 1-2pp 1ndash7 2007

[6] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination of eight synthetic permitted and five commonlyencountered nonpermitted food colors in various foodmatrixesby high-performance liquid chromatographyrdquo Journal of AOACInternational vol 93 no 5 pp 1503ndash1514 2010

[7] S Dixit S K Khanna and M Das ldquoA simple method forsimultaneous determination of basic dyes encountered in foodpreparations by reversed-phase HPLCrdquo Journal of AOAC Inter-national vol 94 no 6 pp 1874ndash1881 2011

[8] N Yoshioka and K Ichihashi ldquoDetermination of 40 syntheticfood colors in drinks and candies by high-performance liquidchromatography using a short column with photodiode arraydetectionrdquo Talanta vol 74 no 5 pp 1408ndash1413 2008

[9] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination of synthetic dyes in selectedfoodstuffs by high performance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 107 no 1 pp 489ndash496 2008

[10] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination of water-soluble and fat-soluble synthetic colorantsin foodstuffbyhigh-performance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal ofChromatography A vol 1103 no 1 pp 170ndash176 2006

[11] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination of food dyes by firstderivative spectrophotometry with sorption onto polyurethanefoamrdquo Analytical Sciences vol 21 no 2 pp 149ndash153 2005

[12] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilfroush ldquoSimultaneous determination of carmoisine andPonceau 4Rrdquo Food Analytical Methods vol 1 no 3 pp 214ndash2192008

[13] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination of carmoisine andbrilliant blue FCF in food samplesrdquo Talanta vol 84 no 1 pp240ndash243 2011

[14] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan andB Asci ldquoAbilities of partial least-squares (PLS-2)multivariate calibration in the analysis of quaternary mixture offood colors (E-110 E-122 E-124 E-131)rdquo Analytical Letters vol40 no 10 pp 1893ndash1903 2007

[15] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectrofluorimetric determination of rhodamine dyein cosmeticsrdquo Talanta vol 75 no 1 pp 135ndash140 2008

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

Submit your manuscripts athttpwwwhindawicom

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CatalystsJournal of

Page 5: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

International Journal of Analytical Chemistry 5

0175

0375

0575

0775

300

350

400

450

500

550

600

650

700

Abso

rban

ce

Wavelength (nm)

C

Mix

P

minus0025

(a)

00002

00022

300

320

340

Firs

t der

ivat

ive

Wavelength (nm)

P

C

00002

00022

475

500

525

550

575

Firs

t der

ivat

ive

Wavelength (nm)

C

P

minus00098

minus00078

minus00058

minus00038

minus00018

minus00098

minus00078

minus00058

minus00038

minus00018

(b)

Figure 1 Zero-order spectra (a) of 10 120583gmLminus1 Carmoisine 10120583gmLminus1 Ponceau 4R and their binary mixture first derivative spectra (b) ofcolorants (Carmoisine(mdash) 2ndash10120583gmLminus1 Ponceau 4R (- -) 2ndash10120583gmLminus1)

transferred to 100mL volumetric flask and the volume wasmade up with distilled water A series of dilution wasprepared quantitatively with water from this solution toobtain standard solutions to reach the concentration ranges ofcalibration curves graphed for each of the proposed methods(DSM RDM and CM) For the DDM method the sameprocedure was used but in this case the final volume wasmade up with 01 N HCI and 01 N NaOH separately forthe determination of Carmoisine and Ponceau 4R HPLCmethod the same sample preparation procedure describedabove was followed using a mixture 1 1 vv of methanolmdashTBA buffer solution After dissolution process preparedsolutions were filtered using 045 120583m disposable membranefilters (Sartrious Minisart 045120583m) by using a syringe Thefinal solutionwas diluted to theworking concentration range

27 Method Validation The validity and suitability of theproposed methods were assessed by accuracy (reported aspercentage recovered) precision (reported as RSD) lin-earity (evaluated by regression equation) limit of detection(LOD) and limit of quantification (LOQ) [21]

LOD and LOQ were based on the standard deviation ofresponse and the slope of the corresponding curve using thefollowing equations

LOD = 3 119904119898 LOQ = 10 119904

119898 (1)

where 119904 the noise estimate is the standard deviation of thederivative amplitude of the blank and 119898 is the slope of therelated calibration graphs [22]

The linearity of the methods was established in theconcentration ranges of 2ndash10120583gmLminus1 for Carmoisine and

Ponceau 4R respectively In this study five concentrationswere analyzed in five replicates The linearity was evaluatedby the least square regression method

In order to demonstrate the validity and suitability ofthe proposed methods intra- and interday variability studieswere performed at three different concentrations (2 6 and10 120583gmLminus1) in the presence of the certain concentration (26 and 10 120583gmLminus1) of the other colorants three times in a dayand also three different days Method efficiency was tested interms of RSDrsquos for both intraday and interday precisions

The precision was ascertained by carrying out five repli-cate determinations of synthetic mixtures at different con-centration ratios of Carmoisine and Ponceau 4R Recoverytest also confirmed the accuracy and applicability of theproposed methods by analyzing several synthetic mixturesof Carmoisine and Ponceau 4R which reproduced differentcomposition ratios within the linearity rangeThese ratios arealso the ratios in drinksThemean percentage recoveries andRSD values were calculated

Recovery of the colorants of interest from a given matrixcan be used as a measure of the accuracy or the bias of themethod The same ranges of concentrations as employed inthe linearity studies were used

In our study the applicability of the derivative spec-trophotometric methods was also validated by comparisonwith the HPLC technique

3 Result and Discussion

Figure 1(a) shows the absorption spectra of CarmoisinePonceau 4R and themixture of them in the wavelength rangeof 300ndash700 nm Zero-order absorption spectra of Carmoisine

6 International Journal of Analytical Chemistry

Table 3 Method validation for the simultaneous determination of Carmoisine and Ponceau 4R in laboratory prepared mixtures by theproposed methods

Method Accuracy (meanlowastplusmn RSD ) Precision repeatabilitya(mean plusmn RSD )

Intermediate precisionb

(mean plusmn RSD )

DSM C (1D331) 1008 plusmn 087 1001 plusmn 096 9991 plusmn 102

P (1D517) 991 plusmn 102 1005 plusmn 105 1001 plusmn 092

RDM C (1D323) 1010 plusmn 095 9998 plusmn 099 1006 plusmn 105

P (1D344) 9825 plusmn 093 9983 plusmn 098 1004 plusmn 096

DDM C (1D366) 1004 plusmn 107 9895 plusmn 105 9992 plusmn 098

P (1D451) 9955 plusmn 095 1005 plusmn 095 1007 plusmn 096

CM C (1D4721D569) 9975 plusmn 102 998 plusmn 101 1001 plusmn 097

P (1D4701D554) 985 plusmn 106 9975 plusmn 103 1003 plusmn 106

aThe intraday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times within the daybThe interday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times in three successive dayslowastThe values of recovery are an average of five replicates of each of five synthetic mixtures at different concentration ratios of C and P (2ndash10 120583gmLminus1)RSD relative standard deviation

and Ponceau 4R in distilled water gave rise to maximumpeaks at 517 nm and 508 nm respectively As can be seenthe spectra of the colorants were strongly overlapped in the400ndash600 nm wavelength region Hence the direct absorp-tion measurement for assaying binary mixture seems to beimpossible On the other hand the absorption spectra of thetwo components within a wavelength range of 300ndash380 nmwere less overlapped than the region 400ndash600 nm So in thiswork the region of 300ndash380 nm was chosen for the analysisof Carmoisine In this spectral range DSM RDM and DDMwere used for the determination of the studied colorants inlaboratory prepared mixtures and soft drink samples Thesemethods are also applied to analyze Ponceau 4R in the region320ndash520 nm

31 Derivative Spectrophotometric Method (DSM) Undercomputer-controlled instrumentation derivative spectro-photometry played a very important role in the resolutionof band overlapping in quantitative analysis [23] Figure 1(b)shows the first derivative spectra of Carmoisine and Ponceau4R As can be seen in Figure 1(b) in the region of 300ndash520 nm the first derivative spectra allowed the simultaneousdetermination of Carmoisine and Ponceau 4R At this regionthe 1D spectra (Figure 1(b)) show a characteristic peak at331 nm for Carmoisine while Ponceau 4R shows no responseTherefore the absolute values of the 1D peak amplitudes at331 nm can be used to quantify Carmoisine in this mixtureOn the other hand Ponceau 4R can be assayed in the presenceof Carmoisine by measuring its 1D response at 517 nm (zero-crossing point of Carmoisine)

Under the experimental conditions described the cal-ibration graphs obtained by plotting the derivative valuesversus concentrations for Carmoisine and Ponceau 4R inthe concentration range stated in Table 2 exhibited linearrelationships Statistical analysis of the data showed thatlinearity of the calibration graphs and compliance withBeerrsquos law were validated and the small values of the inter-cepts as illustrated by the excellent values of correlation

coefficients of the regression equations To prove the valid-ity and applicability of the proposed method laboratory-prepared mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R (Table 3) The obtainedresults for the analysis of these mixtures in five replicates bythe described method showed high accuracy and precisionof the proposed method (Table 3)

32 Ratio Derivative Method (RDM) While the main dis-advantages of the zero-crossing method in derivative spec-trophotometry for resolving a mixture of components withoverlapped spectra are the risk of small drifts of the workingwavelengths and the circumstance that the working wave-lengths generally do not fall in correspondence of peaks ofthe derivative spectrum this may be particularly dangerouswhen the slope of the spectrum is very high with consequentloss of accuracy and precision and the working wavelengthis in proximity of the base of the spectrum which causespoor sensitivity The main advantage of the RDM is thechance of doing easy measurements in correspondence ofpeaks so it permits the use of the wavelength of highestvalue of analytical signals (a maximum or a minimum) andmoreover the presence of a lot of maxima and minima isanother advantage by the fact that these wavelengths give anopportunity for the determination of active compounds in thepresence of other compounds and ingredients which possiblyinterfere in the assay [24ndash27]

This method is based on the use of the first derivativeof the ratio spectra The ratio spectra were obtained bydividing the absorption spectrum of the mixture by thatof one of the components As we depicted above directabsorption measurements are not possible due to spectraloverlap This spectral overlapping was sufficiently enough todemonstrate the resolving power of the proposed methodFigure 2(a) shows the first ratio derivative spectra of differ-ent Carmoisine standard solutions (spectra divided by thespectrum of 6 120583gmLminus1 Ponceau 4R solution) The ratio firstderivative amplitudes at 323 nm (1DD

323) corresponding to

International Journal of Analytical Chemistry 7

001

003

308 328 348 368

Firs

t der

ivat

ive

Carm

oisin

ePo

ncea

u 4R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

Firs

t der

ivat

ive

Ponc

eau

4RC

arm

oisin

e

Wavelength (nm)

fg

hi

j

minus002678

minus001678

minus000678

(b)

Figure 2 First derivative ratio spectra of Carmoisine (a) and Ponceau 4R (b) for different concentrations (Carmoisine (6 120583gmLminus1 Ponceau4R was used as a divisor) a 2 120583gmLminus1 b 4 120583gmLminus1 c 6120583gmLminus1 d 8 120583gmLminus1 e 10120583gmLminus1 Ponceau 4R (6120583gmLminus1 Carmoisine was usedas a divisor) f 2120583gmLminus1 g 4 120583gmLminus1 h 6 120583gmLminus1 i 8120583gmLminus1 j 10120583gmLminus1)

Table 4 Assay results for the determination of Carmoisine and Ponceau 4R in soft drinks using the proposed methods and the referenceHPLC method (NMKL 130)

Methods Analyte Selected wavelengths(nm)

Assay results mean plusmn SDlowast (119905calculated 119865calculated)lowastlowast

Soft drink I (120583g 100mLminus1) Soft drink II (120583g 100mLminus1)

DSM C 1D331 930 plusmn 022 (068 145) 628 plusmn 029 (061 144)

P 1D517 220 plusmn 011 (118 118) 139 plusmn 013 (040 179)

RDM C 1D323 926 plusmn 023 (037 145) 636 plusmn 021 (128 120)

P 1D344 235 plusmn 011 (044 125) 143 plusmn 013 (013 169)

DDM C 1D366 931 plusmn 024 (073 169) 641 plusmn 024 (153 164)

P 1D451 249 plusmn 017 (189 302) 144 plusmn 016 (023 268)

CM C 1D4721D569 930 plusmn 026 (063 188) 636 plusmn 028 (107 211)

P 1D4701D554 230 plusmn 016 (023 256) 143 plusmn 019 (010 368)

HPLC C 520 921 plusmn 018 620 plusmn 019

P 512 232 plusmn 010 142 plusmn 010

lowastResults obtained are the average of five experiments for each method SD standard deviationlowastlowastThe corresponding theoretical value for 119905 and 119865 at 119875 005 (119905theoretical 231 119865theoretical 639)

a maximum wavelength are proportional to the Carmoisineconcentration (Figure 2(a)) To determine the other com-ponent Ponceau 4R an analogues procedure was followedFor determination of Ponceau 4R the stored spectra ofthe mixtures are divided by a standard spectrum of 6mgLminus1 Carmoisine (Figure 2(b)) Standard solutions of Ponceau4R and the content of Ponceau 4R were determined bymeasuring the signals at 344 nm (1DD

344) Figure 2(b)

Under the described experimental conditions the cal-ibration graphs obtained by plotting the derivative valuesof each colorant versus concentration in the concentration

range stated in Table 2 showed linear relationships Con-formity with Beerrsquos law was evident in the concentrationrange of the final dilution cited in Table 2 For this methodthe characteristic parameters of regression equations andcorrelation coefficients are given in Table 2 The correlationcoefficients were 09996 and 09994 indicating good linearityLOD and LOQ were calculated for each colorant and areshown in the same table The detection limits were foundto be 0079120583gmLminus1 for Carmoisine and 0082 120583gmLminus1 forPonceau 4R while the quantification limits were estimatedto be 0263 120583gmLminus1 for Carmoisine and 0273120583gmLminus1 for

8 International Journal of Analytical Chemistry

000988

008988

016988

024988

032988

300 400 500 600 700

Abso

rban

ce

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

Diff

eren

ce fi

rst d

eriv

ativ

e

Wavelength (nm)

C

P

minus00068

minus00028

(b)

Figure 3 Difference absorption spectra of 10 120583gmLminus1 Carmoisine 10 120583gmLminus1 Ponceau 4R and their binary mixture (a) Difference firstderivative spectra (b) of colorants (Carmoisine 2ndash10120583gmLminus1 Ponceau 4R 2ndash10 120583gmLminus1)

Ponceau 4R The proposed method was also applied to thedrink samples (Table 4) The accuracy of RDM was checkedby analyzing the laboratory preparedmixtures of Carmoisineand Ponceau 4R at various concentration ratios ranging from2 to 10 120583gmLminus1 (Table 3) All data represent the average offive determinations Low values of relative standard deviationindicate very good precision Satisfactory recoveries withsmall standard deviations were obtained which indicated thehigh repeatability and accuracy of the method

33 Derivative DifferentialMethod (DDM) Differential spec-trophotometry (ΔD

1) based on pH changes has also been

reported to be useful in the determination of binary mix-tures This method that depends on utilization of differenceabsorption spectra corresponding to the same compoundobtained at two different pHrsquos has been investigated for theanalysis of binary mixtures The procedure is comprised ofthe measurement of ΔD

1food colorantsrsquo in acidic solutions

against their alkaline solutions as blanks The ΔD1has

been successfully used to eliminate interferences from drinksamples There are few reports on utilization of the abovetwo combined techniques (difference and derivative) for theestimation of individual drug substances and for combinedpreparations [28ndash30]

The difference absorption spectra of Carmoisine Pon-ceau 4R and their binary mixture in 01 N NaOH and in01 N HCI are shown in Figure 3(a) Figure 3(b) shows thefirst derivative difference spectra of colorants in the concen-tration range 2ndash10 120583gmLminus1 The spectra of both colorantsin Figure 3(b) offer an advantage for their simultaneousdetermination by having zero-crossing points In particularthe first derivative amplitudes at 366 nm for Carmoisine and

451 nm for Ponceau 4R in the Carmoisine and Ponceau 4Rmixture were considered as the optimum working wave-lengths for their determination By measuring the values ofthe ΔD

1amplitudes at these wavelengths the concentration

of each colorant can be directly calculated since the dif-ferential first derivative measurement cancels the irrelevantabsorbance due to the drinks matrix at these wavelengths

Under the experimental conditions described the cali-bration graphs obtained by plotting the derivative values ofeach colorant in the mixture versus concentration in therange concentration stated in Table 2 show linear relation-ships The correlation coefficients were 09996 and 09998indicating good linearity The LOD were 0071 120583gmLminus1 forCarmoisine and 0081 120583gmLminus1 for Ponceau 4R while theLOQ were 0236 120583gmLminus1 for Carmoisine and 0270 120583gmLminus1for Ponceau 4R The proposed procedure was success-fully applied for the determination of these colorants inlaboratory-prepared mixtures and drink samples The resultsare presented in Tables 3 and 4The values of relative standarddeviation indicate very good reproducibility In additionapplication of the DDMwas found to be correct for the drinkmatrix interference and to enhance the sensitivity

34 CompensationMethod (CM) Thecompensationmethodis a nonmathematical method for the detection and elimi-nation of unwanted absorption during spectrophotometricanalysis [19 20 31 32] The accuracy of the method dependson the evaluation of the balance point Figure 1(b) showsthe first derivative spectra of Carmoisine and Ponceau 4Rin the concentration range 2ndash10 120583gmLminus1 The first derivativespectra were recorded for each reference solution of thecomponents and the ratios of the 1D maximum and 1D

International Journal of Analytical Chemistry 9

000011000211000411000611

401 451 501 551 601Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

Firs

t der

ivat

ive

(a)

000080002800048

400 440 480 520 560 600

Firs

t der

ivat

ive

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)

Figure 4 (a) First derivative spectra between the mixture solution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in the sample cell anddifferent concentrations (2ndash10120583gmLminus1) of Ponceau 4R solutions in the reference cell First derivative spectra between the mixture solution(6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and 6120583gmLminus1 Ponceau 4R in the reference cell at balance points andcomparison of these spectra with 6 120583gmLminus1 concentration of Carmoisine (- - -) spectrum (b) First derivative spectra between the mixturesolution (6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and different concentrations (2ndash10120583gmLminus1) of Carmoisinesolutions in the reference cell Derivative spectra between the mixture solution (6120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in thesample cell and 6 120583gmLminus1 Carmoisine in the reference cell at balance points and comparison of these spectra with 6120583gmLminus1 concentrationof Ponceau 4R (- - -) spectrum

minimum were calculated Table 1 shows the mean valuesof the ratios calculated for five different determinations foreach solutionThe ratios are constant and characteristic of thepure substance independent of concentration and presenceof another component For the determination of Carmoisineconcentrations in Carmoisine-Ponceau 4R binary mixturesand drink samples the sample cell was filled with themixturesolution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R)and the reference cell was filled in succession with aseries of reference Ponceau 4R with solutions in differentconcentrations Ponceau 4R as shown in Figure 4(a) Theratios of the mixture calculated from the recorded 1D spectrawere compared with those of Carmoisine At the exactbalance point the ratio of the mixture corresponds to thatof Carmoisine where the concentration of Carmoisine in themixtures in the sample cell is equal to that of the referencesolution in the reference cell For determining the othercomponent the same steps were followed using solutions ofpure compoundCarmoisine in the reference cell to determineits concentration in the mixture (Figure 4(b)) Conformitywith Beerrsquos law was evident in the concentration range from 2to 10 120583gmLminus1 of Carmoisine and Ponceau 4R In thismethodsynthetic mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R and the recoveries ofthe method were found to be in the range of 9850 plusmn 105ndash9975 plusmn 110 The presented results in Table 3 are in goodagreement with the other proposed methods

35 Application of the Developed Methods to the Soft DrinksThe developed and validated methods were successfullyapplied to the determination of Carmoisine and Ponceau 4Rcontent in the commercial soft drinks The obtained resultsare presented in Table 4 Good agreement was observed forthe assay results of the drinks by application of the fourmethods in this paper (Table 4) The values of standarddeviation indicate very good reproducibility The results ofthe proposed spectrophotometric methods were statisticallycompared with the results of the HPLC method given in

the literature [18] at the 95 confidence level with studentrsquos 119905-test and by the variance ratio 119865-test (Table 4) The calculated119905- and119865-values never exceed the theoretical 119905- and119865-values at005 level of significant difference There was no interferenceof ingredients in the products examined so no additiveextraction or separation procedures were required duringtheir assay The results of all methods were very close toeach other This suggests that the four methods are equallyapplicable

36 Validation of the Proposed Methods

361 Linearity The linearity of the proposed methods wasevaluated under different concentrations of Carmoisine andPonceau 4R within the concentration range stated in Table 2The good linearity of the calibration graphs and negligiblescatter of the experimental points is clearly evident by thevalues of the correlation coefficients and variances around theslope (Table 2)

362 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD is expressed as the analyte concentration cor-responding to the sample blank value plus three standarddeviations and LOQ is the analyte concentration correspond-ing to the sample blank value ten standard deviations [21]All the proposed methods data was presented in Table 2The LOD and LOQ values found were between 0071 and0086 and 0236 and 0286 120583gmLminus1 for Carmoisine and 0081and 0091 and 0270 and 0304 120583gmLminus1 for Ponceau 4Rrespectively The data shows that the methods are sensitivefor the determination of Carmoisine and Ponceau 4R

363 Precision (Repeatability) and Accuracy (Recovery )The precision of the developed methods was expressedas a percentage of relative standard deviation (RSD) forrepeatability (intraday precision) and intermediate precision(interday precision) The data obtained were less than 106(Table 3) indicating reasonable repeatability of the proposed

10 International Journal of Analytical Chemistry

methods The intraday and interday accuracy ranges werefrom 9895 to 1006 for Carmoisine and from 9975 to1007 for Ponceau 4R respectively The results obtainedfrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria To confirm the accuracy of the proposed methodssyntheticmixtures of different concentration ratios consistingof each food colorant were prepared The resulting mixtureswere assayed according to above proposed procedures infive replicates and the average results were calculated asthe percentage of analyte recovered The percentages meanaccuracy for Carmoisine were 1008 plusmn 087 for method DSM1010 plusmn 095 for RDM 10040 plusmn 107 for DDM and 9975 plusmn102 for CM respectively And those of P were 9910 plusmn 102for method DSM 9825 plusmn 093 for RDM 9955 plusmn 095 forDDM and 9850 plusmn 106 for CM The good recovery percent(RSD) assured the high accuracy of the proposed methods(Table 3) The relative standard deviations were found tobe less than 107 and 106 for Carmoisine and Ponceau4R respectively indicating reasonable repeatability of theproposed methods

4 Conclusion

Four derivative spectrophotometric methods for the simul-taneous analysis of two common food colorants have beensuccessfully researched developed applied and compared byreference HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination ofCarmosine and Ponceau 4R in synthetic mixtures and softdrinks without any interference from ingredients presentThe developed methods are simple (as there is no needfor pretreatment) rapid (as they require measurements of998779D1 and D1 values at a single wavelength) and direct (asthey estimate each colorant independently of the other) UVmethods offer a cost effective and time saving alternative toother methods for example colorimetric complexometricand chromatographic analyses The methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetricmethods Also themethodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities of the proposed meth-ods are almost comparable and can be used for the determina-tion of the two colorants in commercial drinks in the absenceof official method

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research has been supported by Scientific ResearchProject Coordination Center of Yildiz Technical University(Project no 2012-01-02-KAP07)

References

[1] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 1989

[2] J Vries Food Safety and Toxicity CRC London UK 1996[3] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination of

food dyes in soft drinks containing natural pigments by liquidchromatography with minimal clean-uprdquo Food Control vol 16no 3 pp 293ndash297 2005

[4] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination of carmoisine allura red and Ponceau4R in sweets and soft drinks by differential pulse polarographyrdquoJournal of Food Composition andAnalysis vol 18 no 6 pp 503ndash515 2005

[5] N Dossi R Toniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem forthe rapid in-channel amperometric detection of synthetic dyesin foodrdquo Journal of Electroanalytical Chemistry vol 601 no 1-2pp 1ndash7 2007

[6] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination of eight synthetic permitted and five commonlyencountered nonpermitted food colors in various foodmatrixesby high-performance liquid chromatographyrdquo Journal of AOACInternational vol 93 no 5 pp 1503ndash1514 2010

[7] S Dixit S K Khanna and M Das ldquoA simple method forsimultaneous determination of basic dyes encountered in foodpreparations by reversed-phase HPLCrdquo Journal of AOAC Inter-national vol 94 no 6 pp 1874ndash1881 2011

[8] N Yoshioka and K Ichihashi ldquoDetermination of 40 syntheticfood colors in drinks and candies by high-performance liquidchromatography using a short column with photodiode arraydetectionrdquo Talanta vol 74 no 5 pp 1408ndash1413 2008

[9] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination of synthetic dyes in selectedfoodstuffs by high performance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 107 no 1 pp 489ndash496 2008

[10] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination of water-soluble and fat-soluble synthetic colorantsin foodstuffbyhigh-performance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal ofChromatography A vol 1103 no 1 pp 170ndash176 2006

[11] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination of food dyes by firstderivative spectrophotometry with sorption onto polyurethanefoamrdquo Analytical Sciences vol 21 no 2 pp 149ndash153 2005

[12] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilfroush ldquoSimultaneous determination of carmoisine andPonceau 4Rrdquo Food Analytical Methods vol 1 no 3 pp 214ndash2192008

[13] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination of carmoisine andbrilliant blue FCF in food samplesrdquo Talanta vol 84 no 1 pp240ndash243 2011

[14] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan andB Asci ldquoAbilities of partial least-squares (PLS-2)multivariate calibration in the analysis of quaternary mixture offood colors (E-110 E-122 E-124 E-131)rdquo Analytical Letters vol40 no 10 pp 1893ndash1903 2007

[15] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectrofluorimetric determination of rhodamine dyein cosmeticsrdquo Talanta vol 75 no 1 pp 135ndash140 2008

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Page 6: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

6 International Journal of Analytical Chemistry

Table 3 Method validation for the simultaneous determination of Carmoisine and Ponceau 4R in laboratory prepared mixtures by theproposed methods

Method Accuracy (meanlowastplusmn RSD ) Precision repeatabilitya(mean plusmn RSD )

Intermediate precisionb

(mean plusmn RSD )

DSM C (1D331) 1008 plusmn 087 1001 plusmn 096 9991 plusmn 102

P (1D517) 991 plusmn 102 1005 plusmn 105 1001 plusmn 092

RDM C (1D323) 1010 plusmn 095 9998 plusmn 099 1006 plusmn 105

P (1D344) 9825 plusmn 093 9983 plusmn 098 1004 plusmn 096

DDM C (1D366) 1004 plusmn 107 9895 plusmn 105 9992 plusmn 098

P (1D451) 9955 plusmn 095 1005 plusmn 095 1007 plusmn 096

CM C (1D4721D569) 9975 plusmn 102 998 plusmn 101 1001 plusmn 097

P (1D4701D554) 985 plusmn 106 9975 plusmn 103 1003 plusmn 106

aThe intraday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times within the daybThe interday (119899 = 3) average of three concentrations (2 6 and 10 120583gmLminus1) for Carmoisine and Ponceau 4R repeated three times in three successive dayslowastThe values of recovery are an average of five replicates of each of five synthetic mixtures at different concentration ratios of C and P (2ndash10 120583gmLminus1)RSD relative standard deviation

and Ponceau 4R in distilled water gave rise to maximumpeaks at 517 nm and 508 nm respectively As can be seenthe spectra of the colorants were strongly overlapped in the400ndash600 nm wavelength region Hence the direct absorp-tion measurement for assaying binary mixture seems to beimpossible On the other hand the absorption spectra of thetwo components within a wavelength range of 300ndash380 nmwere less overlapped than the region 400ndash600 nm So in thiswork the region of 300ndash380 nm was chosen for the analysisof Carmoisine In this spectral range DSM RDM and DDMwere used for the determination of the studied colorants inlaboratory prepared mixtures and soft drink samples Thesemethods are also applied to analyze Ponceau 4R in the region320ndash520 nm

31 Derivative Spectrophotometric Method (DSM) Undercomputer-controlled instrumentation derivative spectro-photometry played a very important role in the resolutionof band overlapping in quantitative analysis [23] Figure 1(b)shows the first derivative spectra of Carmoisine and Ponceau4R As can be seen in Figure 1(b) in the region of 300ndash520 nm the first derivative spectra allowed the simultaneousdetermination of Carmoisine and Ponceau 4R At this regionthe 1D spectra (Figure 1(b)) show a characteristic peak at331 nm for Carmoisine while Ponceau 4R shows no responseTherefore the absolute values of the 1D peak amplitudes at331 nm can be used to quantify Carmoisine in this mixtureOn the other hand Ponceau 4R can be assayed in the presenceof Carmoisine by measuring its 1D response at 517 nm (zero-crossing point of Carmoisine)

Under the experimental conditions described the cal-ibration graphs obtained by plotting the derivative valuesversus concentrations for Carmoisine and Ponceau 4R inthe concentration range stated in Table 2 exhibited linearrelationships Statistical analysis of the data showed thatlinearity of the calibration graphs and compliance withBeerrsquos law were validated and the small values of the inter-cepts as illustrated by the excellent values of correlation

coefficients of the regression equations To prove the valid-ity and applicability of the proposed method laboratory-prepared mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R (Table 3) The obtainedresults for the analysis of these mixtures in five replicates bythe described method showed high accuracy and precisionof the proposed method (Table 3)

32 Ratio Derivative Method (RDM) While the main dis-advantages of the zero-crossing method in derivative spec-trophotometry for resolving a mixture of components withoverlapped spectra are the risk of small drifts of the workingwavelengths and the circumstance that the working wave-lengths generally do not fall in correspondence of peaks ofthe derivative spectrum this may be particularly dangerouswhen the slope of the spectrum is very high with consequentloss of accuracy and precision and the working wavelengthis in proximity of the base of the spectrum which causespoor sensitivity The main advantage of the RDM is thechance of doing easy measurements in correspondence ofpeaks so it permits the use of the wavelength of highestvalue of analytical signals (a maximum or a minimum) andmoreover the presence of a lot of maxima and minima isanother advantage by the fact that these wavelengths give anopportunity for the determination of active compounds in thepresence of other compounds and ingredients which possiblyinterfere in the assay [24ndash27]

This method is based on the use of the first derivativeof the ratio spectra The ratio spectra were obtained bydividing the absorption spectrum of the mixture by thatof one of the components As we depicted above directabsorption measurements are not possible due to spectraloverlap This spectral overlapping was sufficiently enough todemonstrate the resolving power of the proposed methodFigure 2(a) shows the first ratio derivative spectra of differ-ent Carmoisine standard solutions (spectra divided by thespectrum of 6 120583gmLminus1 Ponceau 4R solution) The ratio firstderivative amplitudes at 323 nm (1DD

323) corresponding to

International Journal of Analytical Chemistry 7

001

003

308 328 348 368

Firs

t der

ivat

ive

Carm

oisin

ePo

ncea

u 4R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

Firs

t der

ivat

ive

Ponc

eau

4RC

arm

oisin

e

Wavelength (nm)

fg

hi

j

minus002678

minus001678

minus000678

(b)

Figure 2 First derivative ratio spectra of Carmoisine (a) and Ponceau 4R (b) for different concentrations (Carmoisine (6 120583gmLminus1 Ponceau4R was used as a divisor) a 2 120583gmLminus1 b 4 120583gmLminus1 c 6120583gmLminus1 d 8 120583gmLminus1 e 10120583gmLminus1 Ponceau 4R (6120583gmLminus1 Carmoisine was usedas a divisor) f 2120583gmLminus1 g 4 120583gmLminus1 h 6 120583gmLminus1 i 8120583gmLminus1 j 10120583gmLminus1)

Table 4 Assay results for the determination of Carmoisine and Ponceau 4R in soft drinks using the proposed methods and the referenceHPLC method (NMKL 130)

Methods Analyte Selected wavelengths(nm)

Assay results mean plusmn SDlowast (119905calculated 119865calculated)lowastlowast

Soft drink I (120583g 100mLminus1) Soft drink II (120583g 100mLminus1)

DSM C 1D331 930 plusmn 022 (068 145) 628 plusmn 029 (061 144)

P 1D517 220 plusmn 011 (118 118) 139 plusmn 013 (040 179)

RDM C 1D323 926 plusmn 023 (037 145) 636 plusmn 021 (128 120)

P 1D344 235 plusmn 011 (044 125) 143 plusmn 013 (013 169)

DDM C 1D366 931 plusmn 024 (073 169) 641 plusmn 024 (153 164)

P 1D451 249 plusmn 017 (189 302) 144 plusmn 016 (023 268)

CM C 1D4721D569 930 plusmn 026 (063 188) 636 plusmn 028 (107 211)

P 1D4701D554 230 plusmn 016 (023 256) 143 plusmn 019 (010 368)

HPLC C 520 921 plusmn 018 620 plusmn 019

P 512 232 plusmn 010 142 plusmn 010

lowastResults obtained are the average of five experiments for each method SD standard deviationlowastlowastThe corresponding theoretical value for 119905 and 119865 at 119875 005 (119905theoretical 231 119865theoretical 639)

a maximum wavelength are proportional to the Carmoisineconcentration (Figure 2(a)) To determine the other com-ponent Ponceau 4R an analogues procedure was followedFor determination of Ponceau 4R the stored spectra ofthe mixtures are divided by a standard spectrum of 6mgLminus1 Carmoisine (Figure 2(b)) Standard solutions of Ponceau4R and the content of Ponceau 4R were determined bymeasuring the signals at 344 nm (1DD

344) Figure 2(b)

Under the described experimental conditions the cal-ibration graphs obtained by plotting the derivative valuesof each colorant versus concentration in the concentration

range stated in Table 2 showed linear relationships Con-formity with Beerrsquos law was evident in the concentrationrange of the final dilution cited in Table 2 For this methodthe characteristic parameters of regression equations andcorrelation coefficients are given in Table 2 The correlationcoefficients were 09996 and 09994 indicating good linearityLOD and LOQ were calculated for each colorant and areshown in the same table The detection limits were foundto be 0079120583gmLminus1 for Carmoisine and 0082 120583gmLminus1 forPonceau 4R while the quantification limits were estimatedto be 0263 120583gmLminus1 for Carmoisine and 0273120583gmLminus1 for

8 International Journal of Analytical Chemistry

000988

008988

016988

024988

032988

300 400 500 600 700

Abso

rban

ce

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

Diff

eren

ce fi

rst d

eriv

ativ

e

Wavelength (nm)

C

P

minus00068

minus00028

(b)

Figure 3 Difference absorption spectra of 10 120583gmLminus1 Carmoisine 10 120583gmLminus1 Ponceau 4R and their binary mixture (a) Difference firstderivative spectra (b) of colorants (Carmoisine 2ndash10120583gmLminus1 Ponceau 4R 2ndash10 120583gmLminus1)

Ponceau 4R The proposed method was also applied to thedrink samples (Table 4) The accuracy of RDM was checkedby analyzing the laboratory preparedmixtures of Carmoisineand Ponceau 4R at various concentration ratios ranging from2 to 10 120583gmLminus1 (Table 3) All data represent the average offive determinations Low values of relative standard deviationindicate very good precision Satisfactory recoveries withsmall standard deviations were obtained which indicated thehigh repeatability and accuracy of the method

33 Derivative DifferentialMethod (DDM) Differential spec-trophotometry (ΔD

1) based on pH changes has also been

reported to be useful in the determination of binary mix-tures This method that depends on utilization of differenceabsorption spectra corresponding to the same compoundobtained at two different pHrsquos has been investigated for theanalysis of binary mixtures The procedure is comprised ofthe measurement of ΔD

1food colorantsrsquo in acidic solutions

against their alkaline solutions as blanks The ΔD1has

been successfully used to eliminate interferences from drinksamples There are few reports on utilization of the abovetwo combined techniques (difference and derivative) for theestimation of individual drug substances and for combinedpreparations [28ndash30]

The difference absorption spectra of Carmoisine Pon-ceau 4R and their binary mixture in 01 N NaOH and in01 N HCI are shown in Figure 3(a) Figure 3(b) shows thefirst derivative difference spectra of colorants in the concen-tration range 2ndash10 120583gmLminus1 The spectra of both colorantsin Figure 3(b) offer an advantage for their simultaneousdetermination by having zero-crossing points In particularthe first derivative amplitudes at 366 nm for Carmoisine and

451 nm for Ponceau 4R in the Carmoisine and Ponceau 4Rmixture were considered as the optimum working wave-lengths for their determination By measuring the values ofthe ΔD

1amplitudes at these wavelengths the concentration

of each colorant can be directly calculated since the dif-ferential first derivative measurement cancels the irrelevantabsorbance due to the drinks matrix at these wavelengths

Under the experimental conditions described the cali-bration graphs obtained by plotting the derivative values ofeach colorant in the mixture versus concentration in therange concentration stated in Table 2 show linear relation-ships The correlation coefficients were 09996 and 09998indicating good linearity The LOD were 0071 120583gmLminus1 forCarmoisine and 0081 120583gmLminus1 for Ponceau 4R while theLOQ were 0236 120583gmLminus1 for Carmoisine and 0270 120583gmLminus1for Ponceau 4R The proposed procedure was success-fully applied for the determination of these colorants inlaboratory-prepared mixtures and drink samples The resultsare presented in Tables 3 and 4The values of relative standarddeviation indicate very good reproducibility In additionapplication of the DDMwas found to be correct for the drinkmatrix interference and to enhance the sensitivity

34 CompensationMethod (CM) Thecompensationmethodis a nonmathematical method for the detection and elimi-nation of unwanted absorption during spectrophotometricanalysis [19 20 31 32] The accuracy of the method dependson the evaluation of the balance point Figure 1(b) showsthe first derivative spectra of Carmoisine and Ponceau 4Rin the concentration range 2ndash10 120583gmLminus1 The first derivativespectra were recorded for each reference solution of thecomponents and the ratios of the 1D maximum and 1D

International Journal of Analytical Chemistry 9

000011000211000411000611

401 451 501 551 601Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

Firs

t der

ivat

ive

(a)

000080002800048

400 440 480 520 560 600

Firs

t der

ivat

ive

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)

Figure 4 (a) First derivative spectra between the mixture solution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in the sample cell anddifferent concentrations (2ndash10120583gmLminus1) of Ponceau 4R solutions in the reference cell First derivative spectra between the mixture solution(6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and 6120583gmLminus1 Ponceau 4R in the reference cell at balance points andcomparison of these spectra with 6 120583gmLminus1 concentration of Carmoisine (- - -) spectrum (b) First derivative spectra between the mixturesolution (6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and different concentrations (2ndash10120583gmLminus1) of Carmoisinesolutions in the reference cell Derivative spectra between the mixture solution (6120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in thesample cell and 6 120583gmLminus1 Carmoisine in the reference cell at balance points and comparison of these spectra with 6120583gmLminus1 concentrationof Ponceau 4R (- - -) spectrum

minimum were calculated Table 1 shows the mean valuesof the ratios calculated for five different determinations foreach solutionThe ratios are constant and characteristic of thepure substance independent of concentration and presenceof another component For the determination of Carmoisineconcentrations in Carmoisine-Ponceau 4R binary mixturesand drink samples the sample cell was filled with themixturesolution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R)and the reference cell was filled in succession with aseries of reference Ponceau 4R with solutions in differentconcentrations Ponceau 4R as shown in Figure 4(a) Theratios of the mixture calculated from the recorded 1D spectrawere compared with those of Carmoisine At the exactbalance point the ratio of the mixture corresponds to thatof Carmoisine where the concentration of Carmoisine in themixtures in the sample cell is equal to that of the referencesolution in the reference cell For determining the othercomponent the same steps were followed using solutions ofpure compoundCarmoisine in the reference cell to determineits concentration in the mixture (Figure 4(b)) Conformitywith Beerrsquos law was evident in the concentration range from 2to 10 120583gmLminus1 of Carmoisine and Ponceau 4R In thismethodsynthetic mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R and the recoveries ofthe method were found to be in the range of 9850 plusmn 105ndash9975 plusmn 110 The presented results in Table 3 are in goodagreement with the other proposed methods

35 Application of the Developed Methods to the Soft DrinksThe developed and validated methods were successfullyapplied to the determination of Carmoisine and Ponceau 4Rcontent in the commercial soft drinks The obtained resultsare presented in Table 4 Good agreement was observed forthe assay results of the drinks by application of the fourmethods in this paper (Table 4) The values of standarddeviation indicate very good reproducibility The results ofthe proposed spectrophotometric methods were statisticallycompared with the results of the HPLC method given in

the literature [18] at the 95 confidence level with studentrsquos 119905-test and by the variance ratio 119865-test (Table 4) The calculated119905- and119865-values never exceed the theoretical 119905- and119865-values at005 level of significant difference There was no interferenceof ingredients in the products examined so no additiveextraction or separation procedures were required duringtheir assay The results of all methods were very close toeach other This suggests that the four methods are equallyapplicable

36 Validation of the Proposed Methods

361 Linearity The linearity of the proposed methods wasevaluated under different concentrations of Carmoisine andPonceau 4R within the concentration range stated in Table 2The good linearity of the calibration graphs and negligiblescatter of the experimental points is clearly evident by thevalues of the correlation coefficients and variances around theslope (Table 2)

362 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD is expressed as the analyte concentration cor-responding to the sample blank value plus three standarddeviations and LOQ is the analyte concentration correspond-ing to the sample blank value ten standard deviations [21]All the proposed methods data was presented in Table 2The LOD and LOQ values found were between 0071 and0086 and 0236 and 0286 120583gmLminus1 for Carmoisine and 0081and 0091 and 0270 and 0304 120583gmLminus1 for Ponceau 4Rrespectively The data shows that the methods are sensitivefor the determination of Carmoisine and Ponceau 4R

363 Precision (Repeatability) and Accuracy (Recovery )The precision of the developed methods was expressedas a percentage of relative standard deviation (RSD) forrepeatability (intraday precision) and intermediate precision(interday precision) The data obtained were less than 106(Table 3) indicating reasonable repeatability of the proposed

10 International Journal of Analytical Chemistry

methods The intraday and interday accuracy ranges werefrom 9895 to 1006 for Carmoisine and from 9975 to1007 for Ponceau 4R respectively The results obtainedfrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria To confirm the accuracy of the proposed methodssyntheticmixtures of different concentration ratios consistingof each food colorant were prepared The resulting mixtureswere assayed according to above proposed procedures infive replicates and the average results were calculated asthe percentage of analyte recovered The percentages meanaccuracy for Carmoisine were 1008 plusmn 087 for method DSM1010 plusmn 095 for RDM 10040 plusmn 107 for DDM and 9975 plusmn102 for CM respectively And those of P were 9910 plusmn 102for method DSM 9825 plusmn 093 for RDM 9955 plusmn 095 forDDM and 9850 plusmn 106 for CM The good recovery percent(RSD) assured the high accuracy of the proposed methods(Table 3) The relative standard deviations were found tobe less than 107 and 106 for Carmoisine and Ponceau4R respectively indicating reasonable repeatability of theproposed methods

4 Conclusion

Four derivative spectrophotometric methods for the simul-taneous analysis of two common food colorants have beensuccessfully researched developed applied and compared byreference HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination ofCarmosine and Ponceau 4R in synthetic mixtures and softdrinks without any interference from ingredients presentThe developed methods are simple (as there is no needfor pretreatment) rapid (as they require measurements of998779D1 and D1 values at a single wavelength) and direct (asthey estimate each colorant independently of the other) UVmethods offer a cost effective and time saving alternative toother methods for example colorimetric complexometricand chromatographic analyses The methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetricmethods Also themethodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities of the proposed meth-ods are almost comparable and can be used for the determina-tion of the two colorants in commercial drinks in the absenceof official method

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research has been supported by Scientific ResearchProject Coordination Center of Yildiz Technical University(Project no 2012-01-02-KAP07)

References

[1] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 1989

[2] J Vries Food Safety and Toxicity CRC London UK 1996[3] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination of

food dyes in soft drinks containing natural pigments by liquidchromatography with minimal clean-uprdquo Food Control vol 16no 3 pp 293ndash297 2005

[4] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination of carmoisine allura red and Ponceau4R in sweets and soft drinks by differential pulse polarographyrdquoJournal of Food Composition andAnalysis vol 18 no 6 pp 503ndash515 2005

[5] N Dossi R Toniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem forthe rapid in-channel amperometric detection of synthetic dyesin foodrdquo Journal of Electroanalytical Chemistry vol 601 no 1-2pp 1ndash7 2007

[6] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination of eight synthetic permitted and five commonlyencountered nonpermitted food colors in various foodmatrixesby high-performance liquid chromatographyrdquo Journal of AOACInternational vol 93 no 5 pp 1503ndash1514 2010

[7] S Dixit S K Khanna and M Das ldquoA simple method forsimultaneous determination of basic dyes encountered in foodpreparations by reversed-phase HPLCrdquo Journal of AOAC Inter-national vol 94 no 6 pp 1874ndash1881 2011

[8] N Yoshioka and K Ichihashi ldquoDetermination of 40 syntheticfood colors in drinks and candies by high-performance liquidchromatography using a short column with photodiode arraydetectionrdquo Talanta vol 74 no 5 pp 1408ndash1413 2008

[9] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination of synthetic dyes in selectedfoodstuffs by high performance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 107 no 1 pp 489ndash496 2008

[10] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination of water-soluble and fat-soluble synthetic colorantsin foodstuffbyhigh-performance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal ofChromatography A vol 1103 no 1 pp 170ndash176 2006

[11] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination of food dyes by firstderivative spectrophotometry with sorption onto polyurethanefoamrdquo Analytical Sciences vol 21 no 2 pp 149ndash153 2005

[12] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilfroush ldquoSimultaneous determination of carmoisine andPonceau 4Rrdquo Food Analytical Methods vol 1 no 3 pp 214ndash2192008

[13] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination of carmoisine andbrilliant blue FCF in food samplesrdquo Talanta vol 84 no 1 pp240ndash243 2011

[14] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan andB Asci ldquoAbilities of partial least-squares (PLS-2)multivariate calibration in the analysis of quaternary mixture offood colors (E-110 E-122 E-124 E-131)rdquo Analytical Letters vol40 no 10 pp 1893ndash1903 2007

[15] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectrofluorimetric determination of rhodamine dyein cosmeticsrdquo Talanta vol 75 no 1 pp 135ndash140 2008

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

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CatalystsJournal of

Page 7: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

International Journal of Analytical Chemistry 7

001

003

308 328 348 368

Firs

t der

ivat

ive

Carm

oisin

ePo

ncea

u 4R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

Firs

t der

ivat

ive

Ponc

eau

4RC

arm

oisin

e

Wavelength (nm)

fg

hi

j

minus002678

minus001678

minus000678

(b)

Figure 2 First derivative ratio spectra of Carmoisine (a) and Ponceau 4R (b) for different concentrations (Carmoisine (6 120583gmLminus1 Ponceau4R was used as a divisor) a 2 120583gmLminus1 b 4 120583gmLminus1 c 6120583gmLminus1 d 8 120583gmLminus1 e 10120583gmLminus1 Ponceau 4R (6120583gmLminus1 Carmoisine was usedas a divisor) f 2120583gmLminus1 g 4 120583gmLminus1 h 6 120583gmLminus1 i 8120583gmLminus1 j 10120583gmLminus1)

Table 4 Assay results for the determination of Carmoisine and Ponceau 4R in soft drinks using the proposed methods and the referenceHPLC method (NMKL 130)

Methods Analyte Selected wavelengths(nm)

Assay results mean plusmn SDlowast (119905calculated 119865calculated)lowastlowast

Soft drink I (120583g 100mLminus1) Soft drink II (120583g 100mLminus1)

DSM C 1D331 930 plusmn 022 (068 145) 628 plusmn 029 (061 144)

P 1D517 220 plusmn 011 (118 118) 139 plusmn 013 (040 179)

RDM C 1D323 926 plusmn 023 (037 145) 636 plusmn 021 (128 120)

P 1D344 235 plusmn 011 (044 125) 143 plusmn 013 (013 169)

DDM C 1D366 931 plusmn 024 (073 169) 641 plusmn 024 (153 164)

P 1D451 249 plusmn 017 (189 302) 144 plusmn 016 (023 268)

CM C 1D4721D569 930 plusmn 026 (063 188) 636 plusmn 028 (107 211)

P 1D4701D554 230 plusmn 016 (023 256) 143 plusmn 019 (010 368)

HPLC C 520 921 plusmn 018 620 plusmn 019

P 512 232 plusmn 010 142 plusmn 010

lowastResults obtained are the average of five experiments for each method SD standard deviationlowastlowastThe corresponding theoretical value for 119905 and 119865 at 119875 005 (119905theoretical 231 119865theoretical 639)

a maximum wavelength are proportional to the Carmoisineconcentration (Figure 2(a)) To determine the other com-ponent Ponceau 4R an analogues procedure was followedFor determination of Ponceau 4R the stored spectra ofthe mixtures are divided by a standard spectrum of 6mgLminus1 Carmoisine (Figure 2(b)) Standard solutions of Ponceau4R and the content of Ponceau 4R were determined bymeasuring the signals at 344 nm (1DD

344) Figure 2(b)

Under the described experimental conditions the cal-ibration graphs obtained by plotting the derivative valuesof each colorant versus concentration in the concentration

range stated in Table 2 showed linear relationships Con-formity with Beerrsquos law was evident in the concentrationrange of the final dilution cited in Table 2 For this methodthe characteristic parameters of regression equations andcorrelation coefficients are given in Table 2 The correlationcoefficients were 09996 and 09994 indicating good linearityLOD and LOQ were calculated for each colorant and areshown in the same table The detection limits were foundto be 0079120583gmLminus1 for Carmoisine and 0082 120583gmLminus1 forPonceau 4R while the quantification limits were estimatedto be 0263 120583gmLminus1 for Carmoisine and 0273120583gmLminus1 for

8 International Journal of Analytical Chemistry

000988

008988

016988

024988

032988

300 400 500 600 700

Abso

rban

ce

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

Diff

eren

ce fi

rst d

eriv

ativ

e

Wavelength (nm)

C

P

minus00068

minus00028

(b)

Figure 3 Difference absorption spectra of 10 120583gmLminus1 Carmoisine 10 120583gmLminus1 Ponceau 4R and their binary mixture (a) Difference firstderivative spectra (b) of colorants (Carmoisine 2ndash10120583gmLminus1 Ponceau 4R 2ndash10 120583gmLminus1)

Ponceau 4R The proposed method was also applied to thedrink samples (Table 4) The accuracy of RDM was checkedby analyzing the laboratory preparedmixtures of Carmoisineand Ponceau 4R at various concentration ratios ranging from2 to 10 120583gmLminus1 (Table 3) All data represent the average offive determinations Low values of relative standard deviationindicate very good precision Satisfactory recoveries withsmall standard deviations were obtained which indicated thehigh repeatability and accuracy of the method

33 Derivative DifferentialMethod (DDM) Differential spec-trophotometry (ΔD

1) based on pH changes has also been

reported to be useful in the determination of binary mix-tures This method that depends on utilization of differenceabsorption spectra corresponding to the same compoundobtained at two different pHrsquos has been investigated for theanalysis of binary mixtures The procedure is comprised ofthe measurement of ΔD

1food colorantsrsquo in acidic solutions

against their alkaline solutions as blanks The ΔD1has

been successfully used to eliminate interferences from drinksamples There are few reports on utilization of the abovetwo combined techniques (difference and derivative) for theestimation of individual drug substances and for combinedpreparations [28ndash30]

The difference absorption spectra of Carmoisine Pon-ceau 4R and their binary mixture in 01 N NaOH and in01 N HCI are shown in Figure 3(a) Figure 3(b) shows thefirst derivative difference spectra of colorants in the concen-tration range 2ndash10 120583gmLminus1 The spectra of both colorantsin Figure 3(b) offer an advantage for their simultaneousdetermination by having zero-crossing points In particularthe first derivative amplitudes at 366 nm for Carmoisine and

451 nm for Ponceau 4R in the Carmoisine and Ponceau 4Rmixture were considered as the optimum working wave-lengths for their determination By measuring the values ofthe ΔD

1amplitudes at these wavelengths the concentration

of each colorant can be directly calculated since the dif-ferential first derivative measurement cancels the irrelevantabsorbance due to the drinks matrix at these wavelengths

Under the experimental conditions described the cali-bration graphs obtained by plotting the derivative values ofeach colorant in the mixture versus concentration in therange concentration stated in Table 2 show linear relation-ships The correlation coefficients were 09996 and 09998indicating good linearity The LOD were 0071 120583gmLminus1 forCarmoisine and 0081 120583gmLminus1 for Ponceau 4R while theLOQ were 0236 120583gmLminus1 for Carmoisine and 0270 120583gmLminus1for Ponceau 4R The proposed procedure was success-fully applied for the determination of these colorants inlaboratory-prepared mixtures and drink samples The resultsare presented in Tables 3 and 4The values of relative standarddeviation indicate very good reproducibility In additionapplication of the DDMwas found to be correct for the drinkmatrix interference and to enhance the sensitivity

34 CompensationMethod (CM) Thecompensationmethodis a nonmathematical method for the detection and elimi-nation of unwanted absorption during spectrophotometricanalysis [19 20 31 32] The accuracy of the method dependson the evaluation of the balance point Figure 1(b) showsthe first derivative spectra of Carmoisine and Ponceau 4Rin the concentration range 2ndash10 120583gmLminus1 The first derivativespectra were recorded for each reference solution of thecomponents and the ratios of the 1D maximum and 1D

International Journal of Analytical Chemistry 9

000011000211000411000611

401 451 501 551 601Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

Firs

t der

ivat

ive

(a)

000080002800048

400 440 480 520 560 600

Firs

t der

ivat

ive

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)

Figure 4 (a) First derivative spectra between the mixture solution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in the sample cell anddifferent concentrations (2ndash10120583gmLminus1) of Ponceau 4R solutions in the reference cell First derivative spectra between the mixture solution(6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and 6120583gmLminus1 Ponceau 4R in the reference cell at balance points andcomparison of these spectra with 6 120583gmLminus1 concentration of Carmoisine (- - -) spectrum (b) First derivative spectra between the mixturesolution (6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and different concentrations (2ndash10120583gmLminus1) of Carmoisinesolutions in the reference cell Derivative spectra between the mixture solution (6120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in thesample cell and 6 120583gmLminus1 Carmoisine in the reference cell at balance points and comparison of these spectra with 6120583gmLminus1 concentrationof Ponceau 4R (- - -) spectrum

minimum were calculated Table 1 shows the mean valuesof the ratios calculated for five different determinations foreach solutionThe ratios are constant and characteristic of thepure substance independent of concentration and presenceof another component For the determination of Carmoisineconcentrations in Carmoisine-Ponceau 4R binary mixturesand drink samples the sample cell was filled with themixturesolution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R)and the reference cell was filled in succession with aseries of reference Ponceau 4R with solutions in differentconcentrations Ponceau 4R as shown in Figure 4(a) Theratios of the mixture calculated from the recorded 1D spectrawere compared with those of Carmoisine At the exactbalance point the ratio of the mixture corresponds to thatof Carmoisine where the concentration of Carmoisine in themixtures in the sample cell is equal to that of the referencesolution in the reference cell For determining the othercomponent the same steps were followed using solutions ofpure compoundCarmoisine in the reference cell to determineits concentration in the mixture (Figure 4(b)) Conformitywith Beerrsquos law was evident in the concentration range from 2to 10 120583gmLminus1 of Carmoisine and Ponceau 4R In thismethodsynthetic mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R and the recoveries ofthe method were found to be in the range of 9850 plusmn 105ndash9975 plusmn 110 The presented results in Table 3 are in goodagreement with the other proposed methods

35 Application of the Developed Methods to the Soft DrinksThe developed and validated methods were successfullyapplied to the determination of Carmoisine and Ponceau 4Rcontent in the commercial soft drinks The obtained resultsare presented in Table 4 Good agreement was observed forthe assay results of the drinks by application of the fourmethods in this paper (Table 4) The values of standarddeviation indicate very good reproducibility The results ofthe proposed spectrophotometric methods were statisticallycompared with the results of the HPLC method given in

the literature [18] at the 95 confidence level with studentrsquos 119905-test and by the variance ratio 119865-test (Table 4) The calculated119905- and119865-values never exceed the theoretical 119905- and119865-values at005 level of significant difference There was no interferenceof ingredients in the products examined so no additiveextraction or separation procedures were required duringtheir assay The results of all methods were very close toeach other This suggests that the four methods are equallyapplicable

36 Validation of the Proposed Methods

361 Linearity The linearity of the proposed methods wasevaluated under different concentrations of Carmoisine andPonceau 4R within the concentration range stated in Table 2The good linearity of the calibration graphs and negligiblescatter of the experimental points is clearly evident by thevalues of the correlation coefficients and variances around theslope (Table 2)

362 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD is expressed as the analyte concentration cor-responding to the sample blank value plus three standarddeviations and LOQ is the analyte concentration correspond-ing to the sample blank value ten standard deviations [21]All the proposed methods data was presented in Table 2The LOD and LOQ values found were between 0071 and0086 and 0236 and 0286 120583gmLminus1 for Carmoisine and 0081and 0091 and 0270 and 0304 120583gmLminus1 for Ponceau 4Rrespectively The data shows that the methods are sensitivefor the determination of Carmoisine and Ponceau 4R

363 Precision (Repeatability) and Accuracy (Recovery )The precision of the developed methods was expressedas a percentage of relative standard deviation (RSD) forrepeatability (intraday precision) and intermediate precision(interday precision) The data obtained were less than 106(Table 3) indicating reasonable repeatability of the proposed

10 International Journal of Analytical Chemistry

methods The intraday and interday accuracy ranges werefrom 9895 to 1006 for Carmoisine and from 9975 to1007 for Ponceau 4R respectively The results obtainedfrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria To confirm the accuracy of the proposed methodssyntheticmixtures of different concentration ratios consistingof each food colorant were prepared The resulting mixtureswere assayed according to above proposed procedures infive replicates and the average results were calculated asthe percentage of analyte recovered The percentages meanaccuracy for Carmoisine were 1008 plusmn 087 for method DSM1010 plusmn 095 for RDM 10040 plusmn 107 for DDM and 9975 plusmn102 for CM respectively And those of P were 9910 plusmn 102for method DSM 9825 plusmn 093 for RDM 9955 plusmn 095 forDDM and 9850 plusmn 106 for CM The good recovery percent(RSD) assured the high accuracy of the proposed methods(Table 3) The relative standard deviations were found tobe less than 107 and 106 for Carmoisine and Ponceau4R respectively indicating reasonable repeatability of theproposed methods

4 Conclusion

Four derivative spectrophotometric methods for the simul-taneous analysis of two common food colorants have beensuccessfully researched developed applied and compared byreference HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination ofCarmosine and Ponceau 4R in synthetic mixtures and softdrinks without any interference from ingredients presentThe developed methods are simple (as there is no needfor pretreatment) rapid (as they require measurements of998779D1 and D1 values at a single wavelength) and direct (asthey estimate each colorant independently of the other) UVmethods offer a cost effective and time saving alternative toother methods for example colorimetric complexometricand chromatographic analyses The methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetricmethods Also themethodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities of the proposed meth-ods are almost comparable and can be used for the determina-tion of the two colorants in commercial drinks in the absenceof official method

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research has been supported by Scientific ResearchProject Coordination Center of Yildiz Technical University(Project no 2012-01-02-KAP07)

References

[1] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 1989

[2] J Vries Food Safety and Toxicity CRC London UK 1996[3] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination of

food dyes in soft drinks containing natural pigments by liquidchromatography with minimal clean-uprdquo Food Control vol 16no 3 pp 293ndash297 2005

[4] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination of carmoisine allura red and Ponceau4R in sweets and soft drinks by differential pulse polarographyrdquoJournal of Food Composition andAnalysis vol 18 no 6 pp 503ndash515 2005

[5] N Dossi R Toniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem forthe rapid in-channel amperometric detection of synthetic dyesin foodrdquo Journal of Electroanalytical Chemistry vol 601 no 1-2pp 1ndash7 2007

[6] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination of eight synthetic permitted and five commonlyencountered nonpermitted food colors in various foodmatrixesby high-performance liquid chromatographyrdquo Journal of AOACInternational vol 93 no 5 pp 1503ndash1514 2010

[7] S Dixit S K Khanna and M Das ldquoA simple method forsimultaneous determination of basic dyes encountered in foodpreparations by reversed-phase HPLCrdquo Journal of AOAC Inter-national vol 94 no 6 pp 1874ndash1881 2011

[8] N Yoshioka and K Ichihashi ldquoDetermination of 40 syntheticfood colors in drinks and candies by high-performance liquidchromatography using a short column with photodiode arraydetectionrdquo Talanta vol 74 no 5 pp 1408ndash1413 2008

[9] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination of synthetic dyes in selectedfoodstuffs by high performance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 107 no 1 pp 489ndash496 2008

[10] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination of water-soluble and fat-soluble synthetic colorantsin foodstuffbyhigh-performance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal ofChromatography A vol 1103 no 1 pp 170ndash176 2006

[11] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination of food dyes by firstderivative spectrophotometry with sorption onto polyurethanefoamrdquo Analytical Sciences vol 21 no 2 pp 149ndash153 2005

[12] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilfroush ldquoSimultaneous determination of carmoisine andPonceau 4Rrdquo Food Analytical Methods vol 1 no 3 pp 214ndash2192008

[13] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination of carmoisine andbrilliant blue FCF in food samplesrdquo Talanta vol 84 no 1 pp240ndash243 2011

[14] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan andB Asci ldquoAbilities of partial least-squares (PLS-2)multivariate calibration in the analysis of quaternary mixture offood colors (E-110 E-122 E-124 E-131)rdquo Analytical Letters vol40 no 10 pp 1893ndash1903 2007

[15] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectrofluorimetric determination of rhodamine dyein cosmeticsrdquo Talanta vol 75 no 1 pp 135ndash140 2008

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

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Carbohydrate Chemistry

International Journal of

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Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

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Chromatography Research International

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CatalystsJournal of

Page 8: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

8 International Journal of Analytical Chemistry

000988

008988

016988

024988

032988

300 400 500 600 700

Abso

rban

ce

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

Diff

eren

ce fi

rst d

eriv

ativ

e

Wavelength (nm)

C

P

minus00068

minus00028

(b)

Figure 3 Difference absorption spectra of 10 120583gmLminus1 Carmoisine 10 120583gmLminus1 Ponceau 4R and their binary mixture (a) Difference firstderivative spectra (b) of colorants (Carmoisine 2ndash10120583gmLminus1 Ponceau 4R 2ndash10 120583gmLminus1)

Ponceau 4R The proposed method was also applied to thedrink samples (Table 4) The accuracy of RDM was checkedby analyzing the laboratory preparedmixtures of Carmoisineand Ponceau 4R at various concentration ratios ranging from2 to 10 120583gmLminus1 (Table 3) All data represent the average offive determinations Low values of relative standard deviationindicate very good precision Satisfactory recoveries withsmall standard deviations were obtained which indicated thehigh repeatability and accuracy of the method

33 Derivative DifferentialMethod (DDM) Differential spec-trophotometry (ΔD

1) based on pH changes has also been

reported to be useful in the determination of binary mix-tures This method that depends on utilization of differenceabsorption spectra corresponding to the same compoundobtained at two different pHrsquos has been investigated for theanalysis of binary mixtures The procedure is comprised ofthe measurement of ΔD

1food colorantsrsquo in acidic solutions

against their alkaline solutions as blanks The ΔD1has

been successfully used to eliminate interferences from drinksamples There are few reports on utilization of the abovetwo combined techniques (difference and derivative) for theestimation of individual drug substances and for combinedpreparations [28ndash30]

The difference absorption spectra of Carmoisine Pon-ceau 4R and their binary mixture in 01 N NaOH and in01 N HCI are shown in Figure 3(a) Figure 3(b) shows thefirst derivative difference spectra of colorants in the concen-tration range 2ndash10 120583gmLminus1 The spectra of both colorantsin Figure 3(b) offer an advantage for their simultaneousdetermination by having zero-crossing points In particularthe first derivative amplitudes at 366 nm for Carmoisine and

451 nm for Ponceau 4R in the Carmoisine and Ponceau 4Rmixture were considered as the optimum working wave-lengths for their determination By measuring the values ofthe ΔD

1amplitudes at these wavelengths the concentration

of each colorant can be directly calculated since the dif-ferential first derivative measurement cancels the irrelevantabsorbance due to the drinks matrix at these wavelengths

Under the experimental conditions described the cali-bration graphs obtained by plotting the derivative values ofeach colorant in the mixture versus concentration in therange concentration stated in Table 2 show linear relation-ships The correlation coefficients were 09996 and 09998indicating good linearity The LOD were 0071 120583gmLminus1 forCarmoisine and 0081 120583gmLminus1 for Ponceau 4R while theLOQ were 0236 120583gmLminus1 for Carmoisine and 0270 120583gmLminus1for Ponceau 4R The proposed procedure was success-fully applied for the determination of these colorants inlaboratory-prepared mixtures and drink samples The resultsare presented in Tables 3 and 4The values of relative standarddeviation indicate very good reproducibility In additionapplication of the DDMwas found to be correct for the drinkmatrix interference and to enhance the sensitivity

34 CompensationMethod (CM) Thecompensationmethodis a nonmathematical method for the detection and elimi-nation of unwanted absorption during spectrophotometricanalysis [19 20 31 32] The accuracy of the method dependson the evaluation of the balance point Figure 1(b) showsthe first derivative spectra of Carmoisine and Ponceau 4Rin the concentration range 2ndash10 120583gmLminus1 The first derivativespectra were recorded for each reference solution of thecomponents and the ratios of the 1D maximum and 1D

International Journal of Analytical Chemistry 9

000011000211000411000611

401 451 501 551 601Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

Firs

t der

ivat

ive

(a)

000080002800048

400 440 480 520 560 600

Firs

t der

ivat

ive

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)

Figure 4 (a) First derivative spectra between the mixture solution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in the sample cell anddifferent concentrations (2ndash10120583gmLminus1) of Ponceau 4R solutions in the reference cell First derivative spectra between the mixture solution(6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and 6120583gmLminus1 Ponceau 4R in the reference cell at balance points andcomparison of these spectra with 6 120583gmLminus1 concentration of Carmoisine (- - -) spectrum (b) First derivative spectra between the mixturesolution (6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and different concentrations (2ndash10120583gmLminus1) of Carmoisinesolutions in the reference cell Derivative spectra between the mixture solution (6120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in thesample cell and 6 120583gmLminus1 Carmoisine in the reference cell at balance points and comparison of these spectra with 6120583gmLminus1 concentrationof Ponceau 4R (- - -) spectrum

minimum were calculated Table 1 shows the mean valuesof the ratios calculated for five different determinations foreach solutionThe ratios are constant and characteristic of thepure substance independent of concentration and presenceof another component For the determination of Carmoisineconcentrations in Carmoisine-Ponceau 4R binary mixturesand drink samples the sample cell was filled with themixturesolution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R)and the reference cell was filled in succession with aseries of reference Ponceau 4R with solutions in differentconcentrations Ponceau 4R as shown in Figure 4(a) Theratios of the mixture calculated from the recorded 1D spectrawere compared with those of Carmoisine At the exactbalance point the ratio of the mixture corresponds to thatof Carmoisine where the concentration of Carmoisine in themixtures in the sample cell is equal to that of the referencesolution in the reference cell For determining the othercomponent the same steps were followed using solutions ofpure compoundCarmoisine in the reference cell to determineits concentration in the mixture (Figure 4(b)) Conformitywith Beerrsquos law was evident in the concentration range from 2to 10 120583gmLminus1 of Carmoisine and Ponceau 4R In thismethodsynthetic mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R and the recoveries ofthe method were found to be in the range of 9850 plusmn 105ndash9975 plusmn 110 The presented results in Table 3 are in goodagreement with the other proposed methods

35 Application of the Developed Methods to the Soft DrinksThe developed and validated methods were successfullyapplied to the determination of Carmoisine and Ponceau 4Rcontent in the commercial soft drinks The obtained resultsare presented in Table 4 Good agreement was observed forthe assay results of the drinks by application of the fourmethods in this paper (Table 4) The values of standarddeviation indicate very good reproducibility The results ofthe proposed spectrophotometric methods were statisticallycompared with the results of the HPLC method given in

the literature [18] at the 95 confidence level with studentrsquos 119905-test and by the variance ratio 119865-test (Table 4) The calculated119905- and119865-values never exceed the theoretical 119905- and119865-values at005 level of significant difference There was no interferenceof ingredients in the products examined so no additiveextraction or separation procedures were required duringtheir assay The results of all methods were very close toeach other This suggests that the four methods are equallyapplicable

36 Validation of the Proposed Methods

361 Linearity The linearity of the proposed methods wasevaluated under different concentrations of Carmoisine andPonceau 4R within the concentration range stated in Table 2The good linearity of the calibration graphs and negligiblescatter of the experimental points is clearly evident by thevalues of the correlation coefficients and variances around theslope (Table 2)

362 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD is expressed as the analyte concentration cor-responding to the sample blank value plus three standarddeviations and LOQ is the analyte concentration correspond-ing to the sample blank value ten standard deviations [21]All the proposed methods data was presented in Table 2The LOD and LOQ values found were between 0071 and0086 and 0236 and 0286 120583gmLminus1 for Carmoisine and 0081and 0091 and 0270 and 0304 120583gmLminus1 for Ponceau 4Rrespectively The data shows that the methods are sensitivefor the determination of Carmoisine and Ponceau 4R

363 Precision (Repeatability) and Accuracy (Recovery )The precision of the developed methods was expressedas a percentage of relative standard deviation (RSD) forrepeatability (intraday precision) and intermediate precision(interday precision) The data obtained were less than 106(Table 3) indicating reasonable repeatability of the proposed

10 International Journal of Analytical Chemistry

methods The intraday and interday accuracy ranges werefrom 9895 to 1006 for Carmoisine and from 9975 to1007 for Ponceau 4R respectively The results obtainedfrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria To confirm the accuracy of the proposed methodssyntheticmixtures of different concentration ratios consistingof each food colorant were prepared The resulting mixtureswere assayed according to above proposed procedures infive replicates and the average results were calculated asthe percentage of analyte recovered The percentages meanaccuracy for Carmoisine were 1008 plusmn 087 for method DSM1010 plusmn 095 for RDM 10040 plusmn 107 for DDM and 9975 plusmn102 for CM respectively And those of P were 9910 plusmn 102for method DSM 9825 plusmn 093 for RDM 9955 plusmn 095 forDDM and 9850 plusmn 106 for CM The good recovery percent(RSD) assured the high accuracy of the proposed methods(Table 3) The relative standard deviations were found tobe less than 107 and 106 for Carmoisine and Ponceau4R respectively indicating reasonable repeatability of theproposed methods

4 Conclusion

Four derivative spectrophotometric methods for the simul-taneous analysis of two common food colorants have beensuccessfully researched developed applied and compared byreference HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination ofCarmosine and Ponceau 4R in synthetic mixtures and softdrinks without any interference from ingredients presentThe developed methods are simple (as there is no needfor pretreatment) rapid (as they require measurements of998779D1 and D1 values at a single wavelength) and direct (asthey estimate each colorant independently of the other) UVmethods offer a cost effective and time saving alternative toother methods for example colorimetric complexometricand chromatographic analyses The methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetricmethods Also themethodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities of the proposed meth-ods are almost comparable and can be used for the determina-tion of the two colorants in commercial drinks in the absenceof official method

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research has been supported by Scientific ResearchProject Coordination Center of Yildiz Technical University(Project no 2012-01-02-KAP07)

References

[1] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 1989

[2] J Vries Food Safety and Toxicity CRC London UK 1996[3] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination of

food dyes in soft drinks containing natural pigments by liquidchromatography with minimal clean-uprdquo Food Control vol 16no 3 pp 293ndash297 2005

[4] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination of carmoisine allura red and Ponceau4R in sweets and soft drinks by differential pulse polarographyrdquoJournal of Food Composition andAnalysis vol 18 no 6 pp 503ndash515 2005

[5] N Dossi R Toniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem forthe rapid in-channel amperometric detection of synthetic dyesin foodrdquo Journal of Electroanalytical Chemistry vol 601 no 1-2pp 1ndash7 2007

[6] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination of eight synthetic permitted and five commonlyencountered nonpermitted food colors in various foodmatrixesby high-performance liquid chromatographyrdquo Journal of AOACInternational vol 93 no 5 pp 1503ndash1514 2010

[7] S Dixit S K Khanna and M Das ldquoA simple method forsimultaneous determination of basic dyes encountered in foodpreparations by reversed-phase HPLCrdquo Journal of AOAC Inter-national vol 94 no 6 pp 1874ndash1881 2011

[8] N Yoshioka and K Ichihashi ldquoDetermination of 40 syntheticfood colors in drinks and candies by high-performance liquidchromatography using a short column with photodiode arraydetectionrdquo Talanta vol 74 no 5 pp 1408ndash1413 2008

[9] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination of synthetic dyes in selectedfoodstuffs by high performance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 107 no 1 pp 489ndash496 2008

[10] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination of water-soluble and fat-soluble synthetic colorantsin foodstuffbyhigh-performance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal ofChromatography A vol 1103 no 1 pp 170ndash176 2006

[11] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination of food dyes by firstderivative spectrophotometry with sorption onto polyurethanefoamrdquo Analytical Sciences vol 21 no 2 pp 149ndash153 2005

[12] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilfroush ldquoSimultaneous determination of carmoisine andPonceau 4Rrdquo Food Analytical Methods vol 1 no 3 pp 214ndash2192008

[13] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination of carmoisine andbrilliant blue FCF in food samplesrdquo Talanta vol 84 no 1 pp240ndash243 2011

[14] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan andB Asci ldquoAbilities of partial least-squares (PLS-2)multivariate calibration in the analysis of quaternary mixture offood colors (E-110 E-122 E-124 E-131)rdquo Analytical Letters vol40 no 10 pp 1893ndash1903 2007

[15] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectrofluorimetric determination of rhodamine dyein cosmeticsrdquo Talanta vol 75 no 1 pp 135ndash140 2008

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

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Medicinal ChemistryInternational Journal of

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Chromatography Research International

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CatalystsJournal of

Page 9: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

International Journal of Analytical Chemistry 9

000011000211000411000611

401 451 501 551 601Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

Firs

t der

ivat

ive

(a)

000080002800048

400 440 480 520 560 600

Firs

t der

ivat

ive

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)

Figure 4 (a) First derivative spectra between the mixture solution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in the sample cell anddifferent concentrations (2ndash10120583gmLminus1) of Ponceau 4R solutions in the reference cell First derivative spectra between the mixture solution(6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and 6120583gmLminus1 Ponceau 4R in the reference cell at balance points andcomparison of these spectra with 6 120583gmLminus1 concentration of Carmoisine (- - -) spectrum (b) First derivative spectra between the mixturesolution (6120583gmLminus1 Carmoisine + 6120583gmLminus1 Ponceau 4R) in the sample cell and different concentrations (2ndash10120583gmLminus1) of Carmoisinesolutions in the reference cell Derivative spectra between the mixture solution (6120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R) in thesample cell and 6 120583gmLminus1 Carmoisine in the reference cell at balance points and comparison of these spectra with 6120583gmLminus1 concentrationof Ponceau 4R (- - -) spectrum

minimum were calculated Table 1 shows the mean valuesof the ratios calculated for five different determinations foreach solutionThe ratios are constant and characteristic of thepure substance independent of concentration and presenceof another component For the determination of Carmoisineconcentrations in Carmoisine-Ponceau 4R binary mixturesand drink samples the sample cell was filled with themixturesolution (6 120583gmLminus1 Carmoisine + 6 120583gmLminus1 Ponceau 4R)and the reference cell was filled in succession with aseries of reference Ponceau 4R with solutions in differentconcentrations Ponceau 4R as shown in Figure 4(a) Theratios of the mixture calculated from the recorded 1D spectrawere compared with those of Carmoisine At the exactbalance point the ratio of the mixture corresponds to thatof Carmoisine where the concentration of Carmoisine in themixtures in the sample cell is equal to that of the referencesolution in the reference cell For determining the othercomponent the same steps were followed using solutions ofpure compoundCarmoisine in the reference cell to determineits concentration in the mixture (Figure 4(b)) Conformitywith Beerrsquos law was evident in the concentration range from 2to 10 120583gmLminus1 of Carmoisine and Ponceau 4R In thismethodsynthetic mixtures were prepared at different concentrationratios of Carmoisine and Ponceau 4R and the recoveries ofthe method were found to be in the range of 9850 plusmn 105ndash9975 plusmn 110 The presented results in Table 3 are in goodagreement with the other proposed methods

35 Application of the Developed Methods to the Soft DrinksThe developed and validated methods were successfullyapplied to the determination of Carmoisine and Ponceau 4Rcontent in the commercial soft drinks The obtained resultsare presented in Table 4 Good agreement was observed forthe assay results of the drinks by application of the fourmethods in this paper (Table 4) The values of standarddeviation indicate very good reproducibility The results ofthe proposed spectrophotometric methods were statisticallycompared with the results of the HPLC method given in

the literature [18] at the 95 confidence level with studentrsquos 119905-test and by the variance ratio 119865-test (Table 4) The calculated119905- and119865-values never exceed the theoretical 119905- and119865-values at005 level of significant difference There was no interferenceof ingredients in the products examined so no additiveextraction or separation procedures were required duringtheir assay The results of all methods were very close toeach other This suggests that the four methods are equallyapplicable

36 Validation of the Proposed Methods

361 Linearity The linearity of the proposed methods wasevaluated under different concentrations of Carmoisine andPonceau 4R within the concentration range stated in Table 2The good linearity of the calibration graphs and negligiblescatter of the experimental points is clearly evident by thevalues of the correlation coefficients and variances around theslope (Table 2)

362 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD is expressed as the analyte concentration cor-responding to the sample blank value plus three standarddeviations and LOQ is the analyte concentration correspond-ing to the sample blank value ten standard deviations [21]All the proposed methods data was presented in Table 2The LOD and LOQ values found were between 0071 and0086 and 0236 and 0286 120583gmLminus1 for Carmoisine and 0081and 0091 and 0270 and 0304 120583gmLminus1 for Ponceau 4Rrespectively The data shows that the methods are sensitivefor the determination of Carmoisine and Ponceau 4R

363 Precision (Repeatability) and Accuracy (Recovery )The precision of the developed methods was expressedas a percentage of relative standard deviation (RSD) forrepeatability (intraday precision) and intermediate precision(interday precision) The data obtained were less than 106(Table 3) indicating reasonable repeatability of the proposed

10 International Journal of Analytical Chemistry

methods The intraday and interday accuracy ranges werefrom 9895 to 1006 for Carmoisine and from 9975 to1007 for Ponceau 4R respectively The results obtainedfrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria To confirm the accuracy of the proposed methodssyntheticmixtures of different concentration ratios consistingof each food colorant were prepared The resulting mixtureswere assayed according to above proposed procedures infive replicates and the average results were calculated asthe percentage of analyte recovered The percentages meanaccuracy for Carmoisine were 1008 plusmn 087 for method DSM1010 plusmn 095 for RDM 10040 plusmn 107 for DDM and 9975 plusmn102 for CM respectively And those of P were 9910 plusmn 102for method DSM 9825 plusmn 093 for RDM 9955 plusmn 095 forDDM and 9850 plusmn 106 for CM The good recovery percent(RSD) assured the high accuracy of the proposed methods(Table 3) The relative standard deviations were found tobe less than 107 and 106 for Carmoisine and Ponceau4R respectively indicating reasonable repeatability of theproposed methods

4 Conclusion

Four derivative spectrophotometric methods for the simul-taneous analysis of two common food colorants have beensuccessfully researched developed applied and compared byreference HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination ofCarmosine and Ponceau 4R in synthetic mixtures and softdrinks without any interference from ingredients presentThe developed methods are simple (as there is no needfor pretreatment) rapid (as they require measurements of998779D1 and D1 values at a single wavelength) and direct (asthey estimate each colorant independently of the other) UVmethods offer a cost effective and time saving alternative toother methods for example colorimetric complexometricand chromatographic analyses The methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetricmethods Also themethodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities of the proposed meth-ods are almost comparable and can be used for the determina-tion of the two colorants in commercial drinks in the absenceof official method

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research has been supported by Scientific ResearchProject Coordination Center of Yildiz Technical University(Project no 2012-01-02-KAP07)

References

[1] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 1989

[2] J Vries Food Safety and Toxicity CRC London UK 1996[3] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination of

food dyes in soft drinks containing natural pigments by liquidchromatography with minimal clean-uprdquo Food Control vol 16no 3 pp 293ndash297 2005

[4] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination of carmoisine allura red and Ponceau4R in sweets and soft drinks by differential pulse polarographyrdquoJournal of Food Composition andAnalysis vol 18 no 6 pp 503ndash515 2005

[5] N Dossi R Toniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem forthe rapid in-channel amperometric detection of synthetic dyesin foodrdquo Journal of Electroanalytical Chemistry vol 601 no 1-2pp 1ndash7 2007

[6] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination of eight synthetic permitted and five commonlyencountered nonpermitted food colors in various foodmatrixesby high-performance liquid chromatographyrdquo Journal of AOACInternational vol 93 no 5 pp 1503ndash1514 2010

[7] S Dixit S K Khanna and M Das ldquoA simple method forsimultaneous determination of basic dyes encountered in foodpreparations by reversed-phase HPLCrdquo Journal of AOAC Inter-national vol 94 no 6 pp 1874ndash1881 2011

[8] N Yoshioka and K Ichihashi ldquoDetermination of 40 syntheticfood colors in drinks and candies by high-performance liquidchromatography using a short column with photodiode arraydetectionrdquo Talanta vol 74 no 5 pp 1408ndash1413 2008

[9] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination of synthetic dyes in selectedfoodstuffs by high performance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 107 no 1 pp 489ndash496 2008

[10] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination of water-soluble and fat-soluble synthetic colorantsin foodstuffbyhigh-performance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal ofChromatography A vol 1103 no 1 pp 170ndash176 2006

[11] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination of food dyes by firstderivative spectrophotometry with sorption onto polyurethanefoamrdquo Analytical Sciences vol 21 no 2 pp 149ndash153 2005

[12] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilfroush ldquoSimultaneous determination of carmoisine andPonceau 4Rrdquo Food Analytical Methods vol 1 no 3 pp 214ndash2192008

[13] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination of carmoisine andbrilliant blue FCF in food samplesrdquo Talanta vol 84 no 1 pp240ndash243 2011

[14] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan andB Asci ldquoAbilities of partial least-squares (PLS-2)multivariate calibration in the analysis of quaternary mixture offood colors (E-110 E-122 E-124 E-131)rdquo Analytical Letters vol40 no 10 pp 1893ndash1903 2007

[15] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectrofluorimetric determination of rhodamine dyein cosmeticsrdquo Talanta vol 75 no 1 pp 135ndash140 2008

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Page 10: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

10 International Journal of Analytical Chemistry

methods The intraday and interday accuracy ranges werefrom 9895 to 1006 for Carmoisine and from 9975 to1007 for Ponceau 4R respectively The results obtainedfrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria To confirm the accuracy of the proposed methodssyntheticmixtures of different concentration ratios consistingof each food colorant were prepared The resulting mixtureswere assayed according to above proposed procedures infive replicates and the average results were calculated asthe percentage of analyte recovered The percentages meanaccuracy for Carmoisine were 1008 plusmn 087 for method DSM1010 plusmn 095 for RDM 10040 plusmn 107 for DDM and 9975 plusmn102 for CM respectively And those of P were 9910 plusmn 102for method DSM 9825 plusmn 093 for RDM 9955 plusmn 095 forDDM and 9850 plusmn 106 for CM The good recovery percent(RSD) assured the high accuracy of the proposed methods(Table 3) The relative standard deviations were found tobe less than 107 and 106 for Carmoisine and Ponceau4R respectively indicating reasonable repeatability of theproposed methods

4 Conclusion

Four derivative spectrophotometric methods for the simul-taneous analysis of two common food colorants have beensuccessfully researched developed applied and compared byreference HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination ofCarmosine and Ponceau 4R in synthetic mixtures and softdrinks without any interference from ingredients presentThe developed methods are simple (as there is no needfor pretreatment) rapid (as they require measurements of998779D1 and D1 values at a single wavelength) and direct (asthey estimate each colorant independently of the other) UVmethods offer a cost effective and time saving alternative toother methods for example colorimetric complexometricand chromatographic analyses The methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetricmethods Also themethodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities of the proposed meth-ods are almost comparable and can be used for the determina-tion of the two colorants in commercial drinks in the absenceof official method

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research has been supported by Scientific ResearchProject Coordination Center of Yildiz Technical University(Project no 2012-01-02-KAP07)

References

[1] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 1989

[2] J Vries Food Safety and Toxicity CRC London UK 1996[3] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination of

food dyes in soft drinks containing natural pigments by liquidchromatography with minimal clean-uprdquo Food Control vol 16no 3 pp 293ndash297 2005

[4] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination of carmoisine allura red and Ponceau4R in sweets and soft drinks by differential pulse polarographyrdquoJournal of Food Composition andAnalysis vol 18 no 6 pp 503ndash515 2005

[5] N Dossi R Toniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem forthe rapid in-channel amperometric detection of synthetic dyesin foodrdquo Journal of Electroanalytical Chemistry vol 601 no 1-2pp 1ndash7 2007

[6] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination of eight synthetic permitted and five commonlyencountered nonpermitted food colors in various foodmatrixesby high-performance liquid chromatographyrdquo Journal of AOACInternational vol 93 no 5 pp 1503ndash1514 2010

[7] S Dixit S K Khanna and M Das ldquoA simple method forsimultaneous determination of basic dyes encountered in foodpreparations by reversed-phase HPLCrdquo Journal of AOAC Inter-national vol 94 no 6 pp 1874ndash1881 2011

[8] N Yoshioka and K Ichihashi ldquoDetermination of 40 syntheticfood colors in drinks and candies by high-performance liquidchromatography using a short column with photodiode arraydetectionrdquo Talanta vol 74 no 5 pp 1408ndash1413 2008

[9] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination of synthetic dyes in selectedfoodstuffs by high performance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 107 no 1 pp 489ndash496 2008

[10] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination of water-soluble and fat-soluble synthetic colorantsin foodstuffbyhigh-performance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal ofChromatography A vol 1103 no 1 pp 170ndash176 2006

[11] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination of food dyes by firstderivative spectrophotometry with sorption onto polyurethanefoamrdquo Analytical Sciences vol 21 no 2 pp 149ndash153 2005

[12] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilfroush ldquoSimultaneous determination of carmoisine andPonceau 4Rrdquo Food Analytical Methods vol 1 no 3 pp 214ndash2192008

[13] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination of carmoisine andbrilliant blue FCF in food samplesrdquo Talanta vol 84 no 1 pp240ndash243 2011

[14] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan andB Asci ldquoAbilities of partial least-squares (PLS-2)multivariate calibration in the analysis of quaternary mixture offood colors (E-110 E-122 E-124 E-131)rdquo Analytical Letters vol40 no 10 pp 1893ndash1903 2007

[15] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectrofluorimetric determination of rhodamine dyein cosmeticsrdquo Talanta vol 75 no 1 pp 135ndash140 2008

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Page 11: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

International Journal of Analytical Chemistry 11

[16] N Dossi R Toniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination of additives in softdrinksrdquo Chromatographia vol 63 no 11-12 pp 557ndash562 2006

[17] K S Minioti C F Sakellariou and N S Thomaidis ldquoDeter-mination of 13 synthetic food colorants in water-soluble foodsby reversed-phase high-performance liquid chromatographycoupledwith diode-array detectorrdquoAnalytica ChimicaActa vol583 no 1 pp 103ndash110 2007

[18] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination infoodsrdquo UDC 66728543544 130 1989

[19] A MWahbi O Abdel-Razak A A Gazy H Mahgoub andMS Moneeb ldquoSpectrophotometric determination of omeprazolelansoprazole and pantoprazole in pharmaceutical formula-tionsrdquo Journal of Pharmaceutical and Biomedical Analysis vol30 no 4 pp 1133ndash1142 2002

[20] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectrofluoro-metric and high-performance liquid chromatographic studyfor the quantitative determination of the binary mixturefelodipine and ramipril in pharmaceutical formulationsrdquo Ana-lytical Letters vol 41 no 5 pp 853ndash870 2008

[21] ICHHarmonized Tripartite Guideline ldquoValidation of analyticalprocedures text and methodology Q2 (R1)rdquo in Proceedingsof the International Conference on Harmonization of TechnicalRequırements for Registration of Pharmacetıcals for Human Usepp 1ndash13 2005

[22] D Tarinc and A Golcu ldquoDevelopment and validation ofspectrophotometric methods for determination of somecephalosporin group antibiotic drugsrdquo Journal of AnalyticalChemistry vol 67 no 2 pp 144ndash150 2012

[23] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation of aceclofenac and tramadol withparacetamol in combination solid dosage formsrdquo Indian Journalof Pharmaceutical Sciences vol 69 no 4 pp 540ndash545 2007

[24] F Salinas J J BNevado andA EMansilla ldquoAnew spectropho-tometric method for quantitative multicomponent analysisresolution ofmixtures of salicylic and salicyluric acidsrdquoTalantavol 37 no 3 pp 347ndash351 1990

[25] R Corbella Tena M A Rodrıguez Delgado M J Sanchez andF GarciaMontelongo ldquoComparative study of the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination of atrazine andits degradation product desethylatrazin-2-hydroxy in groundwatersrdquo Talanta vol 44 no 4 pp 673ndash683 1997

[26] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination of benazepril hydrochlo-ride and hydrochlorothiazide in binary mixture using secondderivative second derivative of the ratio spectra and chemo-metric methodsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 25 no 2 pp 299ndash307 2001

[27] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination ofolmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage formrdquo An Official Journal of the Amer-ican Association of Pharmaceutical Scientists vol 10 no 4 pp1200ndash1205 2009

[28] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination ofphenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 44 no 5 pp 917ndash922 1997

[29] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis of binary mixturesrdquo Analytical Letters vol 29 no 14pp 2525ndash2540 1996

[30] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique for the simultaneous determination ofzidovudine and lamivudine in human serumrdquo Pharmazie vol59 no 2 pp 106ndash111 2004

[31] N Erk ldquoSpectrophotometric analysis of valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 35 no 2 pp 283ndash3022002

[32] N Erk ldquoAnalysis of binary mixtures of losartan potassium andhydrochlorothiazide by using high performance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 24 no 4 pp 603ndash611 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Page 12: Research Article Four Derivative Spectrophotometric ...downloads.hindawi.com/journals/ijac/2014/650465.pdf · Research Article Four Derivative Spectrophotometric Methods for the Simultaneous

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of