Analytica Chimica Acta 583 (2007) 103110

Determination of 13 synthetic food colorants in water-soluble foods by reversed-phase high-performance liquid chromatography coupled with diode-array detector
Katerina S. Minioti a , Christina F. Sakellariou b , Nikolaos S. Thomaidis a,
a

Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimioupolis Zografou, 15771 Athens, Greece b General Chemical State Laboratory, D Division of Athens, Section B, An. Tsoha 16, 11521 Athens, Greece Received 1 August 2006; received in revised form 29 September 2006; accepted 2 October 2006 Available online 6 October 2006

Abstract A reversed-phase high performance liquid chromatographic method for the successful separation and determination of 13 synthetic food colorants (Tartrazine E 102, Quinoline Yellow E 104, Sunset Yellow E 110, Carmoisine E 122, Amaranth E 123, Ponceau 4R E 124, Erythrosine E 127, Red 2G E 128, Allura Red AC E 129, Patent Blue V E 131, Indigo Carmine E 132, Brilliant Blue FCF E 133 and Green S E 142) was developed. A C18 stationary phase was used and the mobile phase contained an acetonitrilemethanol (20:80 v/v) mixture and a 1% (m/v) ammonium acetate buffer solution at pH 7.5. Successful separation was obtained for all the compounds using an optimized gradient elution within 29 min. The diode-array detector was used to monitor the colorants between 350 and 800 nm. The method was thoroughly validated. Detection limits for all substances varied between 1.59 (E 142) and 22.1 (E 124) g L1 . The intra-day precision (as R.S.D.r ) ranged from 0.37% (E 122 in fruit avored drink at a concentration of 100 mg L1 ) to 4.8% (E 142 in icing sugar at a level of 0.9 mg kg1 ). The inter-day precision (as R.S.D.R ) was between 0.86% for E 122 in fruit avored drink at 100 mg L1 and 10% for E142 in jam at a concentration of 9 mg kg1 . Satisfactory recoveries, ranging from 94% (E 142 in jam) to 102% (E 131 in sweets), were obtained. The method was applied to the determination of colorants in various watersoluble foods, such as fruit avoured drinks, alcoholic drinks, jams, sugar confectionery and sweets, with simple pre-treatment (dilution or water extraction). 2006 Elsevier B.V. All rights reserved.
Keywords: Liquid chromatography; Diode-array detector; Synthetic dyes; Food analysis; Confectionary; Soft drinks

1. Introduction Synthetic colorants are a very important class of food additives. They are widely used to compensate for the loss of natural colors of food, which are destroyed during processing and storage, and to provide the desired colored appearance. However, some of these substances pose a potential risk to human health, especially if they are excessively consumed. For this reason, safety data, such as the acceptable daily intake, based on toxicological studies on experimental animals and human clinical studies, have been repeatedly determined and evaluated by Food and Agricultural Organization (FAO) and World Health Organization (WHO) [1]. They are divided into ve major colorant

Corresponding author. Tel.: +30 210 7274317; fax: +30 210 7274750. E-mail address: ntho@chem.uoa.gr (N.S. Thomaidis).

classes: the azo compounds (E 102, E 110, E 122, E 123, E 124, E 128 and E 129), the triarylmethane group (E 131, E 133 and E142), the chinophthalon derivative of Quinoline Yellow (E 104), xanthenes as Erythrosine (E 127) and the indigo colorants (Indigo Carmine E 132). The use of synthetic colorants in foods is strictly controlled by legislation and harmonized across the European Union by formulating the directive 94/36/EC [2]. Consequently, accurate and reliable methods for the determination of synthetic colorants are required for the assurance of food safety. Many analytical techniques have been developed for the identication and determination of various synthetic food colorants, such as thin-layer chromatography [3,4], adsorptive voltammetry [5], and differential pulse polarography [6], derivative spectrometry [711] and spectrophotometric methods in combination with chemometrics [12,13], but all of them require time-consuming pretreatment or cannot be applied to complex colorant mixtures.

0003-2670/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.10.002

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Capillary electrophoresis [1418] and micellar electrokinetic capillary chromatography [19] have also been used, but they have sensitivity problems as a result of small injection volume. High-performance ion chromatography [20], reversed-phase liquid chromatography [2123] and ion-pair liquid chromatography [11,19,2429] coupled with UV or diode-array detectors are still the most preferred methods, as they provide unrivalled resolution, sensitivity and selectivity. Both isocratic [24,25,29] and gradient [1923,2628] systems are used, and the latter are preferred for the separation of the more complex mixtures. However, most of the proposed methods were developed for the determination of a small number of colorants in specic group of foodstuffs (i.e. soft drinks) [8,2325] or/and the analysis was time-consuming [22,26]. Recently, analytical methods, based on liquid chromatographymass spectrometry (LCMS) technique, have been developed in order to identify and quantify articial colorants, which are recognized to be carcinogens and are not permitted in foodstuffs under the EU Food Regulations for any purpose and any level (Sudan dyes) [3033]. Selectivity of the MS technique coupled with chromatographic separation provided unambiguous identication and accurate determination of these dangerous compounds in complex matrices at trace levels, without need of laborious clean up procedures. The aim of this study was the development of a reversedphase high-performance liquid chromatography method for the simultaneous determination of 13 synthetic colorants into the minimum run time in various classes of foods. All synthetic food colorants permitted in EU market was efciently separated using an optimized gradient elution in a single run within 29 min. The developed method was validated in different food matrices, such as fruit avored and alcoholic drinks, jam, sweets and sugar confectionary and was successfully applied to the analysis of real samples of water-soluble foods with simple pretreatment that included water extraction for solid samples and water dilution for liquid samples. 2. Experimental 2.1. Apparatus Chromatographic analysis was carried out with a Thermo Finnigan Spectra System liquid chromatograph equipped with a gradient pump Spectra System P 4000 capable for mixing up four solvents, a SCM 1000 vacuum membrane degasser, an Autosampler Spectra System AS 3000, a 100 L loop injector and a UVvis Spectra System UV 6000 LP diode array detector. The chromatographic data were collected and processed using a personal computer running Chrom Quest 4.1 Data System software (Thermo, San Jose, CA, USA). A microprocessor pH-meter (HANNA Instrument, Lisbon, Portugal) equipped with a combined glasscalomel electrode was employed for pH measurements. The determination of colorant purities was carried out with a double beam UV1601 UV/Vis spectrophotometer with 1-cm quartz cells (Shimadzu, Tokyo, Japan). A Transsonic T660/H ultrasonic bath (Elma, Germany) was used for the pretreatment of solid food samples.

2.2. Reagents All solutions were prepared with distilled deionized water and all chemicals were of analytical reagent grade, unless otherwise stated. Ultrapure water from Millipore direct-Q (Milford, MA, USA) was used for the preparation of the aqueous mobile phase. Ammonium acetate was from Riedel-de Ha n (Seelze, Gere many) and sodium hydroxide (NaOH) from Panreac (Barcelona, Spain). The HPLC grade organic solvents methanol (MeOH) and acetonitrile (ACN) were supplied from LAB-SCAN Analytical Sciences (Dublin, Ireland). The colorants Tartrazine (E 102), Sunset Yellow FCF (E 110), Amaranth (E 123) and Erythrosine (E 127) were purchased from Aldrich (Wisconsin, WI, USA). Quinoline Yellow (E 104), Indigo Carmine (E 132) and Green S (E 142) were obtained from Warner Jenkinson Europe (Kings Lynn, Norfolk, UK). Carmoisine (E 122) was from Riedel-arom (Seelze, Germany) and Ponceau 4R (E 124) was from Merck (Darmstadt, Germany). Red 2G (E 128) and Brilliant Blue FCF (E 133) were obtained from Sigma (Steinheim, Germany). Allura Red AC (E 129) was from WS Simpson Ltd. (Caldicot, Monmouthshire, UK) and Patent Blue V (E 131) was from Fluka (Buchs, Switzerland). The chemical structures, common names, European Community numbers (E numbers) and Color Index dominations (CI numbers) are reported in Fig. 1. 2.3. Chromatographic conditions A Discovery C18 (Supelco, Bellefonte, PA, USA) column (250 mm 4.6 mm i.d.) fully-end-capped, with spherical shaped 5- m particles and with carbon load 12% (3 mol m2 ) was used together with a C18 (25 mm 4.6 mm i.d., 5 m) guard column (Supelco). A C18 column was used as the most common column type, available in every routine laboratory. The mobile phase consisted of an aqueous ammonium acetate solution 1% (m/v) (0.13 M), brought to pH 7.5 by drop wise addition of a sodium hydroxide solution 10% (m/v) (mobile phase A) and a mixture of methanol:acetonitrile (80:20 v/v) (mobile phase B). The mobile phase A was ltered by vacuum through a membrane lter with a pore diameter 0.45 m. In order to achieve a successful resolution of all compounds, a number of gradient elution programs were tested, by decreasing gradually the rate of increase of the organic solvent. Retention times (tR ), peak widths (w), the resolution coefcient (Rs ) and the peak purity factor (PP) of each compound were recorded in every optimization experiment. The ow-rate of eluent was always kept constant at 1.5 mL min1 and the injection volume was set at 20 L. The nal, optimized gradient program is given in Table 1. All experiments were carried out at room temperature. The diode-array detector was programmed to monitor the colorants at a range of 350800 nm. The detection and determination of each substance was employed at the appropriate absorbance wavelengths. The chromatographic system was initially conditioned by passing the mobile phase A through the column until a stable baseline signal was obtained (a minimum of 1 h was necessary).

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Fig. 1. Chemical structures, common names, E (European Community) and CI (Colour Index) numbers of synthetic food colorants studied.

Table 1 Optimized gradient program for the separation of 13 colorants by HPLC-DAD, at a ow rate of 1.5 mL min1 t (min) 0.0 2.0 22.0 37.6 40.0 41.0 43.0 A (%) 100 100 47.5 0 0 100 100 B (%) 0 0 52.5 100 100 0 0

2.4. Preparation of colorant standards and sample solutions Individual standard stock solutions containing each color were prepared by dissolving 100 mg unpuried colorant in 100 mL distilled deionized water. The solutions were kept in dark asks. The working standard solutions of each color were prepared by appropriate dilution of stock solutions with water to give concentrations between 0.10 and 60 mg L1 , taking into consideration the purity of the colorants. The mixed standard solutions containing all colorants at concentrations between 0.10

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and 10 mg L1 were also prepared by mixing and dilution of appropriate aliquots from standard stock solution of each substance. All solutions were stored at 4 C in the dark and were stable at least for 2 months. All samples were obtained from market control and included fruit avored drinks, alcoholic drinks, jams, sweets and sugar confectionary. A 10-mL sample of drink was diluted with water in a volumetric ask of 50 mL. The samples were degassed, if necessary, by strong stirring. The solid samples were homogenized. A portion of 10 g of jam, sweet or a confectionary product was accurately weighted and dissolved in 50 mL of water. The sample solution was placed in ultrasonic bath for 15 min for the complete extraction of the colorants. These solutions were ltered through a folded paper lter and the ltrate was collected in volumetric ask of 50 mL. All solutions were injected after ltering through 0.45- m disposable syringe lters. 2.5. Determination of colorant purities Purities of colorants were determined according to the identication method reported in Regulation 95/45/EC [34]. This method is based on spectrophotometric absorbance measurement of a diluted standard solution (i.e. 10 mg L1 ), for which BeerLambert law is valid. The percentage amount of a colorant purity is given by the equation: %purity = DF A 105 A1% C 1 cm (1)

2.6. Validation of the method The instrument calibration was carried out using different concentrations between 0.10 and 60 mg L1 of each compound, with two replicates per concentration, in order to determine the linear region of each colorant. Calibration curves were also prepared with the mixed standards solutions at concentration levels 0.10, 0.20, 0.50, 0.80 and 1.0 mg L1 to check the selectivity of the method. The calibration curve of each colorant was used for the validation experiments and quantication. The limit of detection (LOD) of each compound was calculated as three times the standard deviation of the response of 10 independent replicate analyses of a 0.060 mg L1 standard solution. Intra-day and inter-day precision was assessed by analyzing six replicates of a sample during one day (n = 6, intra-day precision) or two replicates by three analysts in two different days (n = 2, k = 3, j = 2, inter-day precision), correspondingly. Different colorants (E 102, E 110, E 122, E 124, E 129, E 131 and E 142) and food matrices (avoured drinks, liqueurs, cherries, jams, sweets and icing sugar) were chosen for these experiments. In order to evaluate the trueness of the method, recovery experiments were performed. Three different food samples (fruit avoured drink, jam and avoured sweets) were spiked with E 122, E 142 and E 131, respectively, at fortication levels of 10, 50 and 100 mg kg1 and analyzed accordingly (each sample six times at each fortication level). 3. Results and discussion 3.1. Determination of colorant purities All colorant standards were analyzed to determine their colorant content. The procedure using a spectrophotometer enabled easy calculation of colorant purities, presented in Table 2. The purity of the individual colorant standards ranged from 52.5% (E 102) to 97.0% (E 123). The low colorant content is often a consequence of the specic manufacturing process. The by-products are usually inorganic salts, such as NaCl [22]. 3.2. Optimization of the separation A simple method for the determination of all permitted synthetic food colorants is advantageous for practical application. Reversed-phase liquid chromatography (RP-LC) is suitable for the analysis of these compounds, if conditions are chosen in which the ionized analytes are able to form neutral molecules, taking into account their available dissociation constants, shown in Table 2 [35]. Hence, the addition of an ion-pairing reagent, which makes the resolution more complicate and causes a variety of problems [36], is not always necessary. The main characteristics of the colorants that should be taken into account for an efcient separation are their hydrophobicity and the presence of acidic and alkaline groups (Fig. 1). The hydrophobicity of non-azo compounds is weaker than those of azo colorants. In addition, the hydrophobicity of colorants with naphthalene ring is stronger than those with benzene ring [37].

where DF is the dilution factor of measured solution from stock standard solution, A the absorbance of measured solution (A < 1) with reference to water, the expression A1% represents the spe1 cm cic absorbance of an 1% (10 g L1 ) aqueous solution of the colorant at the prescribed wavelength using a path length of 1 cm at 20 1 C, and C is the concentration of stock solution expressed as the mg of the unpuried colorant in 100 mL distilled deionized water. The A1% value for each compound is 1 cm presented in Table 2 [34].
Table 2 Dissociation constants, specic absorbance, A1% , and calculated purities of 1 cm synthetic colorants studied Colorant E 102 E 104 E 110 E 122 E 123 E 124 E 127 E 128 E 129 E 131 E 132 E 133 E 142
a b

pKa S.D.a 9.40 0.01 n.a.b 10.36 0.01 n.a. 10.36 0.02 11.24 0.01 n.a. n.a. 11.35 0.01 7.67 0.02 n.a. n.a. n.a.

A1% 1 cm 530 865 555 510 440 430 1100 620 540 2000 480 1630 1720

Purity (%) 52.5 69.9 89.2 83.6 97.0 56.9 84.2 67.5 84.6 88.1 62.3 60.3 78.5

Dissociation constants standard deviations. n.a.: not available.

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Under a RP-LC separation, the more polar compounds elute rst. However, since the chromatographic determination of the colorants is performed, normally, at pH around 7, the acidic and alkaline groups present in the molecules can change the elution sequence [37]. Thus, under the conditions of the current experiments, colorants are carried along with the organic solvent (mixture of methanol:acetonitrile). The addition of acetonitrile was found to improve signicantly the asymmetry of the peaks, which has also been noticed when used in methanol [38]. However, the addition of an inorganic electrolyte to the mobile phase, as a modier, is necessary in order to improve the separation of ionizable species and to obtain the separation in reasonably short analysis time. Ammonium acetate buffer has been used as a modier for the purication and separation of seven azo colorants by RP-HPLC and preparative liquid chromatography [39]. It was also used (at a pH 5) for the separation and determination of two (E 102 and E 110) [38] or ve azo food dyes (E 102, E 104, E 110, E 122 and E 124) in soft drinks by RP-HPLC [23]. A gradient elution prepared by a mixture of sodium acetate buffer (0.1 M and pH 7) with acetonitrile was used for the successful separation of 14 synthetic food colorants in standard solutions [22]. It was also used for the successful determination of eight azo-colorants in foodstuff by liquid chromatographyelectrospray-mass spectrometry (LCESI-MS), where it was found that excellent separation, with symmetrical peak shapes, was achieved only in the presence of ammonium acetate and acetic acid [40]. The optimum concentration of ammonium acetate in terms of plate number and peak symmetry was reported to be around 0.1 M [39]. At higher concentrations of ammonium acetate, retention times are increased, likely due to an enhanced salting-out effect and so enhance the interaction of the analyte with the stationary phase. This effect was also observed in the separation of Tartrazine (E 102), Brilliant Blue (E 133) and Sunset Yellow (E 110) by a C18 column with the use of phosphate as buffer in the mobile phase [37]. The acetate buffer solution is also stable; it does not interact with the HPLC system and is adequate for UV absorbance measurements. All colorants are present as neutral compounds at pH 7.5, in order a reversed phase mechanism to take place. Decreasing the pH of the aqueous portion of the mobile phase resulted in poor resolution of the LC separation of the azo dyes and the data could not be used for quantitative analysis [37,39]. Therefore, no further pH optimization of the mobile phase A was attempted. A 10 mg L1 mixed standard solution containing the 13 colorants was used to study the optimum conditions of separation. The initial gradient elution program used was as follows. For the initial 2 min, the mobile phase A (ammonium acetate buffer) goes through the column for elution of inorganic compounds. Between 2 and 25 min, a gradient is carried out and for the last 2 min (2527 min) the mobile phase B passes through the column for the elution of possible organic impurities. Finally, a 2-min equilibrium phase of the column is taking place for the next run. The elution of the colorants was observed in a chromatogram that was obtained by scanning the wavelength range from 350 to 800 nm, continuously. From software, the retention time (tR ),

the width (w) and the peak purity factor (PP) of each peak were calculated. The peak purity factor is produced from the comparison of the absorbance spectrum of a compound in mixture with the corresponding spectrum in its individual standard solution. In addition, the resolution coefcient (Rs ) for two successive peaks was determined. Two main resolution problems were observed when the rst gradient elution program was applied. The colorants E 128 and E 142 were co-eluted and, although they were determined in different wavelengths, a mutual interference appeared. The resolution of colorants E 122 and E 133 was also unsatisfactory. However, in this case, only E 133 interfered with the determination of E 122. These conclusions were also veried from the low peak purities values of E 128, E 142 and E 122, the high peak purity value of E 133 and the unsatisfactory Rs values for these two couples of colorants (0.30 and 0.49, respectively). The resolution problems were solved by decreasing gradually the amount of the organic solvent (mobile phase B) in gradient programs. Consequently, the time of analysis increased. Seven experiments were performed for which the rate of increase of solvent B gradually decreased from 4.3% B per min in the rst experiment to 2.6% B per min in the last experiment (data not shown). Finally, a combination of two experiments resulted in the nal optimized gradient program, which is presented in Table 2. Use of this program resulted in an efcient separation with no interferences for all colorants. The chromatogram and the chromatographic characteristics obtained from the optimized gradient program are presented in Fig. 2 and Table 3, respectively. The peak purity (PP) factor is greater than 0.999 for all compounds. The simultaneous determination of the colorants E 128 and E 142 have been achieved and E 133 no longer interferes with the determination of E 122. The elution order of E 133 and E 104III has been changed. These compounds seem to be co-eluted (small Rs value), but from their high PP values and the fact that they have different absorption spectra and they are determined in different wavelengths, obviously, they do not interfere each other. Along with the PP factor (the purity of the absorption spectra), retention times could also be used for qualitative analysis for all the compounds since their repeatability is very good. The R.S.D. of tR (n = 10) was always less than 0.32% (E 102) (Table 3).

Fig. 2. Chromatogram of a mixed standard solution using the optimized gradient program of Table 2. The concentrations of all colorants were 10 mg L1 . Identication of the peaks and their corresponding tR are given in Table 3.

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Table 3 Chromatographic data of the colorants with the optimized gradient program of Table 1 Peak 1 2 3 4 5 6 7 8 9 10 11 13 12 14 15 Colorant E 102 E 123 E 132 E 104I E 124 E 110 E 104II E 129 E 128 E 142 E 122 E 104III E 133 E 131 E 127 max (nm) 427 520 608 417 508 482 417 507 530 630 516 417 624 631 528 tR S.D. (min) (n = 10) 10.90 12.20 12.62 15.35 16.22 16.87 17.55 19.07 19.93 20.38 24.02 24.55 24.68 27.48 28.95 0.035 0.036 0.030 0.040 0.037 0.039 0.042 0.059 0.052 0.047 0.059 0.045 0.032 0.011 0.018 w (min) 0.45 0.47 0.48 0.48 0.43 0.43 0.48 0.4 0.5 0.62 0.5 0.4 0.52 0.6 0.48 Rs 2.83 0.88 5.69 1.91 1.51 1.5 3.45 1.92 0.8 6.49 1.18 0.29 5 2.72 Peak purity 1.0000 0.9990 0.9999 1.0000 0.9999 0.9999 0.9995 0.9999 0.9999 1.0000 0.9999 0.9999 0.9999 0.9999 0.9999

Table 4 Limits of detection, linear range, calibration equations and coefcients of determination (R2 ) of all colorants Colorant E 102 E 104 E 110 E 122 E 123 E 124 E 127 E 128 E 129 E 131 E 132 E 133 E 142 LOD ( g L1 ) 1.87 4.72 4.41 4.35 10.2 22.1 6.68 6.26 7.46 10.5 8.09 2.72 1.59 Linear range (mg L1 ) 0.00621 0.01449 0.01355 0.01354 0.03158 0.06746 0.02034 0.01948 0.02351 0.03227 0.02538 0.00819 0.00532 Calibration equation y = 261 + (30.3 104 )x y = 459 + (26.9 104 )x y = (4.46 103 ) + (19.1 104 )x y = 87.0 + (18.3 104 )x y = 213 + (12.0 104 )x y = (3.22 103 ) + (16.4 104 )x y = 145 + (38.8 104 )x y = 590 + (22.1 104 )x y = 609 + (20.3 104 )x y = 584 + (75.9 104 )x y = 920 + (10.7 104 )x y = (1.46 103 ) + (57.1 104 )x y = (5.09 103 ) + (60.9 104 )x R2 0.9999 0.9999 0.9995 1.0000 1.0000 0.9999 1.0000 0.9999 1.0000 1.0000 0.9999 0.9999 0.9999

Many of colorants have isomers that appear in the chromatogram as small peaks. The colorant Quinoline Yellow (E 104) exhibits three peaks corresponding to isomers, probably disulfo- and monosulfo-derivatives (E 104I, peak 4, E 104II, peak 7 and E 104III, peak 13 in Fig. 2) [22]. The unspecied peaks shown in chromatogram were identied by their absorbance spectra and were found to correspond to isomers of E 132 (15.8 min), E 142 (19.4 min), E 133 (25.8 min), E 127 (26.1 min) and E 131 (26.3 min) (Fig. 2). 3.3. Validation of the method Calibration equations of mixed standard solutions, coefcients of determination (R2 ), linear range and the limits of detection of all colorants are presented in Table 4. Calibration equations were calculated using the peak area of the substances. In case of E 104, the sum of the peak areas of the three isomers was used. The slopes of calibration equations of mixed solutions were compared with t-test with those calculated from the measurement of individual standard solutions (concentration levels 0.1060 mg L1 ) and it was found that they do not differ signicantly. The linear range is different between the analytes. In general, colorants that have high A1% value, such as 1 cm

E 127, E 131, E 133 and E 142, have been characterized from shorter linear ranges. Limits of detection for all substances varied between 1.59 g L1 for E 142 and 22.1 g L1 for E 124. In case of Quinoline Yellow (E 104) only the E 104I isomer was

Table 5 Precision data (intra-day, as R.S.D.r and inter-day, as R.S.D.R ) and concentration levels (C) of seven colorants in various food matrices Colorant Matrix Concentration level (C) 13.6 47.5 97.6 12.4 10.1 0.94 14.4 73.6 4.7 72.2 9 84.5 1.9 0.90 1.4 1.4 4.8 2.4 1.9 2.7 1.9 R.S.D.r (%) 1.2 0.53 0.37 R.S.D.R (%) 3.9 1.1 0.86 2.2 3.9 9.3 3.5 6.5 6.2 3.6 10 3.4

E 122 E 102 E 110 E 142 E 124 E 129 E 131 E 142

Sour cherry avoured drink (mg L1 ) Icing sugar (mg kg1 ) Icing sugar (mg kg1 ) Icing sugar (mg kg1 ) Liqueur (mg kg1 ) Cherries (mg kg1 ) Sweets (mg kg1 ) Jam (mg kg1 )

K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103110 Table 6 Mean recoveries and R.S.D.s (n = 6) of three colorants from spiked food matrices at various fortication levels Colorant Food matrices Concentration level 100 Recovery (%) E 122 E 142 (mg kg1 ) E 131 (mg kg1 ) (mg L1 ) Fruit avoured drink Jam Mint avoured sweets 97.9 94.4 101.8 R.S.D. (%) 0.44 0.90 1.98 50 Recovery (%) 99.6 R.S.D. (%) 0.73 10 Recovery (%) 97.7 95.1

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R.S.D. (%) 1.19 1.87

used for the determination of the LOD. The LODs of the colorants obtained in this study are in the same range as or slightly better than those reported in other studies using ion-pair HPLC methods [2729]. The precision experiments resulted in good R.S.D.s for both intra-day and inter-day precision, for the colorants and food matrices chosen. Precision was determined for colorants with different polarity, which eluted at the beginning (E 102), in the middle (E 110, E 124 and E 129) and near the end of the chromatogram (E 142, E 122 and E 131). Six different food matrices, in which the addition of these colorants is frequent, were chosen (Table 5). The intra-day precision (as R.S.D.r ) ranged from 0.37% for E 122 in fruit avored drink at a concentration of 100 mg L1 , to 4.8% for E 142 in icing sugar at a level of 0.9 mg kg1 . The inter-day precision (as R.S.D.R ) was between 0.86% for E 122 in fruit avored drink at 100 mg L1 and 10% for E142 in jam at a concentration of 9 mg kg1 (Table 5). The results obtained from this work are in general agreement with those obtained from other studies, where maximum R.S.D.s ranged between 2.1% [23] and 6.4% [40]. The results from recovery experiments in three different matrices at levels ranged from 10 to 100 mg kg1 are shown in Table 6. The high level (100 mg kg1 ) was chosen in accordance to the legislation limits [2]. E 122 and E 142, which had shown poor resolution, and E 131, a compound with low polarity (thus, with a possibly low afnity to water extraction), were chosen for recovery experiments. The developed method resulted in satisfactory recoveries for all the tested compounds, ranging from 94% for E 142 in jam to 102% for E 131 in sweets. The recoveries are more than adequate for the developed application and slightly better than those reported in literature [2628]. 3.4. Application to real samples

Table 7 Determination of colorants in water-soluble foodstuffs collected from the Greek market Sample A (mg L1 ) Colorant found E 110 E 122 E 124 E 110 E 122 E 110 E 122 E 124 E 132 E 131 E 104 E 110 E 122 E 129 E 133 E 102 E 104 E 110 E 122 E 129 E 133 E 124 Mean content of colorant 0.35 0.63 0.89 2.7 125 12.2 23.2 15.3 44.9 4.7 15.8 5.9 0.97 18.1 0.5 0.5 10.8 10.8 2.3 27.8 4.8 52 R.S.D. (%) 3.3 7.3 3.9 2.7 1.9 1.5 1.3 1.5 1.2 2.7 1.6 2.1 2.5 1.8 3.1 3.2 1.6 1.2 2.0 1.9 1.7 1.6

B (mg L1 ) C (mg kg1 )

D (mg kg1 ) E (mg kg1 )

F (mg kg1 )

G (mg kg1 )

at low concentrations. In general, colorants were determined in foods collected from the Greek market in a more wide concentration range than those reported in other studies [23,2629].

4. Conclusions The method developed was applied to food samples from market control. The samples were prepared as described in Section 2.4. A fruit avored drink (A), a fruit avored liqueur (B), a sample of icing sugar (C), three samples of sweets (D, E and F) and a blackberry avored jam (G) were analyzed, at least, in duplicate and the results are summarized in Table 7. Data from Tables 5 and 7 show that 10 out of the 13 colorants were detected, at least once. The concentrations of colorants in analyzed foodstuffs ranged from 0.35 mg L1 (E 110 in fruit avoured drink) to 125 mg kg1 (E 122 in fruit avoured liqueur). The low detection limits allowed the accurate determination of colorants in foods An efcient and accurate analytical method for the simultaneous determination of 13 permitted in EU market food colorants in a single run by liquid chromatography-diode array detection was developed and optimized. The proposed method includes a simple pretreatment procedure for the extraction of colorants from food and offers a combination of sensitivity and selectivity, simplicity and relatively short time of analysis. This method permits the detection of colorants at very low concentrations ( g kg1 range). The applicability was veried by the determination of colorants present in various water-soluble foodstuffs.

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