Simultaneous determination of synthetic dyes in foodstuffs and beverages by high-performance liquid chromatography coupled with diode-array detector

Dyes and Pigments 99 (2013) 36e40

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Simultaneous determination of synthetic dyes in foodstuffs and beverages by high-performance liquid chromatography coupled with diode-array detector Stefania Bonan, Giorgio Fedrizzi, Simonetta Menotta, Caprai Elisabetta* Istituto Zooprofilattico Sperimentale Lombardia Emilia Romagna, Chemical Department, Via Pietro Fiorini, 5, 40127 Bologna, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 October 2012 Received in revised form 19 March 2013 Accepted 25 March 2013 Available online 19 April 2013

Synthetic dyes are added to food and drinks to restore their original appearance when color is affected by processing, storage, packaging and distribution. Furthermore colors are used to make food more visually attractive to consumers. The EU Directive 1994/36/CE lists the permitted substances that can be used in foodstuffs. In order to investigate the content of some permitted and non-permitted dyes in food and drinks, a sensitive and helpful method has been developed to determine simultaneously seventeen synthetic colorants by high-performance liquid chromatography coupled with a diode-array detector in solid food matrices and beverages. Substances involved were azorubine (E122), amaranth (E123), cochineal red A (E124), red 2G (E128), allura red (E129), azocarmine B (AZO B), azocarmine G (AZO G), ponceau 2R (P2R), ponceau 6R (P6R), tartrazine (E102), sunset yellow (E110), quinoline yellow (E104), orange II (OR II), metanil yellow (MY), patent blue V (E131), indigo carmine (E132) and brilliant blue FCF (E133). Solid food matrices were extracted by a waterealcohol mixture, cleaned up on a polyamide SPE cartridge and eluted with basic methanol solution. Otherwise a simple dilution and filtration of samples were used for drinks. The method has been validated according to Regulation (2004/882/CE) and could be applied to a concentration range between 5 and 300 mg kg
1 (5e100 mg l
1 for drinks) depending on the dye. The accuracy (precision and trueness) and specificity were assessed. During years 2009e2011 many food samples for the detection of synthetic colorants were analyzed. Most of them were fresh fishery products in which the use of food colorant is banned. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Food additives Synthetic dyes Foodstuffs Beverages HPLC/DAD Multiresidue method

1. Introduction Color is one of the main properties by which food is evaluated. For this reason food colorants (synthetic or natural) are added to foodstuffs in order to make them more visually attractive to consumers and to restore their original appearance when it has been lost during production processes. European Directive 1994/36/EC [1] lists the colors that can be added to food: it also defines foodstuffs to which only certain colorants may be added, their permitted maximum level and their use restrictions as well. The European Food Safety Authority (EFSA) is in charge to reassess all allowed additives according to the Commission Regulation 2010/ 257/CE [2]. In fact, the permitted amount of synthetic dyes is strictly regulated because of their potential risk to human health. The existence of a functional relationship between the ingestion of

* Corresponding author. Tel.: þ39 0514200060; fax: þ39 0514200055. E-mail addresses: [email protected], [email protected] (C. Elisabetta). 0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dyepig.2013.03.029

artificial food colors and an increase both in duration and frequency of hyperactive behaviors in children has been shown [3e5]. Moreover, the chronic consumption of metanil yellow seems to predispose to neurotoxicity in young and adult animals [6]. Some synthetic food colors, in particular azo-dyes, could be reduced by azoreductase enzymes in intestinal bacteria and in liver cells with the release of aromatic amines to the organism [7]. Considering both the potential effects on human health and the need for knowledge of the components of food, detection of synthetic dyes in foodstuffs and beverages is an important issue to deal with. These circumstances are proved by numerous alerts, border rejections and information for attention or follow-up created by RASFF. The European Union (EU) Rapid Alert System for Food and Feed denounces the illegal use of many food colors which include too much high content of some dyes, their undeclared presence or illegal use in certain types of food [8]. In order to prevent food fraud and for food safety reasons it is required to develop an accurate and reliable method for detection of synthetic dyes in foodstuffs and beverages. Many analytical techniques have been developed and

S. Bonan et al. / Dyes and Pigments 99 (2013) 36e40

improved for detection of dyes in food. Determination based exclusively on absorbance measurements at the wavelength corresponding to its maximum absorption can be problematic in dye mixtures because of the overlap of absorption spectra [9]; it may require the use of derivative spectrophotometry [10]. Most developed methods depend separation and consequent absorption detection. Methods include techniques such as thin layer chromatography (TLC) [11,12], high-performance TLC combined with image processing [13], solid phase spectrophotometry [14,15], adsorptive voltammetry [16]; however these methods have a time consuming sample pretreatment. Other methods employ microemulsion electrokinetic chromatography [17] or capillary electrophoresis [9,18,19]: they are suitable for analysis of ionic species and hydrophilic compounds but they have sensitivity problems as a result of small injection volume [25]. There were some methods based on pulse-polarography [20,21] and isotachophoresis [22]. Results from voltammetry techniques [23] were compared to those obtained using liquid chromatography, and they appeared to be in good agreement [21]. However, the most widely used techniques are high-performance liquid chromatography (HPLC) [24e29], ion-pair liquid chromatography [30,31] and high-performance ion chromatography [32]: these techniques allow to reach high sensitivity and resolution of food colorants. Analytical methods based on liquid chromatographyemass spectrometry [30] are particularly suitable for analysis of illegal and toxic dyes since they have been developed to unambiguously identify colorants at trace levels. The aim of the current work was to develop an RP-HPLC method coupled with diode-array detector (DAD) for the determination of 17 synthetic dyes in different types of foodstuffs and drinks. The colorants taken into account were both permitted and nonpermitted for use as food additives in the EU. The analyzed dyes were the azo-compounds tartrazine (E102), sunset yellow (E110), azorubine (E122), amaranth (E123), cochineal red A (E124), red 2G (E128), allura red (E129), metanil yellow (MY), orange II (OR II), ponceau 2R (P2R), ponceau 6R (P6R); the benzophenazine derivatives azocarmine B (AZO B) and azocarmine G (AZO G); the quinophthalone quinoline yellow (E104); the triarylmethane compounds patent blue V (E131) and brilliant blue FCF (E133) and the indigo colorant indigo carmine (E132). The method was validated according to 2004/882/CE Regulation (Annex III) [33] for the detection of yellow, red and blue food dyes in beverages and yellow and red dyes in solid food matrices. 2. Materials and methods

37

a concentration of 1000 mg l
1. Tartrazine and metanil yellow were dissolved in water at the same concentration. Seventeen food colors were divided into 4 groups for working solutions: allowed red colorants (group A), not allowed red colorants (group B), yellow (group C) and blue colorants (group D). Group A includes E122, E123, E124, E128 and E129. Group B contains illegal dyes azocarmine B, azocarmine G, ponceau 2R and ponceau 6R. Group C comprehended E102, E104, E110, metanil yellow and orange II. Group D included E131, E132 and E133. For each group, 100 mg l
1 solutions were disposed as spiking solutions by mixing each stock solution. Calibration curves of the four groups ranged from 1 to 100 mg l
1. The solvent for all dilutions was methanolewater (1:1 v/v). 2.2. Equipments Analyses were performed with HPLC HP 1100 series equipped with G1322A degasser, G1311A quaternary pump, G1313A autosampler, G1315A DAD and HP ChemStation for collect and process data. All instrumentation was provided by Agilent Technologies (Waldbronn, Germany). Ultrasonic bath was from Branson ultrasonic (Danbury, CT, USA). Sample disperser Ultra-Turrax and horizontal shaker were provided by IKA (Staufen Germany). Ultracentrifuge was from Beckman Coulter (Brea CA, USA) and microcentrifuge was from Eppendorf (Hamburg, Germany). 2.3. Sample preparation 2.3.1. Solid food matrices procedure Four grams of sample were accurately weighted. Then 20 ml of ethanolewater (1:1 v/v) were added. The sample was homogenized with a sample disperser and placed into an ultrasonic bath for 15 min. It was shaken for 1 h with a horizontal shaker and centrifuged at 15,000 rpm for 10 min at 0  C. The supernatant was separated and the solid-state sample was extracted once again with a further 20 ml of the solvents mixture. The extracts were collected and H3PO4 was added drop wise to pH 2. The SPE polyamide cartridges were conditioned by 4 ml of MeOH and 2 ml of water. The extract was flowed slowly through the cartridges (1 ml min
1). SPE columns were washed with 2 ml of water and gently vacuum-dried for 30 s. The colorants were eluted with 4.5 ml of a methanol/1% ammonia solution (1:1 v/v) in a graduated test tube. The final extract was made up to a final volume of 5 ml with the same solvent mixture and 20 ml of solution were injected into the HPLC.

2.1. Chemical and reagents Ethanol (EtOH) was purchased from Fluka Analytical (Steinheim, Germany), methanol (MeOH) and phosphoric acid (85% w/v) were from SigmaeAldrich (Steinheim, Germany). Ammonia solution (30% w/v) and acetonitrile (MeCN) were purchased from Carlo Erba Reagents (Rodano, Milan, Italy). Sodium acetate trihydrate (NaOAc) was purchased from Merk KgA (Darmstadt, Germany). Ultrapure water and Ultrafree Centrifugal filter devices were from Millipore (Bedford, MA, USA). Discovery DPA-6S polyamide 6 ml tube, 500 mg SPE cartridges were purchased from Supelco analytical (Bellefonte, PA, USA). Tartrazine, amaranth, azocarmine B and orange II were purchased from Fluka while brilliant blue FCF, indigo carmine, patent blue V, allura red, sunset yellow, ponceau 6R and azocarmine G were from SigmaeAldrich (Steinheim, Germany). Quinoline yellow, cochineal red A, red 2G, azorubine, metanil yellow and ponceau 2R were supplied by Dr. Ehrenstorfer-Schäfers (Augsburg, Germany). Appropriate amounts of powder of each color (except E102 and metanil yellow) were dissolved in methanol/water (1:1 v/v) to give

2.3.2. Beverages procedure Gas in beverages was removed by placing them into an ultrasonic bath for 20 min. Then the beverage was diluted (1:1 v/v) with deionized water. The sample was centrifuged at 15,000 rpm for 10 min at room temperature in a 0.5 ml tube provided with filter device. 2.4. Chromatographic conditions Separation was carried out by Luna C8 column (150 mm  4.6 mm id, 3 mm particle size, Phenomenex, CA, USA) and a binary gradient which included acetonitrile (mobile phase A) and 100 mM sodium acetate buffer at pH 7 (mobile phase B). All experiments were realized at room temperature and flow rate was set at 1 ml min
1 constantly. Time for separation was 35 min. The optimized gradient for dye separation is given in Table 1. Injection volume was 20 ml. Diode-array detector was programmed to monitor the colorants from 240 to 700 nm (515 nm the reds, 420 and 480 nm the yellows and 620 nm the blues).

38

S. Bonan et al. / Dyes and Pigments 99 (2013) 36e40 Table 1 Gradient separation program for the separation of 17 dyes by HPLCeDAD at a flow rate of 1 ml min
1. t (min)

A (%)

B (%)

0.0 2.0 25.0 30.0 35.0

1 1 40 40 1

99 99 60 60 99

2.5. Validation of the analytical procedure The method has been validated according to 2004/882/CE Regulation (Annex III) [33]. Validation was performed by analyzing spiked samples with colorant groups A, B, C and D. Tested solid matrices were meat and fishery products, pastries, cakes, jam, bakery products, fruit and vegetable sauces. Liquid matrices were soft drinks, alcoholic aperitifs, clear fruit juices. The evaluated parameters were the following: linearity of response, applicability range, accuracy as trueness and precision, limit of detection (LOD), limit of quantification (LOQ). All parameters were assessed for both solid matrices and beverages. To test the linearity of standard solutions, 4 replicates of calibration curves at concentrations ranging from 1 to 100 mg l
1 were injected. Five replicates of spiked samples of each group of dye were injected in order to define linear responses of the analytes in matrix. Equations and calibration curves correlation coefficients (r2) were calculated. To evaluate the repeatability of the method, six replicates at three different spiking levels were analyzed in the same day for each dye. The same procedures, performed on three days, were applied to give intermediate repeatability. Identification of every single molecule was carried out comparing both retention time during the chromatographic run and peak area spectrum. For both solids and liquids, twenty blank samples were analyzed to exclude matrix interferences and verify method specificity. For solid matrices spiking levels for each colorant of groups A and C ranged from 10 to 300 mg kg
1 while fortification levels for group B dyes ranged from 5 to 100 mg kg
1. RSDr and RSDR were assessed by processing results coming from 3 spiking levels (50, 100 and 200 mg kg
1). Concentrations, standard deviation, repeatability and intermediate repeatability as percentage variation coefficient (CV%) were calculated for each spiking levels mentioned before. Beverages samples were fortified at levels ranging from 5 to 100 mg l
1 with group A, B, C and D separately. RSDr and RSDR were assessed considering fortification levels of 5, 10 and 25 mg l
1. Concentrations, standard deviation, repeatability and intermediate repeatability as percentage variation coefficient (CV%) were calculated for each spiking levels mentioned before.

3. Results Data regarding 9 red dyes in solid foodstuffs are presented in Table 2 while those from validation of 17 colorants in beverages are shown in Table 3. In the solid matrices all correlation coefficients for matrix calibration curves were above 0.98. The mean recovery of each dye was calculated upon the 3 spiking levels at which reproducibility was assessed. Average recovery values are shown on Table 2. They ranged from 60.4% for E123 to 90.7% for E129. RSDr% ranged from 3.81% to 39.05% while RDSR% ranged from 2.97% to 34.84%. All correlation coefficients of calibration curves for beverages were above 0.99 except for metanil yellow, E131 and E132 that presented quite low r2 (0.92, 0.93 and 0.97 respectively). The average recoveries for beverages ranged from 62.9% for metanil yellow to 101.1% for E124. All calculated RSDr were less than 15% as all the RSDR values. 4. Discussion There is a strong need for an extraction method which covers a wide range of dyes in many matrices, particularly for routine analysis. At the same time, the huge number and different chemical properties of existing colorants ensure a lot of troubles in building up a universal method. The proposed method allows the concurrent analysis of 17 compounds in a variety of matrices (red and yellow for solid matrices and red, yellow and blue for beverages). The method for solid matrices was validated analyzing different foodstuffs, in particular meat and fishery products, pastries, cakes, jam, bakery products, fruit and vegetable sauces. This high heterogeneity, inevitably, did not allow optimal validation performances for all colorants in all matrices (Tables 2 and 3). In fact, quite high variability was exhibited within validation parameters. Repeatability and reproducibility values are related to fortification levels of 100 mg kg
1 and 25 mg kg
1 for group A and B respectively. In particular Table 2 reports validation parameters for red dyes. The same parameters were calculated for yellow dyes. The values were quite similar: E110 (sunset yellow) showed the best validation performances with a mean recovery more than 80% and RSDr and RSDR lower than 20%. On the other hand E102 (tartrazine) had the worst validation performances with a main recovery less than 20%. In solid matrices, among red dyes (Table 2), amaranth analysis showed rather low recoveries (60%) and quite high repeatability and reproducibility values (both of them were over than 30%). The best validation parameters among red dyes were obtained from allura red (E129) that showed a mean recovery of about 90% and repeatability and reproducibility parameters very low. In general, RSD% values for dyes of group B were considerably lower than those resulting from group A. In some cases RSDr values were higher than RSDR parameters: probably the large number of replicates and the ruggedness of the method allowed these apparently anomalous parameters. Rather high values of standard

Table 2 Validation data relating the analysis of solid food matrices. Food color

Detection wavelength (nm)

Equation of calibration curve for solid food matrix

E122 Azorubine E123 Amaranth E124 Cochineal red A E128 Red 2G E129 Allura red Azocarmine B Azocarmine G Ponceau 2R Ponceau 6R

515 515 515 515 515 515 515 515 515

y y y y y y y y y

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

40.61x 39.44x 28.72x 33.43x 40.72x 10.20x 20.68x 22.62x 25.63x

þ þ þ þ þ þ
þ þ

198.48 137.10 142.14 221.16 386.58 25.48 18.27 55.52 52.88

r2

RSDr% (n ¼ 6)

RSDR% (n ¼ 18)

Mean recovery %

0.99 0.99 0.99 0.99 0.99 0.98 0.99 0.99 0.99

30.4 39.0 22.8 24.4 15.1 5.0 3.8 8.5 8.6

24.1 34.8 17.8 19.7 14.2 4.6 2.9 8.4 8.5

78.2 60.4 75.0 84.0 90.7 75.8 64.4 89.3 87.3

S. Bonan et al. / Dyes and Pigments 99 (2013) 36e40

39

Table 3 Validation data related to food dyes in beverages. Food color

Detection wavelength (nm)

Equation of calibration curve for beverages matrix

E122 Azorubine E123 Amaranth E124 Cochineal red A E128 Red 2G E129 Allura red Azocarmine B Azocarmine G Ponceau 2R Ponceau 6R E102 Tartrazine E104 Quinoline yellow E110 Sunset yellow Orange II Metanil yellow E131 Patent blue V E132 Indigo carmine E133 Brilliant blue FCF

515 515 515 515 515 515 515 515 515 420 420 480 480 420 620 620 620

y y y y y y y y y y y y y y y y y

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

33.81x
14.93 18.28x
7.64 22.36x
5.34 14.87x
1.95 31.20x
25.29 4.65x
0.82 14.47x
20.77 14.74x
3.89 17.42x
17.34 1.43x
1.76 31.00x
83.86 28.67x
11.32 25.68x
83.90 20.23x
50.53 59.09x
216.62 8.48x þ 16.32 46.80x
146.68

deviation could be ascribed to the heterogeneity between matrices used for validation procedure. In fact complex matrices, such as food (in particular meat and fishery products), can bind colorants in different sites. Solvent extraction may be not effective enough to remove all dyes from the matrix at optimum levels. Furthermore, the extracts prior to the clean up step are various mixtures containing different substances coming from animal and vegetable tissues. Polyamide resin used in SPE cartridges adsorbs polar compounds containing groups that can be protonated. Since the sample extract is at pH 2 before the SPE step, the dyes are adsorbed to the polyamide stationary phase by Van der Waals’ interactions. Other hydrophilic substances can mask SPE interaction sites by reducing their binding power for the dyes and consequently reducing the capacity of the cartridges. Furthermore, some substances, as amaranth, are strongly restrained by SPE cartridges and the ammonia solution used for elution could be insufficient for its release (low recoveries). For solid food matrices, LOQ values calculated as 10 the standard deviations of blank noise were 10 mg kg
1 for groups AeC and 5 mg kg
1 for group B. For the beverages (Table 3), repeatability and reproducibility values were related to 10 mg l
1 spiking level. RSD% values for drinks are generally lower than those resulting from the solid matrices. This could be due to a poor sample handling. All the recoveries were higher than 75% except for azocarmine G and metanil yellow. However, their recoveries were higher than 60%. This could be explained considering the filtration step before HPLC analysis: perhaps the filter partly retained the dyes. The repeatability and reproducibility were quite low. Optimized gradients for separation of dyes are achieved considering the formation of neutral species from the ionized

RSDr% (n ¼ 6)

RSDR% (n ¼ 6)

Mean recovery %

0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.92 0.93 0.97 0.99

1.3 1.2 0.6 0.4 1.0 0.2 3.6 1.4 0.3 1.4 0.7 0.8 0.9 0.5 0.7 1.1 2.7

1.8 1.8 1.2 0.6 1.1 0.7 3.6 1.8 1.2 1.0 1.3 0.6 1.6 1.2 0.9 1.2 2.3

93.9 98.7 101.1 98.3 86.1 92.7 63.1 77.1 87.0 87.6 75.7 90.8 71.1 62.9 83.2 75.0 66.3

analytes. 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 [26]. At pH 7 all dyes are suitable for reverse phase chromatography since they are neutral. The colorants were eluted from the column according to increasing polarity. The first colorant eluted from the column was the more polar dye tartrazine, while the last was metanil yellow which presents a single sulfonated group and no alcoholic substituents. Quinoline yellow (E104) consists essentially of sodium salts of a mixture of disulfonates (principally), monosulfonates and trisulfonates [34]. It shows three peaks corresponding to relative isomers. The sum of the three peaks was used for determination of recoveries and precision validation data. Figs. 1e3 show chromatograms related to three samples spiked with red (group AeB together), yellow (group C) and blue dyes (group D). DAD detector higher signal was obtained by setting pertinent wavelengths for each group of dyes. For red colorants, wavelength for quantification was fixed at 515 nm as a reasonable average of the maximum absorptions within the red colorants. For yellow dyes 2 different wavelengths were set: E102, E104 and metanil yellow were monitored at 420 nm, while E110 and orange II were observed at 480 nm. For blue dyes DAD wavelength was set at 620 nm. Fortification levels for allowed food colors in foodstuffs were chosen in accordance to legislation limits. For unauthorized dyes in solid matrices, spiking levels was slightly lower. It was estimated that significant quantities of colorant should be added to foodstuffs for changing color appearance. For this reason, validation of illegal red dye in solid matrices was carried out at the ppm order of magnitude.

mAU 50

E129

mAU 140

r2

E110

E104(iii) MY

E128 40

120

E104(i)

E124

100 80

OR II

E102

30

E122

E104(ii)

20

60

E123

40

AZO B

P2R P6R

10

AZO G

20

0

0

-10

0

5

10

15

20

25

30

Fig. 1. Chromatogram of a fish sample spiked with group A red dyes at 10 mg kg group B red dyes at 5 mg kg
1 monitored at 515 nm.

min
1

and

0

5

10

15

20

25

30
1

Fig. 2. Chromatogram of a biscuits sample spiked at 10 mg kg colorants monitored at 420 nm.

min

with group C yellow

40

S. Bonan et al. / Dyes and Pigments 99 (2013) 36e40

mAU 25

E133

E132

E131

20 15 10 5 0 0

5

10

15

20

25

30

min

Fig. 3. Chromatogram of a soft drink sample fortified with group D blue colorants at 5 mg l
1 monitored at 620 nm.

During the years 2009e2011, 202 samples of foodstuffs and drinks were collected for the analysis of synthetic dyes. They included a great variety of matrices such as fishery products (tuna fish, surimi, shrimps, fish roe), meat (aged or smoked sausages, muscles, baby foods), vegetable products (tomato sauce, fruit juices, jam, syrups), bakery products, additives and diet supplements. Most of them were fishery products, in particular tuna fish. In 9 out of 51 fishery samples the illegal presence of E124, cochineal red A, was detected. Colorant concentrations ranged from 6.5 to 57 mg kg
1. Especially at low ppm concentrations, the presence of the colorant itself could not be a matter of concern for food safety. Food dyes could be used fraudulently for masking a lack of food quality due to deterioration processes: however the sample results were not compliant. Extraction and analytical methods could be used to detect brilliant black (E151): the method is not validated but it is applied in routine analysis with good performances. The same extraction method used for red and yellow dyes was tested for detection of blue colorants in solid matrices, but results were not satisfactory. 5. Conclusions The method had a practical application to real samples. It was useful for routine analysis and allowed to process up to 20 samples per day. Furthermore it allowed the simultaneous detection of red and yellow dyes in solid matrices and red, yellow and blue dyes in beverages in a single extraction and chromatographic run. The method showed good linearity, repeatability and reproducibility parameters considering the heterogeneity of analyzed matrices and the large number of analytes. References [1] European Parliament and Council Directive 94/36/EC of 30 June 1994 on colours use in foodstuffs. Off J EC No L237/13 10/9/94. [2] Commission Regulation (EU) 2010/257/CE of 25 March 2010 setting up a programme for the re-evaluation of approved food additives in accordance with Regulation (EC) No 1333/2008 of the European Parliament and of the Council on food additives (text with EEA relevance). Off J EU L80/19 26/3/ 2010. [3] Rose TL. The functional relationship between artificial food colors and hyperactivity. J Appl Behav Anal 1978;11:439e46. [4] McCann D, Barrett A, Cooper A, Crumpler D, Dalen L, Grimshaw K, et al. Food additives and hyperactive behaviour in 3-year-old and 8/9-year-old children in the community: a randomised, double-blinded, placebo-controlled trial. Lancet 2007;370:1560e7. [5] Assessment of the results of the study by McCann, et al. (2007) on the effect of some colours and sodium benzoate on children’s behavior. www.efsa.europa. eu/en/efsajournal/doc/660.pdf [accessed 23.11.11]. [6] Nagaraja TN, Desiraju T. Effects of chronic consumption of metanil yellow by developing and adult rats on brain regional levels of noradrenaline, dopamine and serotonin, on acetylcholine esterase activity and on operant conditioning. Food Chem Toxicol 1993;31(1):41e4. [7] Hildenbrand S, Schmahl W, Wodarz R, Kimmel R, Dartsch PC. Azo dyes and carcinogenic aromatic amines in cell cultures. Int Arch Occup Environ Health 1999;72(3):M52e6.

[8] https://webgate.ec.europa.eu/rasff-window/portal/index.cfm?event¼notifications ListRASFFPortal [accessed 21.11.11]. [9] Ryvolova M, Taborsky P, Vrabel P, Krasensky P, Preisler J. Sensitive determination of erythrosine and other red food colours using capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A 2007;1141: 206e11. [10] Berzas Nevado JJ, Cabanillas CG, Salcedo AMC. Simultaneous spectrophotometric determination of three food dyes by using the first derivative of ratio spectra. Talanta 1995;42:2043e51. [11] Dugar SM, Leibowitz JN, Dyer RH. Identification of synthetic colors in beverage alcohol products by solid-phase extraction and thin layer chromatography. J Assoc Off Anal Chem 1994;77(5):1335e7. [12] Oka H, Ikaia Y, Ohno T, Kawamura N, Hayakawa J, Harada K, et al. Identification of unlawful food dyes by thin-layer chromatographyefast atom bombardment mass spectrometry. J Chromatogr A 1994;674(1e2):301e7. [13] Soponar F, Mot¸ AC, Sârbu C. Quantitative determination of some food dyes using digital processing of images obtained by thin-layer chromatography. J Chromatogr A 2008;1188(2):295e300. [14] Capitan F, Capitan-Vallvey LF, Fernandez MD, de Orbe I, Avidad R. Simultaneous determination of the colorants tartrazine, ponceau 4R and sunset yellow FCF in food stuffs by solid phase spectrophotometry using partial least squares multivariate calibration. Anal Chim Acta 1996;331:141e8. [15] Capitan-Vallvey LF, Fernandez MD, de Orbe I, Avidad R. Determination of colorant matters mixtures in foods by solid-phase spectrophotometry. Talanta 1998;47:861e8. [16] Ni Y, Bai L, Jin L. Multicomponent chemometric determination of colorant mixtures by voltammetry. Anal Lett 1997;30:1761e77. [17] Huang HY, Chuang CL, Chiu CW, Chung. Determination of food colorants by microemulsion electrokinetic chromatography. Electrophoresis 2005;26: 867e77. [18] Kuo KL, Huang HY, Hsieh YZ. High-performance capillary electrophoretic analysis of synthetic food colorants. Chromatographia 1998;47:5e6. [19] Huang HY, Chiu CW, Sue SL, Cheng CF. Analysis of food colorants by capillary electrophoresis with large-volume sample stacking. J Chromatogr A 2003;995:29e36. [20] Fogg G, Yoo KS. Direct differential-pulse polarographic determination of mixtures of the food colouring matters tartrazine-Sunset Yellow FCF, tartrazine-Green S and amaranth-Green S. Analyst 1979;104:723e9. [21] Chanlon S, Joly-Pottuz L, Chatelut M, Vittori O, Cretier JL. Determination of Carmoisine, Allura red and Ponceau 4R in sweets and soft drinks by differential pulse polarography. J Food Comp Anal 2005;18(6):503e15.  [22] Karovi cová J, Polonský J, Príbela A, Simko P. Isotachophoresis of some synthetic colorants in foods. J Chromatogr A 1991;545(2):413e9. [23] Florian M, Yamanaka H, Carneiro PA, Zanoni MVB. Determination of brilliant blue FCF is the presence and absence of erythrosine and quinoline yellow food colours by cathodic stripping voltammetry. Food Addit Contam 2002;19: 803e9. [24] Ma M, Luo X, Chen B, Su S, Yao S. Simultaneous determination of watersoluble and fat-soluble synthetic colorants in foodstuff by high-performance liquid chromatographyediode array detection. J Chromatogr A 2006;1103: 170e6. [25] Minioti KS, Sakellariou CF, Thomaidis. Determination of 13 synthetic food colorants in water-soluble foods by reversed-phase high-performance liquid chromatography coupled with diode-array detector. Anal Chim Acta 2007;583:103e10. [26] Kirschbaum J, Krause C, Pfalzgraf S, Bruckner H. Development and evaluation of an HPLCeDAD method for determination of synthetic food colorants. Chromatographia 2003;57:S115e9. [27] Yoshioka N, Ichihashi K. Determination of 40 synthetic food colors in drinks and candies by high-performance liquid chromatography using a short column with photodiode array detection. Talanta 2008;74:1408e13. [28] Vidotti EC, Costa WF, Oliveira CC. Development of a green chromatographic method for determination of colorants in food samples. Talanta 2006;68: 516e21. [29] Kirschbaum J, Krause C, Bruckner H. Liquid chromatographic quantification of synthetic colorants in fish roe and caviar. Eur Food Res Technol 2006;222: 572e9. [30] Fuh MR, Chia KJ. Determination of sulphonated azo dyes in food by ion-pair liquid chromatography with photodiode array and electrospray mass spectrometry detection. Talanta 2002;56:663e71. [31] Gianotti V, Angioi S, Gosetti F, Marengo E, Gennaro MC. Chemometrically assisted development of IP-RP-HPLC and spectrophotometric methods for the identification and determination of synthetic dyes in commercial. J Liq Chromatogr Relat Technol 2005;28:923e37. [32] Chen Q, Mou S, Hou X, Riviello JM, Ni Z. Determination of eight synthetic food colorants in drinks by high-performance ion chromatography. J Chromatogr A 1998;827:73e81. [33] Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules. [34] Commission Directive 2008/128/EC of 22 December 2008 laying down specific purity criteria concerning colours for use in foodstuffs (codified version) text with EEA relevance. Off J L 006 10/01/2009:0020e0063.