Simultaneous Determination of Tartrazine and Sunset Yellow in Soft Drinks by Liquid Chromatography

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1 Simultaneous Determination of Tartrazine and Sunset Yellow in Soft Drinks by Liquid Chromatography ELENA DIACU*, CORNELIA PETRONELA ENE University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of Analytical Chemistry, 1 Polizu Str., , Bucharest, Romania Food pigments have been used to enhance sensory response and meet the consumers always growing demands. Synthetic pigments present some advantages in comparison with the natural ones, but they can cause adverse toxicological side effects, being considered as unhealthy substances for humans. Therefore, much attention has been given to the development of new advanced analytical techniques for the analysis of synthetic colours in beverages. The main aim of this work was the determination of two colorants, Tartrazine (E-102) and Sunset Yellow (E-110) added to different soft drinks, using an efficient chromatographic method, without an extensive sample pre-treatment. The obtained results for the determinations of Tartrazine and Sunset Yellow on real soft drink samples demonstrated that the described method is rather fast, is accurate and precise, with a very good detection limit (LOD) and quantification limit (LOQ). Keywords: synthetic food pigments, Tartrazine, Sunset Yellow, HPLC, soft drinks Since the visual aspect plays a determinant role for food selection by the consumers, synthetic food dyes stand out as one of the widest used additive class for food industry. Due to their property to keep or to improve the perceived flavor and color of the food, drinks and cosmetics, food pigments have been used for centuries for many purposes, especially to enhance sensory response in concordance with the consumers desire [1, 2]. However, synthetic colorants are suspected to be unhealthy substances for humans and consequently they became perceptible as undesirable or harmful by consumers. Therefore, the synthetic colors have been the subject of numerous toxicological investigations [3-6] and their values are established by national and international legislation, especially for their use in food, dinks, drugs and cosmetics [7-9]. The CEE Directive 94/36 has stipulated a reduced number of synthetic colorants to be used in foodstuffs, specifying their maximum admitted limits [8]. Tartrazine (TAR) - Color Index and Sunset Yellow (SUN) - Color Index 15985, are two synthetic azo dyes among the widest additives used in food and especially in soft drinks, known by consumers also like E102 for TAR and E 110 for SUN. In order to realize all diverse yellow-shades to different food, drinks and drugs, TAR and SUN are added either individually or in binary combination, the TAR offering a lemon yellow color and the SUN a bright orange yellow one. As organic substances, they are the trisodium salt of (4E)-5-oxo-1-(4- sulfonatophenyl)-4-[(4-sulfonatophenyl)hydrazono]-3- pyrazolecarboxylate and disodium salt of 1-psulphophenylazo-2-naphthol-6-sulphonic acid, respectively. The chemical structures of TAR and SUN are represented in figure 1. TAR and SUN are permitted food colorants by international laws in processing in restrictive amounts [8], but the content of TAR and SUN must be declared (when used) and their content must not exceed the limit established by legislation [10]. The maximum accepted level by the European Legislation for TAR and SUN in food is of 100 ppm for TAR and 50 ppm for Sun, when used individually and no more than 100 ppm in binary mixtures [8]. Therefore, efficient analytical techniques able to detect and to analyze the content of different pigments are required. Several analytical methods have been achieved in order to determine TAR and SUN, either separately, in combination or in mixture with other colorants. Here there are included spectrophotometric methods [11-15], chromatographic methods [16-28] and capillary electrophoresis [29]. Due to the fact that using spectrophotometric methods in the analysis of food dyes there is the major inconvenient of the interferences and almost always an additional extractive step is required, HPLC methods are more attractive for this purpose, offering the possibility to analyze synthetic pigments without a prior step, with high accuracy and precision, and with a very good detection limit, as well. The present work deals with the optimisation and application of a rapid and simple HPLC method for simultaneous determination of TAR and SUN from soft drinks without an extensive sample pre-treatment. Experimental part Samples The soft drink samples were filtered in vacuum on a microfiltration membrane (pore size of 0.45 μm) and were placed on an ultrasonic bath for 15 min in order to degas. * e_diacu@chim.upb.ro Fig. 1. The chemical structure of Tartrazine (A) and Sunset Yellow (B) 745

2 Fig. 2. Calibration curves for Tartrazine (A) and Sunset Yellow (B) The ph sample was adjusted at 6.50 value, with a NaOH solution 10%, under potentiometric control, a glass membrane electrode being used as indicator electrode. Chemicals Tartrazine (purity 90%) and Sunset Yellow (purity 90%) were obtained from SIGMA-Alhrich Co. Ltd. (Dorset, UK). Monopotassium phosphate and disodium phosphate were purchased from Scharlow (France); NaOH and all solvents were Chromasolv purity from Merck (Darmstadt Germany). All aqueous solutions were prepared using bidistilled water. Mixed stock standard solution (1000 mg/l) was prepared by weighing g of each colour to 100 ml and its preservation at 2 8 o C temperature and is discarded after 6 months. Mixed working calibration standards were prepared by diluting stock solution taking the appropriate volumes after its equilibration to room temperature. The chromatographic separation of the two synthetic studied colorants was realised in gradient elution system. The two components of the elution system were a mixture of acetonitrile and methanol, 1:4 volumes, as organic modifier (solvent A), and the aqueous constituent (solvent B) consisting from a phosphates buffer (prepared by weighing of g Na 2 HPO g KH 2 PO 4 and diluted to 1000 ml with water). Apparatus The measurements for the separation and determination of TAR and SUN content from soft drinks samples were performed using an HPLC system Agilent Series 1100 equipped with diode array detection - DAD, on reverse column and auto-sampler. For TAR and SUN analysis, it has been chosen a stationary phase as Hypersil 5 μmos C 8 on a column oven of 250 x 4.60 mm. The flow rate was set up at 1.0 ml/min and the injection volume was 10 µl. The detection was achieved at 470 nm, as the optimum wavelength for monitoring both analytes. Results and discussions The calibration curves for TAR and SUN were plotted on the basis of peak areas of various concentrations of working standard solutions, daily prepared from the mixed stock solution of 1000 mg/l, after equilibration to room temperature. The mobile phases gradient programme was appointed as follows: 0-1 min isocratic elution at 5%A:95%B 1-15 min linear gradient from 5%A:95%B to 45%A:55%B min linear gradient to recover initial conditions of 5%A:95%B min conditioning step at 5%A:95%B The retention times were for TAR 6.4 ± 0.2 min and 12.9 ± 0.2 min for SUN. The calibration curves obtained by fortification of a real sample (lemon juice) with TAR and SUN are presented in figure 2 A and B, and it can be remarked that these are very similar. A very good linearity for both colorants in the range of mg/l concentrations, with an excellent regression factor ( and ). Under the optimized experimental conditions, the HPLC method was applied to study the TAR and SUN content in real soft drink samples. The two pigments were identified on the chromatograms of the samples by comparison of their retention times with those of standard synthetic pigments solutions. A total of fourteen liquid soft drinks samples were purchased randomly from Romanian markets and analyzed. The different kind of soft drinks were seven orange juices, three peach juices, one lemon juice one apple juice and two carrot juice, according to the incidence of TAR and SUN in different types of soft drinks. The results for quantitative determination of TAR and SUN separately, and for their sum, are exhibited in table 1, with a measurement uncertainty for TAR, U TAR = 1.12 mg/ L and U SUN = 1.03 mg/l. For each soft drink sample, two 746

3 Fig. 3. Typical chromatogram for an orange juice sample Table 1 TARTRAZINE AND SUNSET YELLOW CONTENT IN DIFFERENT KINDS OF SOFT DRINKS HPLC analyses have been performed, in the table 1 being showed the mean value of the colorant content. Because, usually into many kind of soft drinks, different artificial adjuvant are present, interferences due to the presence of these substances in TAR and SUN determinations from soft drinks have been studied. No interferences of citrate, sodium benzoate, saccharin, aspartame and sorbic acid have been detected under the special experimental conditions of the present HPLC method. In the case of a soft drink natural coloring (like carrot juice) it was observed that the pigmentation was retained on the microfilter and resulted a colourless solution. The selectivity of this chromatographic method was confirmed by spectral purity, depicted in figure 4 for one soft drink sample (orange juice). It can be seen that the purity factors for both colorants exceed the calculated thresholds, being for TAR and for SUN. Detection limit (LOD) and quantification limit (LOQ) of the applied method have been established taking into account the characteristics of HPLC techniques and the specific literature for analytical performances of analytical methods [30, 31]. The found LOD is 100 µg/l for both colorants, and 300 μg/l for LOQ, respectively. 747

4 Fig. 4. Spectral purity of Tartrazine and Sunset Yellow The recovery percentage was determined by fortification of different soft drink samples with TAR and SUN and was ranged between 92-97%. Conclusions This paper proposes a simple, selective and rather fast HPLC method for the determination of Tartrazine and Sunset Yellow in commercial soft drinks. Practically the HPLC analysis is complete within 15 min, after the sample filtration and sonication. Under the established values of experimental parameters, the method is adequate for the simultaneous determination of the two soluble synthetic pigments, Tartrazine and Sunset Yellow, in soft drinks with good analytical performances. The obtained results showed that the Tartrazine and Sunset Yellow concentrations varied between different kinds of soft drinks, with values for the sum of Tartrazine and Sunset Yellow in the range of 0 and mg/l, these levels being lower than the maximum values established by national and international legislation. References 1. MACRAE,R., ROBINSON,R.K., SADLER,M.J., (Eds.), Encyclopaedia of Food Science, Food Technology and Nutrition, Vol. II, Academic Press, San Diego, CA, GHORPADE, V.M.,. DESHPANDE, S.S.,. SALUNKHE,D.K., in: J.A. Maga, A.T. Tu (Editors), Food Additive Toxicology, Marcel Dekker, New York, GOLKA,K., KOPPS, S., W. MYSLAK, Z.W.,Toxicology Letters, 2004, 151, 1, p PUTTEMANS, M.,. DRYON, L., MASSART,D., Journal - Association of Official Analytical Chemist., 1982, 65, p ALI, M.A.,.BASHIER,S.A., Food Additives & Contaminants 2006, 1, p.1 6. W.DANIEL, J.W.,Toxicology and Aplied Pharmacology, 1962, 4, 5, p *** ORDIN Nr. 438/295/2002, Monitorul Oficial R.A., nr. 722/ *** Council Directive 94/34, European Parliament, Official Journal of the European Communities, 1994, No. L *** Commission Regulation (EC) No 884/2007, European Parliament, Official Journal of theeuropean Communities, 2007, No.L *** Council Directive Directiva 2006/33, European Parliament, Official Journal of the European Communities, 2006, No. L CAPITAN, F., CAPITAN-VALLVEY, L.F., FERNANDEZ,M.D., DE ORBE, I., AVIDAD,R., Analytica Chimica Acta, 1996, 331, p CAPITAN-VALLVEY, L.F., FERNANDEZ,M.D., DE ORBE, I., AVIDAD,R., Talanta, 1998, 47, 4, p NI, Y.,. GONG,X., Analytica Chimica Acta, 1997, 354, p BERZAS NEVADO, J.J., RODRÍGUEZ FLORES,J.,.VILLASEÑOR LLERENA,M.J., Fresenius Journal of Analytical Chemistry., 1998, 361, p MILIANI,C., ROMANI, A., FAVARO,G., Spectrochim. Acta, A: Molecular and Biomolecular Spectroscopy, 1998, 54, 4, p NAGA,S., KOIKE,H., Aanalyt. Sci., 1990, 6, p GENNARO,M.C., ABRIGO C., CIPOLLA, G., Journal of chromatography. A, 1994, 15, 674(1-2), p

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