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Determination of Synthetic Food Colors, Caffeine, Sodium Benzoate and Potassium Sorbate in Sports Drinks

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Determination of Synthetic Food Colors, Caffeine, Sodium Benzoate and Potassium Sorbate in Sports Drinks Moghaddam et al Tropical Journal of Pharmaceutical Research January 2016; 15 (1): 183-188 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v15i1.25 Original Research Article Determination of Synthetic Food Colors, Caffeine, Sodium Benzoate and Potassium Sorbate in Sports Drinks Fatemeh Zamani Mazdeh1, Zeinab Moradi1, Ghazaleh Moghaddam1, Zhila Moradi-Khatoonabadi1, Farideh Esmaeili Aftabdari1, Parnaz Badaei2 and Mannan Hajimahmoodi1,2* 1Food and Drug Administration, 2Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran *For correspondence: Email: Received: 26 February 2015 Revised accepted: 11 November 2015 Abstract Purpose: To employ high performance liquid chromatography (HPLC) techniques to determine edible additives in commercial sports drinks. Methods: A total of 105 samples including 21 different brands of sports drinks were purchased from markets in Tehran, Iran. The process conditions included a C18 column, a mobile phase consisting of aqueous ammonium acetate buffer (pH = 4.2) and acetonitrile (80:20 v/v) at a flow rate of 0.8 ml min–1. The injection volume was 20 μL and the UV detector was set at 225 nm. Results: Mean recovery was between 95 and 106 %. The mean concentrations of sodium benzoate and potassium sorbate in Iranian brands (147.72 and 11.54 ppm, respectively) were significantly higher than in foreign brands (19.43 and 4.91 ppm, respectively, p < 0.05). The maximum amount of sodium benzoate and potassium sorbate was 251.50 ± 18.69 and 96.38 ± 38.56 ppm, respectively, while caffeine content was in the range of 293.48 ± 14.33 - 607.32 ± 135.33 ppm. Conclusion: HPLC permits the detection of sodium benzoate and potassium sorbate at very low concentrations. Furthermore, caffeine was detected in all the sport drinks. Only Brilliant Blue and Allura Red were detected in the drinks. Keywords: Edible additives, Sports drinks, Sodium benzoate, Potassium sorbate, Caffeine, Brilliant Blue, Allura Red Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION as additives in energy and sport drinks [3-5]. Benzoic acid is sodium salt of benzene Recently, sport drinks have gained popularity carboxylic acid and phenilcarboxylic acid, is 180 based on the attribution of energy-giving times more soluble than benzoic acid in water properties. Caffeine which is almost certainly the [6,7]. The additives may also cause allergic most widely consumed psychoactive substance reactions in sensitive individuals as well as in the world is a well-known stimulant effect over hyperactivity color. However, potassium solubility the central nervous system [1]. The US Food and is more than 50 % in foods [6]. In Europe drug Administration (FDA) limits the maximum sorbates and benzoates are permitted in amount in carbonated beverages to 6 mg/oz. [2]. beverages [8]. On the other hand, preservatives are permitted Trop J Pharm Res, January 2016; 15(1): 183 Moghaddam et al For safety reasons, there have been recent chromatograph equipped with a gradient pump reductions in the number of permitted food colors capable of mixing four solvents, a vacuum but because of their low price, effectiveness and membrane degasser, a 20 μL loop injector and a stability they are still being used [9–11]. The lists UV Detector (Agilent Technologies, Santa Clara, of permitted food colors is definitely different CA, USA). Analysis was performed on an Eclipse between countries [12]. The beverage - XDB C18 column (150 × 4.6 mm, 5 μm). The processing industry uses several types of food mobile phase consisted of an aqueous colors, but to minimize potential toxicity, the ammonium acetate buffer (pH = 4.2) and amounts of permitted synthetic colors used are acetonitrile (80:20 v/v) with a flow rate of 0.8 ml min – 1. The injection volume was 20 μL and the strictly limited [11,13]. So they are permitted and UV detector was set at 225 nm. The frequently used in EU countries. However, they chromatographic system was initially conditioned are not permitted in other countries, including by the mobile phase until a stable baseline signal Japan and USA [14]. was obtained, which needed at least 1 h. The main objectives of this work were to evaluate To evaluate the synthetic colors, the mobile the levels of mentioned additives compounds in phase consisted of ammonium acetate solution 105 sport drinks and by comparing the levels of (0.1 mol L-1, pH = 6.7), as solvent A and these compounds with the current available methanol - acetonitrile (50:50, v/v), as solvent B. standards. A cellulose membrane with a pore diameter 0.45 μm was used to filter mobile phase A. In order to EXPERIMENTAL achieve a successful resolution, gradient elution programs were tested and the flow rate was A total number of 105 samples were purchased always kept constant at 1 ml min-1. The final in accordance with the market availability, in optimized gradient program was 3 % solvent B Tehran - Iran. The samples were collected as the initial step; then it increased linearly to 60 between December, 2013 and January. 2014. % in 18 min and was held for 2 min. Analysis The studied commodities were 21 different was performed on the UV detector with two brands of sports drinks. All stages of the optimized conditions. First, the fixed wavelength experiment were done before expiry date of the was set at 250 nm within the whole runtime to drinks. The analyses were carried out in triplicate make it easy to use for amateur operators; next, for each sample. it was programmed at 415 nm for tartrazine, 460 nm for Quinoline yellow, Ponceau 4R, and Standards and chemicals Sunset yellow, 500 nm for Allura red AC and Carmosine, and 600 nm for Indigo carmin and All solvents/chemicals used were of analytical Brilliant blue for different colorants [11]. grade. In the study, high purity standards of potassium sorbate (> 99 %), sodium benzoate (> Data analysis 99 %), sodium salicylate (> 99.5 %), Caffeine (> 99 %), ammonium acetate (98 %), acetic acid All measurements were replicated three times to glacial (100 %) and HPLC - grade acetonitrile improve the reliability of the results. Data were were purchased from Merck (Darmstadt, analysed using statistical program for social Germany). Deionised water was prepared by a sciences (SPSS), version 21 (IBM SPSS Inc., Branstead Easypure II system (Thermo Fisher Chicago, USA), Data are expressed as mean ± Scientific Inc., Waltham, MA, USA). Methanol SD. One way analysis of variances (ANOVA - (Merck) was HPLC quality. Acetic acid (Merck, d Dunnett T3) was used for determining significant = 1.05) that was used for acetate buffer and difference which was set at p < 0.05. NaOH (Merck, 5 mol/L) that was used for regulating pH were of analytical purity. Millipore Method validation Milli - Q water was used in all stages of the assay. The LOD and the LOQ were calculated as LOD = 3.3 σ/S and LOQ = 10 σ/S, where σ is the Chromatographic conditions standard deviation of the response and S is the slope of the calibration curve. The estimate of σ Sport drinks were filtered through 0.45 µm was carried out by analyzing blank samples membrane filter and ultrasonicated, before HPLC seven times, measuring of the magnitude of the analysis. Analytical separation of the caffeine, analytical background response and then sodium benzoate and potassium sorbate was calculating the standard deviation of the carried out by reverse phase liquid response. Recoveries were determined by chromatography of an Agilent 1200 series liquid spiking a sport drink known to be free of all Trop J Pharm Res, January 2016; 15(1): 184 Moghaddam et al additives, in triplicate, with known amounts of and also caffeine, while in Figure 2 the synthetic studied additives at final concentrations between. dyes resolution are illustrated. RESULTS The concentrations of preservatives quantified from sports- drink samples are shown in Table 2. The analytical methodology revealed good Sodium benzoate was found in 14 brands at linearity, sensitivity, exactitude and precision. levels ranging between 12.50 and 251.50 ppm, Correlation coefficients (r2) were comprised (Table 2). between 0.998 for caffeine acid and 0.999 for the other studied factors. Limits of qualification and Moreover, 15 brands didn’t have any potassium quantification are presented in Table 1 sorbate. The levels of sodium benzoate and respectively. potassium sorbate in Iranian brands (147.72 and 11.54 ppm, respectively) were significantly more Recovery values were between 95 % and 106 % than foreign studied brands (19.43 and 4.91 ppm for studied additives at determined spiked levels. respectively). The developed method was successfully in the evaluation of the mentioned additives. Figure 1 Also, methods to evaluate levels of coloring in shows the chromatograms of a standard solution foods for quality control are currently made by containing sodium benzoate, potassium sorbate the TLC method. The mean concentration of colors in each sample group is presented in Table 3. Table 1: Recovery data for the additives
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