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

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 injecti on 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.


INTRODUCTION
Recently, sport drinks have gained popularity based on the attribution of energy-giving properties.Caffeine which is almost certainly the most widely consumed psychoactive substance in the world is a well-known stimulant effect over the central nervous system [1].The US Food and drug Administration (FDA) limits the maximum amount in carbonated beverages to 6 mg/oz.[2].On the other hand, preservatives are permitted as additives in energy and sport drinks [3][4][5].Benzoic acid is sodium salt of benzene carboxylic acid and phenilcarboxylic acid, is 180 times more soluble than benzoic acid in water [6,7].The additives may also cause allergic reactions in sensitive individuals as well as hyperactivity color.However, potassium solubility is more than 50 % in foods [6].In Europe sorbates and benzoates are permitted in beverages [8].
For safety reasons, there have been recent reductions in the number of permitted food colors but because of their low price, effectiveness and stability they are still being used [9][10][11].The lists of permitted food colors is definitely different between countries [12].The beverage processing industry uses several types of food colors, but to minimize potential toxicity, the amounts of permitted synthetic colors used are strictly limited [11,13].So they are permitted and frequently used in EU countries.However, they are not permitted in other countries, including Japan and USA [14].
The main objectives of this work were to evaluate the levels of mentioned additives compounds in 105 sport drinks and by comparing the levels of these compounds with the current available standards.

EXPERIMENTAL
A total number of 105 samples were purchased in accordance with the market availability, in Tehran -Iran.The samples were collected between December, 2013 and January.2014.The studied commodities were 21 different brands of sports drinks.All stages of the experiment were done before expiry date of the drinks.The analyses were carried out in triplicate for each sample.

Chromatographic conditions
Sport drinks were filtered through 0.45 µm membrane filter and ultrasonicated, before HPLC analysis.Analytical separation of the caffeine, sodium benzoate and potassium sorbate was carried out by reverse phase liquid chromatography of an Agilent 1200 series liquid chromatograph equipped with a gradient pump capable of mixing four solvents, a vacuum membrane degasser, a 20 μL loop injector and a UV Detector (Agilent Technologies, Santa Clara, CA, USA).Analysis was performed on an Eclipse -XDB C18 column (150 × 4.6 mm, 5 μm).The mobile phase consisted of an aqueous ammonium acetate buffer (pH = 4.2) and acetonitrile (80:20 v/v) with a flow rate of 0.8 ml min -1.The injection volume was 20 μL and the UV detector was set at 225 nm.The chromatographic system was initially conditioned by the mobile phase until a stable baseline signal was obtained, which needed at least 1 h.
To evaluate the synthetic colors, the mobile phase consisted of ammonium acetate solution (0.1 mol L-1, pH = 6.7), as solvent A and methanol -acetonitrile (50:50, v/v), as solvent B. A cellulose membrane with a pore diameter 0.45 μm was used to filter mobile phase A. In order to achieve a successful resolution, gradient elution programs were tested and the flow rate was always kept constant at 1 ml min-1.The final optimized gradient program was 3 % solvent B as the initial step; then it increased linearly to 60 % in 18 min and was held for 2 min.Analysis was performed on the UV detector with two optimized conditions.First, the fixed wavelength was set at 250 nm within the whole runtime to make it easy to use for amateur operators; next, it was programmed at 415 nm for tartrazine, 460 nm for Quinoline yellow, Ponceau 4R, and Sunset yellow, 500 nm for Allura red AC and Carmosine, and 600 nm for Indigo carmin and Brilliant blue for different colorants [11].

Data analysis
All measurements were replicated three times to improve the reliability of the results.Data were analysed using statistical program for social sciences (SPSS), version 21 (IBM SPSS Inc., Chicago, USA), Data are expressed as mean ± SD.One way analysis of variances (ANOVA -Dunnett T3) was used for determining significant difference which was set at p < 0.05.

Method validation
The LOD and the LOQ were calculated as LOD = 3.3 σ/S and LOQ = 10 σ/S, where σ is the standard deviation of the response and S is the slope of the calibration curve.The estimate of σ was carried out by analyzing blank samples seven times, measuring of the magnitude of the analytical background response and then calculating the standard deviation of the response.Recoveries were determined by spiking a sport drink known to be free of all additives, in triplicate, with known amounts of studied additives at final concentrations between.

RESULTS
The analytical methodology revealed good linearity, sensitivity, exactitude and precision.Correlation coefficients (r 2 ) were comprised between 0.998 for caffeine acid and 0.999 for the other studied factors.Limits of qualification and quantification are presented in Table 1 respectively.
Recovery values were between 95 % and 106 % for studied additives at determined spiked levels.The developed method was successfully in the evaluation of the mentioned additives.Figure 1 shows the chromatograms of a standard solution containing sodium benzoate, potassium sorbate and also caffeine, while in Figure 2 the synthetic dyes resolution are illustrated.
The concentrations of preservatives quantified from sports-drink samples are shown in Table 2. Sodium benzoate was found in 14 brands at levels ranging between 12.50 and 251.50 ppm, (Table 2).Moreover, 15 brands didn't have any potassium sorbate.The levels of sodium benzoate and potassium sorbate in Iranian brands (147.72 and 11.54 ppm, respectively) were significantly more than foreign studied brands (19.43 and 4.91 ppm respectively).Also, methods to evaluate levels of coloring in foods for quality control are currently made by the TLC method.The mean concentration of colors in each sample group is presented in Table 3.

DISCUSSION
Additives perform a variety of useful functions in foods that consumers often take for granted.Some additives could be eliminated if we were willing to grow our own food, harvest and grind it, spend many hours cooking and canning, or accept increased risks of food spoilage.Iranian National Standards [15] have some stringent rules for regulating the use of food additives.The level of sodium benzoate and potassium sorbate in Iranian standard (6693) [15] is 150 and 500 ppm, respectively.Therefore, based on the achieved results, 26.66 % of samples had higher sodium benzoate but all samples were in the range of sorbate.

Fig 1 :
Fig 1: Chromatograms of a standard solution containing sodium benzoate, potassium sorbate and also caffeine.

Table 1 :
Recovery data for the additives in sports drinks

Table 2 :
Sodium benzoate and potassium sorbate concentrations (ppm) in different brands of sports-drink

Table 3 :
Caffeine and color concentrations (ppm) in different brands of sports-drink