1. INTRODUCTION:

Artificial high-intensity sweeteners (also called non-nutritive sweeteners) form an important class of food additives, which are commonly used in the foods, beverages, confectionery and pharmaceuticals. They provide the sensation of sweetness, but with little or no intake of food energy. There are a large number of known intense sweeteners, but only very few are allowed to be used in modern food industry. The food industry is heavily promoting its artificially-sweetened products (frequently called ‘‘diet’’ or ‘‘light’’), highlighting their benefits. Low-calorie or reduced-calorie food products and beverages can help in treatment of obesity, maintaining body weight and management of diabetes. Last, but not least, artificial sweeteners are not fermented by the microflora of the dental plaque, which makes them tooth-friendly.

Sweeteners may be used separately or in combination with other sweeteners, as so called blends. Now a day, the common trend in food industry is to use sweetener blends, because some of the sweeteners impart side tastes and aftertastes that can limit their applications in foods and beverages¹. It was found that mixing one sweetener with the other frequently yields a blend which does not have unwanted side or aftertastes but also is sweeter than the algebraic sum of the components. A very well-known example of such a mixture is saccharin-aspartame (Fig-1) blend used in syrups and beverages². Simultaneously (due to synergistic effect), the sweetening power of the mixture increases. Properly formulated sweetener blends can precisely reproduce the texture and the sweetness profile of traditional sugar-containing products, create new products characterized by an original sweetness profile and improve taste stability³. Artificial high-intensity sweeteners, intensely promoted by the food industry are among the most controversial food additives due to suspicions of adverse health effects⁴.

Saccharin Aspartame

Fig 1: Structures of Saccharin and Aspartame

Based upon the study related to adverse effects of artificial sweeteners the Food and Drug Administration Authority (FDA) and World Health Organization (WHO) have approved several sweeteners for daily use but have given the daily intake limit for these sweeteners. Acceptable daily intake of saccharin and aspartame permitted by FDA and WHO are 2.5 mg /kg body weight per day and 50 mg/kg body weight per day respectively^5,6. Saccharin and aspartame were simultaneously determined by high performance liquid chromatography⁷, Ion chromatography^8,9, capillary electrophoresis^10,11, flow injection analysis¹² and visible spectrophotometry¹³. Except the visible spectrophotometry the other methods described above^7-12 are expensive, tedious and require time consuming sample preparation and in one method there was a probable interference with reagents¹³. So, there is a need of simple and cheap methods for the simultaneous determination of saccharin and aspartame.

In this paper we described a simple method for the simultaneous determination of saccharin and aspartame in drinks using UV spectrophotometry.

2. EXPERIMENTAL:

2.1 Material and methods:

All solutions were prepared with double distilled water. Buffer solution (pH 4.5): 385 ml of 0.2 M di-sodium hydrogen phosphate (S.d. Fine Chemicals, India) was mixed with 615 ml of 0.1 M acetic acid (Qualigens, India). All the chemicals used were of analytical reagent grade. Standard saccharin was supplied by Kare Labs (Goa, India) where as standard Aspartame was purchased from Sigma-Aldrich (India). Cola drinks, carbonated soda, energy drink and lemon juices were purchased from local market.

2.2 Apparatus:

A Chemito Spectroscan UV 2600 double beam spectrophotometer (Thermofisher Scientific, India) with a computer system and Spectrum^TM 6.89.8 software was used.

2.3 Preparation of standards and sample:

Standard solution:

Standard stock solutions of saccharin (SAC) and aspartame (ASP) were prepared individually in buffer solution having concentration of 1 gL^-1. These solutions of both sweeteners were scanned in the range of UV 200 nm to 400 nm against blank solution to determine wavelength (λ_max). To determine the limit of detection (LOD) and linearity aliquot portions of the stock solutions were diluted individually with buffer to get final concentration of 0.001 gL^-1 and 0.01 gL^-1 for saccharin and aspartame respectively. For drawing calibration curve five standard solutions containing mixture of saccharin and aspartame in the concentration range of 0.007 gL^-1 to 0.035 gL^-1 and 0.05 gL^-1 to 0.25 gL^-1 respectively. The method has been validated according to ICH guidelines for analytical method validation.

Sample preparation:

Cola drink, carbonated soda, lemon juice and energy drink samples of each were spiked with saccharin and aspartame (from standard stock solution) to produce the solutions having the concentration of saccharin 0.035 gL^-1 and aspartame 0.25 gL^-1 respectively. These solutions were further diluted with buffer solution to give three different concentrations within the calibration range.

3. PROCEDURE:

A suitable aliquot of the sample is taken and diluted with 4.5 pH buffer to give a final concentration in the range of 0.007 gL^-1 to 0.035 gL^-1 for saccharin and 0.05 gL^-1 to 0.25 gL^-1 for aspartame respectively. The absorbance’s of these sample solutions and standard were measured at 235 nm (for saccharin) and at 257 nm (for aspartame) (Fig-2). Simultaneous estimation of these sweeteners was made by multicomponent analysis provision provided in the Spectrum^TM 6.89.8 software. The concentration of the sample can also be calculated by the formula:

(C_SAC), Cu= (Au/As) x Cs

Cu = Concentration of unknown, Cs = Concentration of SAC in standard

As = Absorbance of standard at 235 nm

Au = Absorbance of unknown sample at 235 nm

(C_ASP), Cu= (Au/As) x Cs

Cu = Concentration of unknown, Cs = Concentration of ASP in standard

As = Absorbance of standard at 257 nm

Au = Absorbance of unknown sample at 257 nm

Fig 2: Showing the UV spectra obtained for saccharin and Aspartame.

4. RESULTS AND DISCUSSION:

By the proposed method saccharin and aspartame were determined simultaneously in drinks. Simultaneous estimation of these sweeteners was made by multicomponent analysis provision provided in the Spectrum^TM 6.89.8 software. Interference of other components such as dyes would be minimized to a negligible level by dilution of the samples with buffer solution (Table-1). The method was validated accordingly to ICH guidelines and several parameters like LOD of this method was found to be 0.0015 gL^-1 and 0.04 gL^-1 for saccharin and aspartame respectively. Saccharin and aspartame obeys Beer Lambert’s law in the concentration range of 0.002 gL^-1 to 0.04 gL^-1 and 0.06 gL^-1 to 0.28 gL^-1 respectively (Fig-3and4). Molar absorptivity of the proposed method for saccharin and aspartame was found to be 5.679 x 10³ L/mol/cm and 1.86 x 10³ L/mol/cm respectively. The other parameters like slope, intercept of the calibration curve was also studied (Table-2). The coefficient correlation (r²) value for saccharin and aspartame was found to be 0.9997 and 0.9999 respectively. The recovery values of saccharin and aspartame in different samples like cola drinks, carbonated soda, lemon juice and energy drinks were found to be better than 92.91, 101.66, 96.25, 93.75 and 94.44, 101.66, 98.33, 93.33 respectively with the standard deviation values of better than +0.97, +2.06, +1.51, +1.31 and +2.1, +1.6, +1.73, +1.51 respectively (Table-3). Intraday and interday repeatability studies were performed by spiking the samples with known concentrations of these sweeteners and samples were analyzed 5 times in a day and consequently for continuous three days. Interday and intraday determination of saccharin and aspartame in cola drinks, carbonated soda, lemon juice and energy drinks are (described) shown in Table-4.

Fig 3: Showing the Calibration curve obtained for saccharin with the regression equation of (y=32.114x – 0.0036) and coefficient correlation value of r² = 0.9997.

Table-1: Data showing the interference of other additives on absorbance measurement.

Sample	Measurement at 235 nm		Measurement at 257 nm
Sample	Blank	Spiked	Blank	Spiked
Standard in Buffer	-0.001	0.316	-0.003	0.576
Cola drink	0.016	0.339	0.026	0.589
Carbonated soda	-0.004	0.302	-0.001	0.558
Lemon juice	-0.001	0.321	0.003	0.561
Energy drink	0.021	0.351	0.007	0.601

Table-2: Quantitative parameters/Optical characteristics of proposed method.

Parameter	Saccharin	Aspartame
Beer’s Law limit (gL-1)	0.002 – 0.04	0.06 – 0.28
Limit of detection (gL-1)	0.0015	0.04
Slope	32.114	6.294
Intercept	-0.0036	0.0053
Correlation coefficient (r2)	0.9997	0.999
Molar absorptivity (L/mol/cm)	5.679 x 10³	1.860 x 10³

Fig 4: Showing the Calibration curve obtained for aspartame with the regression equation of (y=6.294x + 0.0053) and coefficient correlation value of r² = 0.9999.

Table-3: Showing Recovery data of samples spiked with saccharin and aspartame.

Components	% Recovery Mean + SD (n=5)
Components	Cola drink	Carbonated soda	Lemon juice	Energy drink
Amount of Saccharin spiked (gL^-1)
0.012	99.16+0.86	105.83+1.52	100.83+1.51	96.66+1.01
0.018	97.77+0.31	101.66+2.06	105+1.07	96.11+1.31
0.024	92.91+0.97	102.08+1.37	96.25+0.56	93.75+0.78
Amount of Aspartame spiked (gL^-1)
0.06	98.33+0.73	108.33+1.11	101.66+0.83	93.33+0.43
0.12	96.66+1.30	104.16+0.96	98.33+1.73	94.16+0.54
0.18	94.44+2.1	101.66+1.6	102.77+1.13	93.88+1.51

Table-4: Showing the intraday and interday repeatability data.

Sample

Intraday recovery data

Mean + SD (n=5)

Interday recovery data

Mean + SD (n=3)

Saccharin

Aspartame

Saccharin

Aspartame

Cola drink

99.16+

0.86

98.33

+0.73

98.21

+0.9

98.26

+0.93

Carbonated soda

105.83+

1.52

108.33

+1.11

103.77

+2.1

105.52

+2.77

Lemon juice

100.83+

1.51

101.66

+0.83

101.45

+0.66

101.23

+1.18

Energy drink

96.66+

1.01

93.33

+0.48

96.66

+0.69

94.62

+1.22

5. CONCLUSION:

The developed method was found to be simple, precise, accurate, cost effective and large number of samples can be analyzed with in short period of time. And can be useful for food processing laboratories, food regulatory laboratories and forensic science laboratories for determining the level of saccharin and aspartame in drinks.

6. ACKNOWLEDGEMENT:

One of the authors (MI) is thankful to Dr. C.N. Bhattacharya, Incharge-Director-cum-Chief Forensic Scientist, Directorate of Forensic Science, Ministry of Home Affairs, Govt. of India for awarding him research fellowship. Authors are also thankful to Mr. A.K. Ganjoo, Director, Central Forensic Science Laboratory, Hyderabad, India for providing necessary facility for research work. Thanks also due to Kare Labs (Goa, India) for gifting reference sample of saccharin.

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Received on 14.01.2011 Modified on 16.02.2011

Asian J. Research Chem. 4(5): May, 2011; Page 737-740