Simultenious Estimation of Sodium Benzoate and Caffeine in Soft Drinks by UV Spectroscopy

 

Manda Pravalika1*, Jorige Archana2

1Department of Pharmaceutical Analysis, RBVRR Women’s College of Pharmacy,

Hyderabad, Telangana, India. 500027.

2Department of Pharmacology, RBVRR Women’s College of Pharmacy, Hyderabad, Telangana, India, 500027.

*Corresponding Author E-mail: achanacology@gmail.com

 

ABSTRACT:

A  simple spectrophotometric methods have been developed for the simultaneous estimation of sodium benzoate  and  caffeine  from the food products Method-I simultaneous equation method involves the measurement of absorbance at two wavelengths 224nm (λ max of sodium benzoate) and 274nm (λ max of caffeine) using Water as diluent. Method – II Absorption ratio method involves the measurement of absorbance at two wavelengths 274nm (λ max of caffeine) and 241nm (λ max of Isobestic point). The method was validated as per ICH guideline rules in terms of accuracy (recovery study), precision, ruggedness, robustness and the validation parameters were within acceptable limits. The percentage recovery at various concentration levels varied from 99.5 to 100.6% for sodium benzoate and 99.3 to 98.3% for caffeine. Two popular soft drink brands were purchased from supermarket and evaluated for sodium benzoate and caffeine by simultaneous equation method and Q-absorption ratio method. The amount of sodium benzoate and caffeine found in sample 1is 150µg/ml and139µg/ml and in sample 2 is 179µg/ml and 190µg/ml respectively.

 

KEYWORDS: Sodium benzoate, Caffeine, Soft drinks, Simultaneous equation method, Q - absorption method, UV spectrophotometry.

 

 


INTRODUCTION: 

Spectrophotometric analysis is the most widely used analytical tool in the field of compound analysis, both in simple and complicated mixtures. Multicomponent analysis, on the other hand, is the result of different software improvements in spectrophotometers.1 The UV-Vis spectrophotometer (ultraviolet-visible light spectrophotometer) is well-known in the food industry for its rapid analytical potential and ease of use.2 Food quality and safety are two of the most significant factors for consumers in the food sector. preservatives are permitted as additives in energy and sport drinks. Energy drinks are consumed because they claim to boost energy, promote weight loss, increase endurance, and improve attention. Energy drinks frequently contain caffeine.7

 

 

Caffeine (CAF), chemically 1,3,7-trimethyl-xanthin is widely used in foods and beverages as stimulant.. It is soluble partially in water because of its moderate polarity.3 Caffeine is the most versatile compound in that it is consumed in a variety of beverages and medicines by practically everyone. Caffeine is commonly used as a flavouring component in soft drinks, and it is added with the intent to make people addicted to it.  Caffeine acts as a stimulant to the central nervous system and heart and also increases the activity of brain through its adenosine antagonist action. Soft drinks, pickles, bread products, soy sauces, meals, and beverages commonly contain sodium benzoate as a preservative. Food additives are compounds that are added to or stored with food. With the development in the manufacture of processed and convenience foods. Food preservatives have become an increasingly essential practise in modern food technology they are frequently used in processed foods to prevent the growth of bacteria, yeast or other nasty microorganisms that could spoil food4 Several Analytical methods have been deployed for the estimation of these preservatives.5,6. These preservatives are added to foods to prevent or postpone nutritional losses caused by microbiological, enzymatic, or chemical changes, as well as to extend shelf life and improve food quality7

 

There were very few analytical methods for simultaneous estimation of caffeine and sodium benzoate in Beverages.8,9,10

 

 

Figure 1. Structure of sodium benzoate 

 

Figure 2. Structure of caffeine

 

MATERIALS AND METHODS:

Chemicals:

Standard sodium benzoate (Ganesh Bezoplast Ltd.), standard caffeine of SD fine chemicals, India. Soft drinks distilled water.

 

Instruments:

Double beam UV spectrophotometer; Model: SL 210; Make: ELICO. The data was obtained using Spectra Treats 3.11.01Rel 2b software, CONTECH electronic balance.

 

UV-Visible Spectrophotometric method:

Preparation of standard solution (1000µg/ml):

Standard solution of sodium benzoate was prepared by taking 10mg in 10ml volumetric flask containing distilled water and the volume was made up to the mark with distilled water (stock solution). To get 1000µg/ml.

 

Preparation of standard solution (100µg/ml):

From the above 1000µg/ml the standard solutions of sodium benzoate and caffeine, Pipette out 1ml of sodium benzoate and 1.ml of caffeine standard solution to 10.0ml volumetric flask and dilute up to the mark with distilled water. To get 100µg/ml.

 

Preparation of working standard solution (10µg/ml):

From the above 100µg/ml the standard solutions of sodium benzoate and caffeine, Pipette out 1ml of sodium benzoate and 1ml of caffeine standard solution to 10.0ml volumetric flask and dilute up to the mark with distilled water. To get 10µg/ml.

 

Preparation of sample solution:

Four popular soft drink brands were purchased from retail market. 20ml of sample was taken into a 125ml Erlenmeyer flask and sonicated for 5 minutes to remove dissolved CO2. 1ml sample was taken into 10ml volumetric flask and diluted.

 

Determination of wavelength of maximum absorption:

From the above the working standard solution of sodium benzoate and caffeine were scanned in UV spectrophotometer from 200-400nm. from resulting spectra λ max for sodium benzoate and caffeine was calculated respectively. The overlay spectra of sodium benzoate and caffeine were also recorded. [18]From the overlay spectra iso-bestic   point of sodium benzoate and caffeine was determined

 

Method A- simultaneous equation method:

This method is based on the absorption of drugs sodium benzoate and caffeine at their wavelength maxima's.  The two-wavelength selected for the development of simultaneous equation are 224nm and 274nm. the absorptivity values are determined. These values were substituted in the fallowing equation.

 

Cx =A2ay1-A1ay2/ax2ay1-ax1ay2

Cy =A1ax2-A2ay2/ax2ay1-ax1ay2

 

Method B Q- Absorbance ratio method20:

This method is based on the absorbance at two selected wavelength one is an iso bestic point and other being the wavelength of maxima absorption of one of the components. From the overlay spectra 241 nm selected as the isobestic point and 274 nm (λ max of caffeine) is selected for Q- absorption ratio method. The absorptivity values determined for sodium benzoate and caffeine at 241nm.these values are substituted in the fallowing   equation

 

Cx = (Qm - Qy) / (Qx – Qy)*A/a1

Cy = (Qm -Qx) / (Qy-Qx)*A/a2

 

RESULTS:

 

Figure 3. UV overlay Spectrum of sodium benzoate and caffeine using distilled water

Validation parameters of UV-visible spectrophotometer for two methods:

Linearity:

It is the ability to obtain test results which are directly proportional to the concentration of analyte in the sample. A series of dilutions were prepared and their absorbance was measured at 224nm. Aliquots ranging from 1-40µg/ml were found to be linear after plotting concentration on X-axis and absorbance on Y-axis.   The correlation coefficient (r2) was found be equivalent to 0.9999.

 

Calibration curves were drawn by plotting concentration vs absorption and Linearity of the curves was validated by the value of correlation coefficients (r2= 0.9989) shown in (Figure 4, 5)

 

Precision:

Precision of the method was performed by the analysing the sample solution six times. Precision studies were evaluated in the terms of

 

% RSD and the acceptable limits is ˂2

 

The %Relative Standard Deviation (%RSD) was found to be 1.8 , 1.2 for method I and 0.41 for method II. (Table-1).

 

Ruggedness:

It is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of condition, such as different laboratories or different analyst .ruggedness were evaluated in the terms of  % RSD and the acceptable limits is ˂2

 

The % Relative Standard Deviation (%RSD)   was found to be 0.07 and 0.006.

 

 

Figure 4. Lineatity graph of sodium benzoate

 

 

Figure 5. Linearity graph of caffiene


 

Table 1. Precision study data for method I and method II

Simultaneous method  I

Q-absorption ratio method II

Concentration

224nm

274nm

274nm

241 nm

10ppm

0.559

0.8445

0.8445

0.2901

10 ppm

0.586

0.8448

0.8448

0.2902

10 ppm

0.572

0.8270

0.8270

0.2891

10 ppm

0.573

0.8487

0.8487

0.2872

10 ppm

0.562

0.8637

0.8637

0.2901

10ppm

0.560

0.8637

0.8637

0.2902

Mean

0.568

0.61814

0.61814

0.28949

SD

0.0104

0.007572

0.007572

0.001205

%RSD

1.8

1.22

1.22

0.41

 


 

Table 2. Ruggedness study data for method I and method II

 

Simultaneous equation method I

 

224nm

274nm

 

Analyst 1

Analyst 2

Analyst 1

Analyst 2

1

0.586

0.581

0.8470

0.8476

2

0.582

0.580

0.8487

0.8481

3

0.593

0.589

0.8403

0.8403

Mean

0.5870

0.5833333

0.84533333

0.88453333

SD

0.58515±0.000449

0.84533333±0.00005303

%RSD

0.076732416

0.0062732

 

 

Q absorption ratio  method  II

 

Analyst 1

Analyst 2

Analyst 1

Analyst 2

1

0.8403

0.8476

0.2810

0.2879

2

0.8470

0.8481

0.2822

0.2861

3

0.8487

0.8403

0.2831

0.2814

Mean

0.84533333

0.88453333

0.2821

0.283633

SD

0.84533333±0.00005303

0.2578665±0.001868

%

RSD

0.0062732

0.72440

 

 

Robustness:

Robustness was performed by changing the wavelength from the fixed wavelength. Robustness were evaluated in the terms of % RSD and the acceptable limits is ˂2

 

The %Relative Standard Deviation (%RSD)   was found to be 0.2 and 0.33. (Table-3).

 

Table 3. Robustness study data for method I and method II

 

Simultaneous equation method  I

Q-absorption ratio method II

 

224nm

274nm

274nm

241 nm

223

0.586

0.849

0.849

0.2902

224

0.582

0.855

0.855

0.2890

225

0.573

0.847

0.847

0.2873

Mean±SD

0.7153±0.001764

0.569565±0.001913

%RSD

0.2

0.33

 

Accuracy:

Accuracy is the closeness of test results obtained by the procedure to the true values. The accuracy of the method was determined by performing recovery studies by spiking the standard solution to that of sample solution at three different levels i.e., 50%, 100%, 150%. (Table-4).

 

Estimation of Sodium benzoate and Caffeine in Soft drink Samples:

Simultaneous estimation of Sodium benzoate and Caffeine was done from following observations in Two popular cool drink brands in India using the above said two methods. (Table-5).


 

Table 4. Accuracy study data for method I and method II

Samples + standard

224nm

Recovery

Mean % recovery

274nm

Recovery

Mean % recovery

10 +5

0.875

0.878

0.879

99.3

99.6

99.7

99. ±0.20%

1.106

1.109

1.110

99.2

99.4

99.5

99. ±0.15%

10+10

1.142

1.148

1.149

99.6

100.1

100.2

99.9±0.32%

1.301

1.311

1.315

98.1

98.8

99.1

98.6±0.5%

 

10+15

1.1961

1.1972

1.1981

100.5

100.6

100.8

100.6±0.15%

1.470

1.471

1.473

98.2

98.3

98.4

98.3±0.1%

 

 

Table 5. Simultaneous equation Method.

 

Brand  1

Brand  2

DRUG

Absorbance maxima

( Λ1-224nm)

Absorbance maxima

(Λ1-274nm)

Absorbance maxima

(Λ1-224nm)

Absorbance maxima

(Λ1-274nm)

Sample

0.553 (A1)

0.7859 (A2)

0.8834 (A1)

0.7414 ](A2)

Absorbance of sodium benzoate

0.574

0.467

0.574

0.467

Absorbance of caffeine

0.792

0. 861

0.792

0. 861

Absorptivity of sodium benzoate

0.0574 (ax1)

0.0467 (ax2)

0.0574 (ax1)

0.0467 (ax2)

Absorptivity of caffeine

0.0792 (ay1)

0.0861(ay2)

0.0792(ay1)

0.0861 (ay2)

Sodium benzoate

150 mg/L

139mg/L

Caffeine

181mg/L

185 mg/L

 

Table 6. Q-absorption ratio Method

 

Brand 1

Brand 2

Drug

274nm

241nm (Isobestic)

274nm

241nm (Isobestic)

Sample

0.7859

0.7450(A)

0.7414

0.7450(A)

Absorbance of sodium benzoate

0.073

0.2124

0.467

0.2124

Absorbance of caffeine

0.3317

0.2366

0.861

0.2366

Absorptivity of sodium benzoate

0.0073

0.02124 (a1)

0.0467

0.02124(a1)

Absorptivity of caffeine

0.03317

0.02366 (a2)

0.0861

0.02366(a2)

Sodium benzoate

150mg/ L

139 mg/ L

Caffeine

179 mg/ L

190mg/ L

 


DISCUSSION:

Preservatives  are those  substances  that are  added into pharmaceutical formulations  to increase  its  shelf  life and are estimated by various analytical methods11. The estimation of food samples using UV-visible spectroscopy is commonly employed in food quality control.12 UV spectroscopic methods for simultaneous measurement of sodium benzoate and caffeine in soft drinks were devised and validated in the current study. The absorption maxima of sodium benzoate, caffeine, and isobestic point were found to be 224,274 and 241nm, respectively, in these procedures using distilled water as the solvent. This were in line with the previous studies.13 The findings of these procedures were analysed and validated on commercial samples using set criteria. Both approaches were essentially identical in their quantification of two components.  As a result, the developed method can be applied for routine quality control and analysis of two components in soft drinks. The amount of caffeine found in the second brand is slightly higher than that found in the first, and the amount of sodium benzoate found in the second brand is slightly lower than that found in the first, but both are within allowed limits as found in ealier studies14 Up to 400 milligrams (mg) of caffeine a day appears to be safe for most healthy adults15 and the levels of Sodium benzoate was within permissible limits mentioned by Food safety  and standards authority of India (FSSAI). FSSAI standard applauds sodium benzoate concentration in ready in carbonated water it should not exceed 120mg/kg.16

 

Statistical validation was done it shows that the method was reproducible and accurate. Also the various parameters were calculated such as standard deviation and percentage relative standard deviation.17

 

CONCLUSION:

Based on the result obtained new, simple, rapid; precise UV spectrophotometric method was developed for the simultaneous estimation of sodium benzoate and caffeine. There is a need of a new, simple, rapid analytical technique that can detect additives and over the limit additives in various food samples. The suggested method for binary mixture resolution does not require any advanced mathematical analysis of the absorption data, and it has various advantages over previous Spectrophotometric methods. As a result, the methodology given here is suitable for routine quality control examination of binary mixtures.

 

CONFLICT OF INTEREST:

The authors declare that they don’t have any conflict of interest.

 

ACKNOWLEDGMENT:

We are thankful to RBVRR Women’s College of Pharmacy for supporting and encouraging us to carry out this research.

 

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17.   Sumithra M. Yuvanesh P. Mistry A. Analytical method development and validation of ambroxol hydrochloride by UV spectroscopy and forced degradation study and detection of stability. Research Journal of Pharmacy and Technology. 2016; 9(7): 794-800. DOI: 10.5958/0974-360X.2016.00152.9 

 

 

 

Received on 10.10.2021            Modified on 19.04.2022

Accepted on 25.10.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(5):2436-2440.

DOI: 10.52711/0974-360X.2023.00401