INTRODUCTION:

Soft drinks are one of the most widely consumed non-alcoholic beverages in the world, and they have serious public health implications. Due to the current trend of increased diet drink usage, it has become a highly visible and debated public health concern.¹

The ever-increasing quantity of samples to be analysed, together with the growing concern about food safety, necessitates methods that are easy, dependable, inexpensive, and, above all, take into consideration chemical laboratory waste issues.

Due to heightened food safety regulations, laboratories must be cautious when selecting approach for detecting the harmful components.²To provide increased shelf life and enhancement of appearance, flavour and aroma in soft drinks, synthetic colourants, preservatives and sweeteners are widely used in these products.

The sodium salt of benzoic acid, often known as benzoate of soda, is sodium benzoate. Sodium benzoate is more effective and preferable than benzoic acid since it is 200 times more soluble.³ It is an aromatic molecule with the molecular weight of 144.11 and the chemical formula C₇H₅NaO₂. Sodium benzoate is created by mixing sodium hydroxide with benzoic acid in a chemical reaction. Sodium benzoate is an odourless, colourless chemical with a sweet, astringent flavour that is water soluble. Because of its antibacterial properties, as well as its low toxicity and taste, benzoate is commonly used as a preservative all over the world. When the pH of a system is reduced, it becomes more effective, with the optimal functioning range being between pH 2.5 to 4.0.⁴ Benzoic acid and sodium benzoate are used to preserve fruit juices, apple syrup, perishable foods, fizzy drinks, baked flour goods, salad sauces, margarine, ketchup, fruit salads, jams, and jellies.^5-7 The FDA allows a maximum amount of benzoate in food with less than 0.1 percent.⁸

In both animals and humans, long-term usage of benzoate has been associated to a number of negative effects. If increasing amounts of benzoate are used in soft drinks, the taste may change. Concentrations of less than 0.010 percent, on the other hand, are unlikely to be inhibitory. As a result, a comprehensive testing technique is needed to confirm that the benzoate concentration is within acceptable limits.⁹

Structure of sodium benzoate:

Different analytical procedures, such as TLC¹⁰, HPLC-DAD¹¹, and GC-MS¹², can be used to quantify preservatives. Spectrophotometry is frequently chosen over other methods due to its lower operational expenses and ease of maintenance. The absorption of a monochromatic light in the near ultraviolet region (200-380nm) by a colourless compound is measured using the specttrophotometric approach.¹³

UV spectroscopy is an absorption spectroscopy in which ultra violet light which having a range of 200-400nm is absorbed by the molecule. Every molecule has either n, π or σ or electrons. Either bonding (σ and π) and non-bonding (n) electrons absorb the radiation and undergo transition from ground state to excited state.¹⁴ By the absorption peaks obtained for a particular molecule and the nature of the electron present in that particular molecule, the molecular structure of a molecule can be elucidated.¹⁵

MATERIALS AND METHODS:

Chemicals:

Standard sodium benzoate (Ganesh Bezoplast Ltd.), 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 .

Method:

Preparation of standard solution:

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). From the above solution 1ml was pipetted out into 10ml volumetric flask and made up to the mark with water to get 100μg/ml.

Selection of wavelength:

Solutions of 10μg/ml of sodium benzoate were prepared and the solution was scanned in the spectrum mode from 200nm to 400nm. The maximum absorbance of sodium benzoate was observed at 224nm

Figure 1: UV Spectrum of sodium benzoate

RESULTS:

Analytical method validation plays a fundamental role in pharmaceutical industry for releasing the commercial batch and long term stability data. Hence the data must be produced to acceptable scientific standards. Therefore the need to satisfy regulatory authority requirements all analytical methods should be properly validated and documented.¹⁶The developed method was validated according to ICH guidelines.¹⁷ The proposed method was Validated in terms of linearity, precision, accuracy, robustness, ruggedness, LOD and LOQ.

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.

Precision:

Degree of agreement between a series of measurement obtained from multiple sampling of same homogenous sample.

Figure2: Calibration plot of Sodium Benzoate

Intra-day precision:

Intra-day precision was evaluated by analysing six replicates of the standard solution in a day

Inter-day precision:

Inter-day precision was evaluated by analysing single aliquot of the standard solution on 5 different days. The % relative standard deviation (%RSD) for intraday and inter day precision were calculated by using the formula

SD of measurement

%RSD = ----------------------------- x 100

Mean value of measurement

Table 2: Intra-Day Precision and Inter-Day Precision Study Data

Intraday precision		Inter day precision
S. No	*Absorbance at 224nm*	*Day*	*Absorbance at 224 nm*
1	0.5873	Day 1	0.5853
2	0.5861	Day 2	0.5844
3	0.5867	Day 3	0.5863
4	0.5870	Day 4	0.5856
5	0.5872	Day 5	0.5861
6	0.5874	Day 6	0.5873
Mean ±SD	0.58695 ± 0.000485		0.585833±0.000983
% RSD	0.0826305477		0.167795259

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%

Amount found

% Recovery = -----------------------------x 100

Amount added

Table 3: Accuracy Study Data

Level	Standard +sample	Absorbance	% recovery	Mean % recovery
50%	5+10 µg/ml	0.872 0.876 0.876	99.0 99.4 99.4	99.3± 0.23%
100%	10+10 µg/ml	1.141 1.140 1.138	99.6 99.5 99.3	99.5 ±0.15 %
150%	15+10 µg/ml	1.376 1.378 1.373	99.5 99.6 99.3	99.5 ±0.15 %

Robustness:

The robustness of the proposed method was evaluated by changing wavelength. The %RSD was calculated. The low values of %RSD obtained after small deliberate changes in method indicates that the method was robust and the results were presented in Table. 4.

% Relative Standard Deviation (% RSD) was calculated by using the formula

Standard deviation of the measurement

% RSD = ------------------------------------------------ × 100

Mean value of measurement

Table 4: Robustness data

Conc	223nm	224 nm	225nm
10ppm	0.587	0.583	0.577
	0.587	0.580	0.579
	0.586	0.579	0.576
Mean	0.587	0.581	0.577
Mean ±SD	0.582 ± 0.005
% RSD	0.86

Ruggedness:

The degree of reproducibility of the results obtained by the analysis of the sample under a variety of conditions such as different analyst and different instrument is called ruggedness. The ruggedness of the proposed method was evaluated by varying conditions different analyst and different instrument (“ELICO SL 210" and "SYSTRONIC 2203"). The %RSD was less than 2%.

Table 5: Ruggedness data

Concentration	Analyst 1	Analyst 2
10 ppm	0.568	0.565
	0.567	0.569
	0.566	0.568
Mean	0.567	0.5673
Mean ±SD	0.5672± 0.0002
% RSD	0.035

% Relative Standard Deviation (% RSD) was calculated by using the formula

Standard Deviation of the measurement

% RSD = ------------------------------------------------ × 100

Mean value of measurement

LOD:

It is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated under the stated experimental condition. It was calculated using following equation and was found to be 0.03.

3.3 x SD

LOD =-------------

Slope.

Where,

σ =Standard deviation of the response and

S = Slope of the corresponding calibration curve

LOQ:

Definition: It is the lowest amount of analyte in the sample which can be quantitatively with acceptable precision and accuracy under the stated experimental conditions. LoQ is calculated as 0.093 from the following equation.

10 x SD

LOQ =------------------

Slope

Where,

σ = Standard deviation of the response and

S = Slope of the corresponding calibration curve

Application of proposed method for Soft drinks:

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. For colored samples, chloroform (0.8ml) and HCl (0.2ml) were added to 1ml sample and vetexed. Chloroform layer was collected and evaporated to dryness, diluted with water to check the absorbance.

Table 6. Concentration of Sodium benzoate in Soft drink samples

S. No	Concentration(mg/L)
Sample 1	279.3
Sample 2	151
Sample 3	69.7
Sample 4	37.8

DISCUSSION:

The UV spectrophotometric approach is simple, precise, and cost-effective. The estimation of food samples using UV-visible spectroscopy is commonly employed in food quality control. The proposed method can also be used to estimate sodium benzoate in soft drinks in a simple and accurate manner. The study's findings revealed that sodium benzoate concentrations ranged from 37.8 to 279.3mg/L. All the four samples should Sodium benzoate within permissible limits. These findings are in line with previous findings.¹⁸ According to FAO/WHO Expert Committee on Food Additives (JECFA), the acceptable daily intake (ADI) of sodium benzoate is 0-5 mg/kg body weight.¹⁹ However consumption of large volumes of soft drinks with high concentration of sodium benzoate can lead to deleterious health effects. The current analytical method has been validated in accordance with the ICH Q2(R1) guideline and meets specified acceptance requirements. The analytical model was considered to be specific, precise, linear, accurate, robust, and reliable, according to the findings. The current analytical approach can be applied to its intended purpose.

CONCLUSION:

The obtained results demonstrate that the proposed UV spectrophotometric method is simple, rapid, accurate and precise. Therefore, this method can be used for routine quality control of sodium benzoate bulk and in dosage formulation without interference.

ACKNOWLEDGMENT:

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

CONFLICT OF INTEREST:

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

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Received on 05.07.2021 Modified on 16.11.2021

Asian J. Pharm. Ana. 2022; 12(1):13-16.

DOI: 10.52711/2231-5675.2022.00003