1. INTRODUCTION:

Food additives are the substances that placed in food during storage or manufacturing¹, which are of synthetic or natural origin². It's known as any substance not commonly used as a food by itself³ Which becomes part of food through processing, packaging or manufacturing⁴.

The most important of food additives are preservatives, which have become progressively important in epochal food technology and play a vital role; due to the rising consumption of packaged foods; it stops the progression of damage and chemical decomposition of food constituents by microorganisms. The use of preservatives is important in food technology to preserve the nutritional value and quality of food^1,5,6. Food additives extend the shelf life of food and are used in accurate amounts to comply with daily intake range⁷. The most commonly used preservatives are benzoic acid (Figure1-a) and its various salts such as sodium benzoate (Figure 1- b), calcium benzoate and potassium benzoate.

a) b)

Fig. 1. Chemical structure of: (a) Benzoic acid, (b) Sodium benzoate

Benzoic acid is a colourless crystalline solid substance, discovered in the 16th century and has antifungal properties^8,9. Is found naturally in prunes, cinnamon, cranberries, apple and ripe cloves¹⁰. Permissible levels to use benzoic acid and its several salts as preservatives are between 0.005% and 0.1 %^11,12. The most commonly used salt of benzoic acid is sodium benzoate, which can be converted into benzoic acid that have bacteriostatic and fungi static features¹³ It is resulting by blending benzoic acid and sodium hydroxide in a chemical reaction¹⁴. Sodium benzoate is added to commercially available foods and beverages such as pickles, salads, sauces, bakery products, jams, jellies, juices, soft drinks, tomato paste¹⁵. It is more active in an acidic media (pH≤ 4.5)¹⁶. And it is 200 times extra soluble in water than acid. Sodium benzoate is a substance that dissolves in water at normally room temperature¹. It can also be used in pharmaceutical and cosmetics products for its antimicrobial properties^17,18.

Some harmful effects resulting from excessive use of benzoic acid and its several salts, such as meTableolic acidosis, hyperventilation, and convulsions, have been observed in some humans and experimental animals that were given high concentrations of these substances. The development of allergic reactions such as urticarial and asthma has been observed¹⁹. Other studies have also shown that sodium benzoate induces oxidative stress and has negative effects on the kidneys, liver, testicular and immune system^20,21. It can motivate fetal deformity in pregnant mice²². Preservatives like sodium, calcium and potassium benzoates are harmless in themselves, but under certain conditions, they can form dangerous chemicals^15,23. Many previous studies have focused on monitoring and identifying benzoates using different methods: like a study conducted in Bangladesh in 2017 using HPLC to determine sodium benzoate in several types of mango juices accessible in the commercial market at a wavelength of 254nm found that some of the studied brands used excessive amounts of benzoate above the recommended limits²⁴. In another study conducted in Libya in 2020, using a spectrophotometer to determine sodium benzoate in some soft drinks accessible in regional markets in the city of Al-Bayda, the study was conducted on local and imported samples, and the results showed that some brands did not contain benzoate despite its presence on the label, and that the imported samples contained benzoate in higher concentrations than the local ones, and the remaining samples were within the permissible limits according to the World Health Organization¹. In addition, a study was conducted in Yemen in 2023 using high-performance liquid chromatography to determine benzoic acid in some commercial baby foods available on the local market, such as cheese, milk, juices, and cakes. The results showed that all samples tested contained benzoates at permissible levels. The amount of sodium benzoate in several food products was evaluated by a simple and inexpensive analytical method²⁵. In 2018 the amount of benzoates in some food products was evaluated using HPLC-RP at a wavelength of 235nm, the results showed that some samples exceeded the permissible boundaries determined by the Indian food safety authority²⁶. Various methods have been used to analyse sodium benzoate in food samples including: thin layer chromatography, UV Spectrophotometer, Gas and High-performance liquid chromatography.

In our research we used spectrophotometric analysis because it is the most commonly used methods, and the most generally analytical tool in the range of molecular analysis^27,28. It is considered a sensitive, less expensive and time-consuming technique than other methods²⁹.

Food are products that are often supplemented with additives like preservatives, and because of most people are adapted to eat processed food especially in cities according to many WHO reports, the food safety has become an essential concern in the current era^30,31,32.

The aim of this study was to evaluate the percentage of benzoates present in several food products in the Syrian market using a simple and inexpensive method differs from the previously mentioned methods. To do this: the highly consumed ready to serve samples with different brand products were collected from local markets in Aleppo, Syria.

2. MATERIALS AND METHODS:

2.1 Solvents and Chemicals:

Benzoic acid (99.5% purity) was obtained from Tammedia, Titan Biotech LTD, India, Sodium Benzoate (99%) was acquired from Qualikems fine chemicals Pvt. Ltd., New Delhi; India, Hexane (95%) was obtained from Chem Lab NV, Belgium, Phosphoric acid (85%), Methanol of analytical grade (Merck, Germany), Dichloro methane, Dichloro ethane, Heptane, Ethyl acetate, Ethanol, Methanol, Chloroform, Diethyl ether, Petroleum ether, Deionized water.

Hydrochloride acid, Sulphuric Acid and Nitric acid (Analytical grade) was procured from Loba Chemie Pvt. Ltd., India. Analytical grade sodium hydroxide and potassium Dihydrogen Phosphate was obtained from Merck, Mumbai, India.

2.2 Instruments:

The ultra-violet spectrophotometric instrument is T80+UV/V Spectrophotometer Instrument Ltd (UK). That is connected to a computer. Using 1-cm width quartz cells. A device of weighing which is an analytical balance (Sartorius, model 2474, from Germany). Other apparatus and instruments used to do the work are an ultra-sonic bath (Power sonic, model 405, from Korea), Heater, Thermometer, volumetric flasks, and various scales of glass pipettes.

2.3 Solution Preparation:

2.3.1 Standard Solution Preparation:

The stock solution of Sodium Benzoate was prepared individually by weighing 0.1g then we transferred it into 100mL volumetric flask; then we filled it with deionized water to the calibration mark. We prepared the working solution by taking a particular volume of stock solution; put it into 10mL glass volumetric flasks, then diluting it with purified deionized water to the marked line, to reach the required concentration.

2.3.2 Sample Preparation:

The beverages were prepared by weighing 1g of each sample and put it into 50 mL glass volumetric flasks. Then diluting it to 50 mL with deionized water and the gases were removed by an ultrasonic bath for 15 minutes in room temperature. The gases were removed from soft drinks for 5 minutes using an ultrasonic bath. We took a volume of 1 mL from the sample and diluted it to 10 mL with deionized water. Several products of soft drinks and beverages were filtered through a 0.45 µm nylon syringe filter in advance.

3. RESULTS AND DISCUSSION:

The method used was based on converting sodium benzoate present in the studied food samples into benzoic acid using a suiTablele acid, then extracting it with an organic solvent and measuring the resulting extracts using a spectrophotometer to determine the amount of benzoate, evaluate its proportions and the extent to which it meets the permitted.

3.1 Selection of wavelength detection

The wavelength selection was done with the help of UV spectrum of standard benzoic acid in the range of (200-400) nm, the maximum absorbance was observed at wavelength of 230 nm figure (2) And Sodium Benzoate does not have absorption in the ultraviolet region. Therefor, 230 nm was used to determinate the benzoic acid in the analysed samples.

Fig (2): UV-spectra of: a- Benzoic acid (100 µg/ml) in hexane; b- Benzoic acid (100µg/ml) and Sodium Benzoate (100 µg/ml) in hexane; c- Sodium Benzoate (100µg/ml) in hexane

3.2 The optimal conditions for extraction:

Study and determine the optimal conditions for extraction Sodium Benzoate including solvent, acid, acid concentration, amount of acid added, temperature, and volume of the solvent

3.2.1 Selection of the best solvent:

We took a food sample approved to contain sodium benzoate and converted it to benzoic acid by H₃PO₄ and extracted it by initially setting all the conditions and changing the solution with the aim of choosing the most suiTablele solution (about 10 solvents have been tested). The results shown in the corresponding spectrums showed that hexane is a suiTablele solution for extraction with a good absorption value in comparison to the rest of the solutions, in addition to being available and inexpensive, and since sodium benzoate is a polar substance that is difficult to dissolve in organic solvents and does not have absorption in hexane figure (3). Therefore, Hexane was chosen in this study.

Fig (3): Extraction of Sodium Benzoate and converted it to benzoic acid by H₃PO₄ in food sample (x) with different solvents

3.2.2 Selection of Acid:

Four acids were tested to select the most suiTablele acid: Hydrochloric acid, Sulphuric acid, Phosphoric acid and Nitric acid (we used a concentration of 150µg/ml of standard sodium benzoates to do this and the following assays, by initially fixing all conditions including acid concentration and its quantity. The spectrums corresponding to the extraction showed that Phosphoric acid is a suiTablele acid with good absorbance value compared to nitric acid and sulphuric acid and that it is almost identical to Hydrochloric acid in value. This acid was relied upon in our study as new acid that has not been used before in the extraction of sodium benzoate

3.2.3 Selection of Acid Concentration:

All conditions were fixed and the acid concentration was studied from 0.5 molar to 8 molars. A gradual increase in the absorbance value was observed after extraction with the increase in the molar concentration until 6 M was the highest absorbance value and remained constant no matter how much we increased the concentration. The pH of the previous solutions was studied before extraction with the organic solution using a pH meter device in a manner similar to that in the reference [33], the stock solution of phosphoric acid was variable from (0.5- 8 M) and we took a fixed volume from it and added to it 3 mL of the studied standard, then the pH was measured, the resulting values showed sTableility in the pH value after a concentration of 6 M from the stock solution, which indicates that the entire amount of benzoate was converted into benzoic acid at this concentration.

3.2.4 Selection of acid Volume:

By studying the change in the volume of Phosphoric acid (0.2 ,0.4,0.6 and 1mL), it was found that the added volume had no effect on the extraction process because the absorbance value remained constant no matter how much we increased the volume. So, in this study we depended on a volume of 0.4 ml according to references.

3.2.5 Effect of temperature:

· With H₃PO₄:

To study the effect of temperature on extraction, we put equal amounts of sodium benzoate with phosphoric acid, and put them in ultrasonic at different temperatures (20, 40, 60 ,80 and 100) °C for half an hour, then extracted them with hexane. The resulting spectrums gave almost equal absorbance values, and thus the effect of temperature in the presence of acid on extraction is almost non-existent.

· Without H₃PO₄:

The purpose of this test was to monitor the impact of temperature on the decomposition of benzoates present in the studied samples without the presence of acid. To study this effect, several flasks containing sodium benzoate with distilled water instead of acid in exactly the same quantities were taken and exposed to different temperatures using a convection oven or a water bath (ultrasonic). The results shown in the corresponding spectrum showed that at laboratory temperature, the benzoates remained the same. Upon exposure to increasing temperatures, we observed that the decomposition of benzoates under the influence of heat into benzoic acid begins at 20°C, increases to completely decompose at 40°C, it is completely converted to benzoic acid. As the temperature increases further, we observed that the absorbance value decreases again. This is due to the formed benzoic acid beginning to decompose into water and carbon dioxide gas. This decomposition increases with increasing temperature until it completely decomposes at 90-100°C.

3.2.6 Effect of Hexane Volume on extraction:

We studied the following volumes based on some references (10 ml to 50 ml). Absorbance increases with increasing hexane volume to a certain extent. In our study, we relied on a volume of 30 ml of the solution sufficient to extract the full amount of benzoic acid for the studied concentrations. The relationship between the volume of the solution and the absorbance value was studied, and the volume corresponding to the intersection was taken, which is 30 ml. Increasing the volume above 30 ml is accompanied by a slight increase in absorbance that is almost constant, as shown in the following Figure (4):

3.3 The optimal spectrophotometric conditions of analysis:

The measurement was done in the ultraviolet range (200-400) nm because the studied material has absorption in this range.

Fig (4): Effect of Hexane Volume (ml) on extraction of Sodium Benzoate (150 µg /ml)

3.4 Method validation:

3.4.1 Linearity:

A series of dilutions were prepared (5-150 µg/ml) and their absorbance’s was measured at 230 nm. Aliquots ranging from (10-100 µg/ml) were found to be linear after plotting concentration on x-axis and absorbance on y-axis, as the following figure (5) shows:

Fig (5): Calibration curve of sodium benzoate after extraction (benzoic acid)

Where SD is the standard deviation of y-intercepts (a) of regression lines and (b) is the slope of the equitation of calibration curve, y = a + b x.

In Table (1) shows statistical data for calibration graphs.

Table (1): Statistical data for calibration graphs.

Parameters	Value
λ _max (nm)	230
Beer’s law limits (μg/mL)	10-100
Temperature of solution	25 ± 5^oC
Solvent	Hexane
Hexane, V (mL)	30mL
C (H₃PO₄), M	0.70 M
pH	0.15
Regression equation	Y = 0.0021X + 0.0106
Slope (b)	0.0021
Intercept (a)	0.0106
R²	0.9995
LOD (µg/mL)	1.48
LOQ (µg/mL)	4.5

The limit of detection and the limit of quantitation were calculated by repeating the extraction of the approved concentrations within the linear range six times, then measuring the organic extracts and forming the corresponding linear equations, as shown in the following Table (2):

Table (2): Calculate the LOD and LOQ

Concentrations ug/ml	Linear equation	R²
10-100	Y= 0.0021x+ 0.0106	0.9995
10-100	Y= 0.002x + 0.0097	0.9972
10-100	Y= 0.0021x + 0.0084	0.9987
10-100	Y= 0.0021x + 0.0078	0.9993
10-100	Y= 0.0021x + 0.009	0.9989
10-100	Y= 0.0021x + 0.0088	0.9951

The limit of detection was calculated using the equation:

3.3 * SD/S

And the limit of quantitation was calculated using the equation:

10 * SD/S

Considering that SD is the standard deviation of the points of intersection of the straight line with the Y-axis from the previous equations in the Table (2) and S is the average slope of the straight line for each of the previous equations.

3.4.2 Accuracy:

Accuracy was studied by measuring three concentrations with three replicates for each concentration and then calculating the average of the recoveries and the relative standard deviation for each concentration. The analytical results were among the accepted range (99.39 - 100.35) % and RSD% was less than 2% summarized in Table 3.

Table (3): Results of the study of accuracy of the proposed method

Conc. of Sodium benzoate taken, µg/ml	Absorbance	Conc. of Sodium benzoate found*, µg/ml	R%	RSD%
50	0.117 0.116 0.115	50.15	100.30%	1.12%
75	0.17 0.169 0.167	75.26	100.35%	0.96%
100	0.219 0.22 0.219	99.39	99.39%	0.27%

*n=3

3.4.3 Precision:

Precision was studied at two levels (Repeatability, Intermediate Precision) at wavelength 230nm: By measuring three concentrations with three replicates for each concentration on the same day for first level (Repeatability) and calculating their relative standard deviation (RSD%). Then the process was repeated on each of the following two days for the study of the second level (Intermediate Precision) and then calculating the RSD% summarized in Table- 4.

3.4.4 Robustness:

The robustness of the method was estimated at the studied wavelength for two concentrations (25, 50 µg/ml), with three replicates for each concentration. This way done by testing the stability of the method results by:

1. Changing the spectral scanning speed from the one used in the method, which is medium, to high speed and to slow speed then calculating the recoveries for each of the two concentrations and calculating the relative standard deviation for each concentration, which did not exceed 2%.

2. Changing the range of wavelengths studied by the user in the method, which is (200-400) nm to (210-300) nm, and calculating the recovery of each of the two concentrations and their relative standard deviation.

3. Changing the wavelength λ max studied by the user in the method, which is (230) nm to (229/231) nm, and calculating the recovery of each of the two concentrations and their relative standard deviation.

3.5. Application of the proposed method on some beverages (carbonated and non-carbonated drinks)

We applied the analytical method using the previous conditions on various samples. A Total of 45 sample from 17 brands were collected from all over the city of Aleppo, out of 45 samples, 35 samples contained sodium benzoate, which was declared to be present, 6 samples did not contain sodium benzoate, although it was not present on the label. And 4 samples were declared to contain benzoates, but it did not contain it.

Seven samples from different brands were excluded due to spectral overlap as they contained the sweetener acesulfame k, which has an absorbance in the same range as benzoic acid. The results of the previous samples are shown in Table (4), taking into consideration that the acceptable limits of sodium benzoate according to the Syrian standard specifications for beverages are (200 mg/l), and it is (150 mg/l) for flavored beverages.

3,5,1. Sample preparation:

3,5,1,1. Carbonated Drinks (Soft and flavored Drinks):

The CO₂ gas was removed from the sample using the ultrasonic, and then it was filtered using a filter (0.45 µm). A suitable amount was taken from it + 0.4 mL H₃PO₄ (0.70 M) and extracted it with 30 ml of hexane in 6 stages (5 mL each time). Then we measured the organic layer using a spectrophotometer.

3,5,1,2. Non carbonated Drinks (Fruit juice):

The same steps were applied to prepare carbonated drinks, except for the removal of gases.

Table (6): summary of the concentration of sodium benzoate (mg/ml) in soft drink samples

Soft and Flavoured Drink samples	Concentration of sodium benzoate (mg/l)	Compare to the allowed limit 200mg/ml
Brand 1	147.04	Aacceptable
Brand 2	180.94	Acceptable
Brand 3	161.31	Acceptable
Brand 4 (flavoured)	189.9	Unacceptable
Brand 5 (flavoured)	170.85	Unacceptable
Brand 6	185.52	Acceptable
Brand 7	198.85	Acceptable
Brand 8	206.47	Unacceptable
Brand 9	104.19	Acceptable
Brand 10	161.31	Acceptable

The previous results showed that: two brands (5,4) exceeded the permissible limits (150 mg/l for flavored beverages). And one soft drink brand also exceeded the permissible value.

For juices, 2 brands did not mention sodium benzoate on their labels and did not contain it. Another brand (3 samples) did have sodium benzoate on its label, but analysis showed that it was not present in these samples.

4. DISCUSSION:

In this article, we studied the optimal conditions for extracting sodium benzoate from food samples. It was shown that the suitable solvent is hexane because of its availability and low cost compared to other solutions, and the most suitable acid is phosphoric acid 0.70 M as it gave a higher absorption compared to HCL and HNO₃ and thus contributed to a greater extraction process of benzoates from the food sample, and it is safer than H₂SO₄. We also found that temperature affects extraction only in the absence of acid, as it leads to the gradual conversion of benzoate to benzoic acid as we raise the temperature until it is completely converted, and then the benzoic acid decomposes again with the continued rise in temperature. Our analysis also showed that some of the samples studied exceed the permissible limits, while others were considered adulterated because it contained sodium benzoate on the label, although it was not present in the product during the analysis. This suggests the importance of monitoring these substances to control these values and thus preserve the health of consumers.

5. CONCLUSION:

This study resulted in the most appropriate conditions for extracting sodium benzoate from food samples. This aimed to ensure complete extraction of these substances and thus analyse and determine their quantities correctly, which enables monitoring their levels and ensuring their safety for use. This method (with the new conditions) was suitable for extracting most food samples containing sodium benzoate and available in local markets. It is also simple, fast, and low-cost compared to other methods, with an R² value of 0.9995.

6. ACKNOWLEDGEMENT:

The University of Tartous in Syria financially and technically supported this work through department of analytical and food chemistry, Faculty of Pharmacy, Aleppo University, Syria.

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Received on 17.07.2025 Revised on 09.08.2025

Accepted on 28.08.2025 Published on 30.09.2025

Available online from October 07, 2025

Asian J. Research Chem.2025; 18(5):303-310.

DOI: 10.52711/0974-4150.2025.00046

This work is licensed under a Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License. Creative Commons License.

Concentration	RSD% of the 12.5 µg/ml concentration	RSD% of the 25 µg/ml concentration	RSD % of the 50µg/ml concentration
Repeatability in the first day	1.85	1.87	0.94
Intermediate precision in the second day	1.08	1.087	0.93
Intermediate precision in the third day	1.10	1.09	0.55

The variable factor	Concentration	R% ± RSD%	Average of the 6 repetitions of the two concentrations
Medium to high scanning speed	25 µg/ml	100.76 ± 0.94	99.93 ± 1.42
	50 µg/ml	99.11 ± 1.46
Medium to slow scanning speed	25 µg/ml	100.4 ± 1.09	101.52 ± 1.62
	50 µg/ml	102.06 ± 1.41
Spectrum scanning range (210-300) nm	25 µg/ml	101.07 ±1.08	100.25 ± 1.28
	50 µg/ml	99.42 ± 0.95
Spectrum scanning range (220-250) nm	25 µg/ml	100.12 ± 1.45	100.09 ± 0.99
	50 µg/ml	100.06 ± 0.54
Wavelength λ max= 230nm -1 nm	25 µg/ml	98.41 ± 1.24	98.28 ± 0.9
	50 µg/ml	98.15 ± 0.56
Wavelength λ max =230nm + 1 nm	25 µg/ml	98.60 ± 1.06	98.30 ± 0.77
	50 µg/ml	98.00 ± 0.48