Study on the identification and quantification of sodium benzoate in different brands of jelly by high performance liquid chromatography

A reversed - phase high - performance liquid chromatography analysis was carried out for rapid determination and quantification of sodium benzoate in different jelly products. Sodium benzoate is allowed food additive by universal laws in processing in restrictive amounts


Introduction
Jelly is a dessert prepared from gelatin (Yusof et al., 2019).Jelly originates from the Old French gelee, "jelly" and also "frost," from the verb geler, "to congeal," with its Latin root gelare, "to freeze (Rabilloud, 2002).Using food preservation methods has been conjoint both naturally and chemically for the past 1000 to 8,000 years (Knbab et al., 2016).A large number of chemical compounds are dynamic food preservatives, yet because of authoritarian laws on food safety that have been ratified by the FDA and to a lower extent due to the fact that all compounds display antimicrobial effects, adding these compounds to some food products has no upshot and only a few of them are acceptable for use in food products (Bhunia et al., 2013;Makwana et al., 2014).Food preservatives must be within allowable safety limits, which must not exceed the maximum allowed concentration of sodium benzoate and potassium sorbate 0.1% and 0.2%, respectively (Smith and Pell, 2003;Gören et al., 2015).Although sodium benzoic is in the set of safe additives, the snags of synthetic preservatives such as sodium benzoate on human health have been earlier stated (Yolmeh et al., 2014).There are many analytical methods used in qualitative and quantitative appreciation of sodium benzoate in foods such as UV-vis spectrophotometry, HPLC methods, gas chromatography, capillary electrophoresis and polarography (Michael et al., 2005).Conserved foods help people to carry a variety in their diet, thereby declining nutritional inadequacies (Alghamdi, 2005).Preservatives can be used at a relatively low level to indorse that the product does well over time, which is usually one to three years (Sivakumar and Ghosh, 2017).Sodium benzoate is also trained as an animal food additive at up to 0.1%, according to AFCO's official publication (Khoshnoud et al., 2018).Sodium benzoate is secondhand as an action for urea cycle complaints due to its capacity to bind amino acids (Häberle et al., 2012), this clues to the flow of these amino acids and a FULL PAPER reduction in ammonia levels.Sodium benzoate has been publicized to stop the progression of Parkinson's in mice (Wilcken, 2004).So, the objective of this study was to develop a simple method that provides accurate results for sodium benzoate in different brands of jelly available in Bangladesh.

Chemicals
HPLC grade sodium acetate (97%) and acetonitrile were purchased from Merck, Darmstradt, Germany; glacial acetic acid and anhydrous sodium benzoate were purchased from Siga Chemical Co., Germany.The different brands, as well as different batches of jellies, were purchased from the market and supermarket in Tangail town.A total of twenty-seven samples were collected for the experiment of sodium benzoate analysis.The expiry date of all samples was within the study period.The volumes of the samples were 250 mL.

HPLC system
The chromatographic system consisted of a Shimadzu isocratic pump, a degasser, column, Oven; a UV-V is detector, an LC Workstation Class-VP for data acquisition and analysis (Saad et al., 2005;Antakli et al., 2010;Khade and Mirgane, 2014).An aliquot of 20 µL of the sample was injected into the injector.A Luna 5 µ C18 (2) 100A column (length 250×4.60 mm) was used for the chromatographic analysis and the column temperature was set at 40°C (Rana et al., 2011).The sodium benzoate analysis was performed with isocratic solvent system sodium acetate and acetic acid buffer (pH= 4.0): acetonitrile-70:30 with a flow rate of 0.8 mL/ min.chromatograms were recorded at 254 nm.

Mobile phase preparation
The mobile phase comprising 80% acetate buffer with 20% HPLC grade acetonitrile was prepared using the modified method (Pylypiw and Grether, 2000).About 1 mL of glacial acetic acid and 1000 mg of sodium acetate were taken in a 1000 mL volumetric flask containing about 50 mL de-ionized water and shaken well.After that, the deionized water was added up to the mark to make 1000 mL and was mixed well.About 20 mL of acetonitrile was added to 80 mL of the acetate buffer solution and mixed well.The mixture was then filtered with a nylon-66 (pore size 0.2 μm) filter membrane.

Preparation of standard solution
Approximately 50 mg of anhydrous sodium benzoate and 20 mL 50% aqueous acetonitrile were taken in a volumetric flask up to mark and shake well.The solution was then filtered through a syringe filter and labelled as standard stock solution-1 (1 mg/mL).After that, 1 mL of stock solution-1 was taken in a 50 mL volumetric flask and added mobile phase up to the mark and labelled as standard solution-2 (20 µg/mL standard solution).Aliquot of 0.0, 31.25,62.5, 250 and 500 µL of each standard solution-2 was taken into Eppendorf tubes and diluted to volume 1 mL with mobile phase and mixed well with a vortex mixer.

Preparation of sample solution
Approximately 5 g of jelly was taken in a 50 mL volumetric flask.Aqueous 50% acetonitrile was added up to the mark of 50 mL volumetric flask and mixed well.10 mL of the solution was taken in another 50 mL volumetric flask and added the same solvent up to the mark.About 5 mL of the solution was filtered with a sample filter; pore size 0.2 µm and 20 µL was injected into the column.

Experimental analysis of sodium benzoate
A high-performance liquid chromatography technique was used to determine the concentrations of sodium benzoate in the samples by using the modified procedures described by (Pylypiw and Grether, 2000).Each of the samples of 1 mL was diluted 1:5 with the mobile phase.The diluted sample was again diluted 1:10 with the mobile phase.The clear aqueous solution was filtered through a PTFE syringe filter.Then the solution was transferred to the dry HPLC vials and was injected onto the column for detection and quantification.

Limit of detection and quantification
The limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected but not necessarily quantified as an exact value while the limit of quantification (LOQ) refers to the lowest level of analyte which can be determined with an acceptable degree of confidence.In this work, the detection limit (LOD) and quantification limit (LOQ) values were calculated based on a standard deviation of the response and the slope of the calibration curve (ICH, 1996).The concentration was multiplied by 3 and 10 to obtain the limit of detection and quantification, respectively.

Recovery study
In order to verify the accuracy and precision of the analytical procedure, recovery studies were carried out by spiking some samples with very low levels of sodium benzoate (2.0 µg/mL, 4.0 µg/mL and 8.0 µg/mL) from a known standard.In this study, 2.0 mL of each sample mixture and 2.0 mL of 25.0 mg/L standards were taken, mixed together and injected.Due to the dilution, the FULL PAPER actual concentration becomes 12.5 mg/L.The observed concentration and the known concentration are divided and then multiply by 100 to obtain the % of recoveries.

Statistical analysis
All the sample analyses were performed in triplicate and descriptive statistics were analyzed by using SPSS software package version 16.0 (SPSS Inc., Chicago, IL, USA) for all variables.The significance of the differences between the means of the two groups was determined by independent sample Student's t-test.Differences were considered to be significant at p<0.05.

Analysis of chromatogram
HPLC is the most convenient and accurate technique for the analysis bulk and finished pharmaceutical products.An RP-HPLC method has been developed and validated as per ICH, USP and FDA guidelines for the determination of the sodium benzoate by using the mobile phase comprising of sodium acetate buffer (pH = 4) and acetonitrile in the ratio of 70:30 (v/v) over C-18 column at 40°C.The flow rate was at 0.8 mL/min and the eluent was monitored by a UV detector at 254 nm.The retention time of sodium benzoate was 7.886±0.1025mins (Figures 1 and 2).The calibration curve (Figure 3) for sodium benzoate was obtained by plotting the peak areas of different concentrations of working standard solutions prepared from the stock solutions.Very good linearity for sodium benzoate was obtained as it is presented in Figure 3 with an excellent regression factor (0.985).A linear regression line was obtained y = 9131.3x(Table 1).

Analysis of sodium benzoate in jellies sample
Table 2 shows the concentration of sodium benzoate in all jellies content.There was a significant difference between the label of sodium benzoate in Brand 1, Brand 2 and Brand 3 which were 3.27±0.39,11.45±1.4,and 53±1.9 mg/100 mL respectively.This concentration is quite similar to the findings (2.6972 mg/100 mL) recorded by (Sarower et al., 2015).Table 3 shows that sodium benzoate concentration from all brands of jellies was within the range according to the US FDA standard range of 0.1% (100 mg/100 mL).But according to the BSTI standard range-150 ppm (15 mg/100 mL) brand 3 jelly exceeds the level of sodium benzoate as was 25.53 mg/100 mL (Figure 4).Table 4 shows the % recovery of brand 2 jelly.The known amount of Sodium Benzoate was added to Brand 2 jelly at three different levels of concentration considered as low (2.0 μg/mL), medium (4.0 μg/mL) and high (8.0 μg/mL).The % recovery of three concentrations was 94.18±2.9,95.17±3.9 and 93.24±4.8.

Conclusion
The outcome of the present study revealed that brand 1 and brand 2 jelly contained sodium benzoate within the permitted range that is set by the international body FDA but brand 3 exceeds the permitted range of the National Authority BSTI.Government-authorized agencies such as BSTI should take control and regular monitoring to check the level of Sodium Benzoate in all brands of jelly.Gören, A.C., Bilsel, G., Şimşek, A., Bilsel, M., Akçadağ, F., Topal, K. and Ozgen, H. (2015)

Table 1 .
Analytical characteristics of HPLC method Figure 1.Chromatogram of 20 µl/L sodium benzoate standard solution Figure 2. Chromatogram of Brand 1 jelly containing sodium benzoate Figure 3. Calibration curve for the sodium benzoate standard

Table 2 .
Concentration of sodium benzoate from different brands of jelly

Table 3 .
Concentration of sodium benzoate from different brands of jelly in comparison with BSTI and FDA Values are presented as mean±SD.P a value compared with BSTI, P b value compared with FDA (Food and Drug Administration).
. HPLC and LC-MS/MS methods for determination of sodium benzoate and potassium sorbate in food and beverages: Performances of local accredited laboratories via proficiency tests in Turkey.Food

Table 4 .
Percentage recovery of sodium benzoate from spiked sample Figure 4. Overall comparison of sodium benzoate concentration among different brands of jelly with a standard range