Analysis and validation of food additives with HPLC

In this thesis, an HPLC method is developed for analysis of sodium benzoate, potassium sorbate, which are added to preprocessed foods as preservatives, sunset yellow and quinoline, which are added as dyestuffs. First of all, several solvent systems were carried out to separate these substances chromatographically. Then analytical conditions were optimized by using experimental design. pH, flow rate and mobile phase ratio were selected as optimization factors in the experimental design. Effects of these factors and the interactions between them on resolution between peaks in HPLC chromatogram were analyzed. Box-Behnken design was used for analysis of the 3 factors. 3 levels were selected for each factor. These levels were determined as pH 6-6.5-7, flow rate 0.8-1-1.2 mobile phase ratio 80-85-90. The values; -1, for low, 0, for middle, and +1, for high levels of the factors were used. 15 experiments were done in total. Regression analysis was used for evaluation of the results. It was determined that the second effect of mobile phase ratio is the most efficient factor; flow rate, pH and pH-flow rate interaction are also effective. ANOVA was performed to test reliability of the results. According to the data obtained a model was created with respect to the resolution. Resolution values were obtained by using the model. The relationship between these values and the empirical values has been examined. Regression coefficient was found 0.9066 at the end of the examination. And it was decided that the model is appropriate. When the model was used, the optimum conditions for the simultaneous analysis of sodium benzoate, potassium sorbate, sunset yellow and quinoline were found as pH 6, flow rate 0.8 mL min-1 and mobile phase combination 15/85 asetonitrile-acetate buffer. Chromatograms of the substances were recorded by using spectrophotometric detector, at the optimum conditions found in the experimental design, as potassium sorbate and sodium benzoate 230 nm, quinoline and sunset yellow at 460 nm. Analytical method validation was made by the examination of the stability parameters as linearity, repeatability, accuracy, limit of quantification and detection. The correlation between the peak areas and the concentrations of potassium sorbate, sodium benzoate, quinoline and sunset yellow was examined for linearity parameter. The correlation coefficients (R2) were found 0.9979; 0.9980; 0.9979; 0.9993 respectively by regression analysis of the results. The solutions which were prepared at 3 different concentrations of each substance were analyzed 3 times within the same day and 4 times on different days for the repeatability parameter. In the analysis, RSD% values were obtained in the analysis performed for potassium sorbate, sodium benzoate, quinoline and sunset yellow, respectively, on the same day 0.862-1.236; 0.200-1.447; 1.379-1.576; 0.287-1.275, on different days 1.026-1.244; 0.228-0.638; 1.158-1.446; 0.854-1.933. In order to calculate accuracy, analyses were performed by the addition of 3 different concentrations of potassium sorbate, sodium benzoate, sunset yellow and quinoline into the examined samples which contain the substances. The recovery percentage values were obtained respectively, 97.600-103.350; 104.280-104.730; 95.600-104.600; 98.600-100.260. The limit of detection values were calculated as 0.082; 0.154; 0.044; 0.069 μg mL-1 and the limit of quantification values were calculated as 0.273; 0.513; 0.146; 0.230 μg mL-1 for sodium benzoate, quinoline and sunset yellow respectively. In order to determine stabilities of the substances, they were analyzed both in water and the mobile phase and both at 40C and room temperature. It was determined that potassium sorbate, quinoline and sunset yellow are stable at any condition and sodium benzoate is stable at 40C at least one month. Some lemon juice and drink samples were analyzed by using the developed method. It was determined that these samples contain 55.69-267.79 mg mL-1 potassium sorbate, 105.47-159.34 mg mL-1 sodium benzoate, 2.39-15.02 mg mL-1 quinoline, 0.86-0.92 mg mL-1 sunset yellow. The RSD% values were obtained between 0.746-2.629 from the sample analysis.