Simultaneous determination of active components and additive components in pharmaceutical preparations by HPLC

In this study, a gradient HPLC method was developed for simultaneous analysis of ephedrine hydrochloride, guaiphenesin used as active ingredients and methylparaben, propylparaben, ponceau 4R, saccharin used as additives in various syrup preparations. The optimization process was carried out using 3 levels 4 factors Box-Behnken design. pH, flow rate, mobile phase ratio (ratio of phosphate buffer (H2PO4-/HPO42-) in mobile phase system consisted of phosphate buffer containing tetrabutylammoniumhydroxid(TBAH) and methanol) and TBAH% were selected as factors. The levels were determined as pH 6,5-7,0-7,5, flow rate 0,6-0,8-1,0, phosphate buffer % 45-50-55 and TBAH% 0,1-0,2-0,3. The codes; -1, for low, 0, for middle, and +1, for high levels of the factors were used. 27 experimental runs were randomly carried out. Effects of these factors and the interactions between them on resolution between peaks in HPLC chromatogram were analyzed. Regression analysis was performed on data obtained by the optimization design. ANOVA was performed to test reliability of the results. In studies, it was determined that pH (A), flow rate (B), mobile phase ratio (C) and quadratic contribution of mobile phase ratio and pH (AA and CC) can influence the resolution significantly. A mathematical model related to resolution was obtained using regression results. According to the data obtained a model was created with respect to the resolution . Theoretical resolution values were obtained from the model. The relationship between these values and the experimental values has been examined to test the suitability of the model. Regression coefficient was found 0,9195 and it was decided that the model is appropriate. According the model chromatographic conditions were found as pH 6,5; flow rate 0,6 mL min-1 and the mobile phase system phosphate buffer (pH = 6.5) containing 0.15 % TBAH/ methanol (52:48). Gradient programme was applied that 0-8,5 min. %52 phosphate buffer; 8,5-25 min. %40 phosphate buffer. Chromatograms of the substances at the determined conditions were recorded by using spectrophotometric detector at 210 nm. Analytical method validation was made by the examination of linearity, repeatability, accuracy(Recovery %), limit of quantification-limit of detection and stability parameters. In studies for linearity, the regression coefficients (R2) for ephedrine hydrochloride, guaiphenesin, methylparaben, propylparaben, ponceau 4R and saccharin were found 0,9981; 0,9995; 0,9994; 0,9970; 0,9993; 0,9999 respectively. The RSD% values of the intra-day repeatability analysis for ephedrine hydrochloride, guaiphenesin, methylparaben, propylparaben, ponceau 4R, saccharin were found 0,56-1.62; 0,09-0,89; 1,20-1,60; 0,40-099; 0,59-1,02; 0,77-1,60 and inter-day repeatability analysis were found 0,78-1,89; 1,02-1,90; 1,44-1,88; 0,80-1,34; 0,98-1,20 1,32-1,85 respectively. In order to calculate accuracy, analyses were performed by the addition of 3 different concentrations of ephedrine hydrochloride, guaiphenesin, methylparaben, propylparaben, ponceau 4R and saccharin, into the examined samples which contain the substances. The recovery percentage values were obtained respectively, %100,86-%102,50; %96,47-%101,22; %99,00-%101,20; %99,67-%101,80; %95,00-%99,80; %100,70-%102,58. The limit of detection values were calculated as 0,25; 0,02; 0,18; 0,12; 0,11; 0,008 μg/ mL, and the limit of quantification values were calculated as ; 0,83; 0,07; 0,60; 0,40; 0,38; 0, μg/ mL for ephedrine hydrochloride, guaiphenesin, methylparaben, propylparaben, ponceau 4R and saccharin. In order to determine stabilities of the solutions of substances, they were analyzed both in water and the mobile phase and both at 40C and room temperature. The developed HPLC method for the simultaneous determination of ephedrine hydrochloride, guaiphenesin, methylparaben, propylparaben, ponceau 4R and saccharin was applied to Brodil and Broksin syrup preparations, successfully. It was determined that these samples contain 1,044 -1,320mg/ mL methylparaben, 0,139 mg/ mL propylparaben, 0,153-0,154 mg/ mL ponceau 4R, 3,223-5,095 mg /mL saccharin. The RSD% values for the active componenets were found between 99,39-103,82 from the sample analysis.