Three analytical methods for determination of Epinastine ...

Ramzia I. El-Bagary et al J. Chem. Pharm. Res., 2012, 4(2):1361-1369______________________________________________________________________________For the determination of the optimum UV detector wavelength, variable wavelength values were set for detection ofthe eluted drug and its internal standard. The optimum wavelength with ideal sensitivity and low noise was found tobe 262 nm .An accurate aliquot (0.2 ml) of EPH standard stock solution was transferred into a 10 ml volumetric flask followedby 0.1 ml of methylparaben standard tock solution then the volume was completed with methanol. When 20 µlaliquot of this mixture solution was subjected to the previously mentioned chromatographic conditions, a typicalchromatogram of both compounds in Fig (4) showing well resolved peaks of EPH at 4.004 min and its internalstandard at 5.309 min. The total run time for a complete quantification of the drug and its internal standard wasabout 6.5 min.Fig.(4): HPLC chromatogram of EPH (20 µg.ml -1 ) and methyl paraben (1 µg.ml -1 )3.4.Method validation :The proposed methods of analysis were validated as per the recommendations of ICH (IQ1,IQ2)[14] for theparameters like accuracy, linearity, precision, specificity, detection limit and quantitation limit. The regressionanalysis for the three proposed methods using the method of least squares was applied for the slope (a) and theintercept (b) where results are summarized in Table (2).Table (2): Method validation and analytical parameters obtained by the proposed methodsParameters Difference method Colorimetric method HPLC methodLinearity range (µg.ml -1 ) 16-96 20-220 20-100Regression equation *:Intercept (a)Slope (b)Y=0.0096X-0.0191 Eq.(1)-0.01910.0096Y=0.0046X+0.0257 Eq.(2)+0.0257.0.0046Y=0.0249X-0.0221 Eq.(3)-0.0221.0.0249Limit of detection (LOD) 1.31 3.25 0.76Limit of quantitation(LOQ) 3.96 9.84 2.32SD of slope S b 0.000061 0.0000082 0.0000226SD of intercept S a 0.0038 0.004528 0.0576444Correlation coefficient, r 0.9994 0.9992 0.9998Confidence limit of intercept ** -0.0191±0.008103 0.0257±0.009645 0.0221±0.122782Confidence limit of slope ** 0.0096±0.0001299 0.0046±0.0000174 0.0249±0.000048* is regression equation: Y= a + bX , where Y is the measured response , X is the concentration of the drug (µg.ml -1 )**95% confidence level.3.4.1.Linearity and Range:Linearity was established by performing the proposed methods using a minimum of six concentrations on threedifferent days. Linear relationships were observed over the concentration ranges of EPH stated in Table (2).3.4.2. Accuracy & Precision:Accuracy of the methods was determined by performing recovery studies via the standard addition technique inwhich pre-analyzed samples were taken and known quantities of standard EPH were added. Results are shown inTable (3).Interday & Intraday precisions were performed at three concentrations levels (32, 64, 96 µg.ml -1 ) for differencemethod, (40,100,160 µg.ml -1 )for colorimetric method and (30,50,70 µg.ml -1 ) for HPLC analysis. The determinations1366