Three analytical methods for determination of Epinastine ...

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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
Ramzia I. El-Bagary et al J. Chem. Pharm. Res., 2012, 4(2):1361-1369______________________________________________________________________________were repeated three times in a day and on three different days at each concentration level as shown in tabulatedresults in Tables (4-6).Table (3): Results of determination of EPH in “Relestat” eyedropsParametersDifference method Colorimetric method HPLC methodLabeled amount Added standard Labeled amount Labeled amount Added standard Labeled amountMean 99.94 99.61 99.47 100.46 98.90 99.98±SD 0.73 1.19 1.09 1.26 0.80 1.12± SE 0.37 0.42 0.54 0.36 0.40 0.35RSD% 0.73 1.20 1.09 1.25 0.80 1.12Table (4): Assessment of accuracy and repeatability of the difference methodConcentration ofDay 1* Day 2* Day 3* Interday statisticsEPHMeanMeanMeanMean(µg.ml -1 RSD%RSD%RSD%) recovery%recovery%recovery%recovery%RSD%32 101.37 0.12 101.40 0.08 101.33 0.07 101.37 0.0964 99.28 0.05 99.53 0.05 100.16 0.05 99.66 0.0596 100.95 0.02 99.56 0.34 100.41 0.23 100.31 0.20*n=3x3=9 , regression equation: Y= 0.0096 X – 0.0191Table (5): Assessment of accuracy and repeatability of the colorimetric methodConcentration ofDay 1* Day 2* Day 3* Interday statisticsEPHMeanMeanMeanMean(µg.ml -1 RSD%RSD%RSD%) recovery%recovery%recovery%recovery%40 100.70 0.27 99.08 0.28 99.62 0.00 99.80 0.28100 101.59 1.41 100.93 1.71 99.84 0.21 100.79 1.11160 100.14 0.13 101.94 0.13 101.53 0.00 101.20 0.13*n=3x3=9 , regression equation: Y= 0.0046 X + 0.0257Concentration ofEPH(µg.ml -1 )Table (6): Assessment of accuracy and repeatability of the HPLC methodDay 1* Day 2* Day 3* Interday statisticsMeanMeanMeanRSD%RSD%recovery%recovery%recovery%RSD%30 101.22 0.41 101.62 0.16 100.01 0.57 100.95 0.3850 101.37 1.10 100.17 0.76 99.93 0.08 100.49 0.6570 10.75 0.09 100.64 0.70 100.98 0.66 100.79 0.48*n=3x3=9 , regression equation: Y= 0.0249 X – 0.0221Meanrecovery%3.4.3. LOD and LOQThe calculated LOD and LOQ values, listed in Table (2), confirmed good sensitivity for the proposed methods andconsequently their capability to determine low amounts of the investigated drug.3.4.4. System suitability for HPLC:System suitability test parameters were calculated under the optimized experimental conditions. EPH and theinternal standard could be successfully eluted in the form of symmetrical peaks quite away from each other. Theretention time values of the peaks together with other chromatographic parameters are collected in Table (7) .Thetable describes the calculated resolution value(R s ) as well as selectivity factor(α)which insures complete or 100%separation of the compounds under investigation. The Tailing factor of the drug peak also revealed linear isothermpeak elution without tailing.3.2.5. Specificity for HPLC:Specificity is the ability of proposed method to accurately measure the analyte response in the presence of allpotential sample components. Complete separation and resolution of internal standard from EPH with good peakshapes and without any apparent shoulders confirm specificity of the method (HPLC chromatogram in Fig (4).1367
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