hplc system ( waters 2489 , milford , usa ) consisting of quaternary gradient pump ( tm 600 ) , rheodyne manual injector with 20 l loop , column oven and uv detector was employed for analysis .
working standards of atenolol and lercanidipine were provided by emcure pharmaceuticals , pune , india . a tablet containing 50 mg ate and 10 mg ler ( lotensyl at ;
hplc grade acetonitrile and orthophosphoric acid were obtained from merck india limited , mumbai . analytical grade hydrochloric acid , sodium hydroxide pellets and hydrogen peroxide solution 30% ( v / v )
were obtained from ranbaxy fine chemical ( new delhi , india ) and 0.45 m membrane filter was obtained from pall life sciences ( mumbai , india ) .
high purity deionised water was obtained from millipore , milli - q ( milford , usa ) purification system .
phenomenex gemini c18 ( 2504.6 mm , 5 m ) column used as a stationary phase .
the isocratic mobile phase consisting of acetonitrile and buffer ( 20 mm potassium dihydrogen phosphate , ph 3.5 adjusted with orthophosphoric acid ) in the ratio of ( 55:45 , v / v ) was used throughout the analysis .
standard stock solution containing ate ( 500 g / ml ) and ler ( 100 g / ml ) was prepared by transferring 50 mg ate and 10 mg ler working standard into a 100 ml volumetric flask . a 40 ml portion of diluent ( acetonitrile - water 50:50 , v / v ) was added , sonicated and cooled to room temperature .
standard solution containing ate ( 100 g / ml ) and ler ( 20 g / ml ) was prepared by pipetting 10 ml stock solution into a 50 ml volumetric flask and diluting to volume with diluent .
the tablets were crushed with a mortar and pestle for 10 min . a portion of powder equivalent to the weight of one tablet was accurately weighed and transferred to a 100 ml volumetric flask .
approximately 50 ml diluent was added and the mixture was sonicated for 15 min with intermittent shaking .
the contents were restored to room temperature and diluted to volume with diluent to furnish stock test solution .
the stock solution was filtered through 0.45 m membrane filters and 10 ml of the filtered solution was transferred to a 50 ml volumetric flask and diluted to volume with diluents to give test solution containing 100 g / ml ate and 20 g / ml ler .
tablet powder equivalent to the weight of one tablet was transferred to 250 ml round bottomed flask and treated under acidic , alkaline , oxidizing , thermal and photolytic stress conditions .
when degradation was complete , the solution were left to equilibrate to room temperature and diluted with diluents to furnish solutions of concentration equivalent to 100 g / ml ate and 20 g / ml ler .
was heated under reflux with 1 m hydrochloric acid for 30 min at 80 on oil bath and the drug was treated with 0.1n naoh at room temperature for 2 h in alkaline degradation .
the drug was treated with 2% ( v / v ) h2o2 at room temperature for 2 h in oxidative degradation .
thermal degradation was performed by exposing the solid drug to dry heat in a convection oven at 70 for 72 h and photolytic degradation was performed by exposing the drug to sunlight for 72 h. in the system suitability standard solution of 100 g/ ml ate and 20 g / ml ler ( n=5 ) was prepared and injected . then system suitability parameters like retention time , theoretical plates , peak asymmetry and resolution were calculated from the chromatogram .
the specificity of the method was evaluated by assessing interference from excipients in the pharmaceutical dosage form prepared as a placebo solution .
the specificity of the method for the drug was also established by checking for interference with drug quantification from degradation products formed during the forced degradation study .
test solutions for assessment of the linearity of the method where prepared at seven concentrations from 40 to 160% of the analyte concentration in the assay ( i.e. 40 , 60 , 80 , 100 , 120 , 140 and 160 g / ml for ate and 8 , 12 , 16 , 20 , 24 , 28 and 32 g / ml for ler ) .
the precision of the method , as intra - day repeatability was evaluated by performing six independent assays of the test sample preparation and calculating the rsd % .
the intermediate ( interday ) precision of the method was checked by performing same procedure on different days by another person under the same experimental conditions .
accuracy was studied by adding three different amounts ( corresponding to 50 , 100 and 150% of the test preparation concentrations ) of ate and ler to the placebo preparation and comparing the actual and measured concentrations . for each level , three solutions were prepared and each was injected in duplicate .
the robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions .
the conditions studied were flow rate ( altered by 0.1/min ) , mobile phase composition ( by using 57:43 and 53:47 v / v acetonitrile : buffer ph 3.5 ) , buffer ph ( altered by 0.2 ) and use of hplc columns from different batches .
sample solution stability was evaluated by shorting the solution at ambient temperature and at 2 - 5 and analysis after 6 , 12 , 24 , 36 and 48 h. the responses from the aged solutions were compared with those from freshly prepared standard solution .
phenomenex gemini c18 ( 2504.6 mm , 5 m ) column used as a stationary phase . the isocratic mobile phase consisting of acetonitrile and buffer ( 20 mm potassium dihydrogen phosphate , ph 3.5 adjusted with orthophosphoric acid ) in the ratio of ( 55:45 , v / v )
standard stock solution containing ate ( 500 g / ml ) and ler ( 100 g / ml ) was prepared by transferring 50 mg ate and 10 mg ler working standard into a 100 ml volumetric flask . a 40 ml portion of diluent ( acetonitrile - water 50:50 , v / v ) was added , sonicated and cooled to room temperature .
standard solution containing ate ( 100 g / ml ) and ler ( 20 g / ml ) was prepared by pipetting 10 ml stock solution into a 50 ml volumetric flask and diluting to volume with diluent .
the tablets were crushed with a mortar and pestle for 10 min . a portion of powder equivalent to the weight of one tablet was accurately weighed and transferred to a 100 ml volumetric flask .
approximately 50 ml diluent was added and the mixture was sonicated for 15 min with intermittent shaking .
the contents were restored to room temperature and diluted to volume with diluent to furnish stock test solution .
the stock solution was filtered through 0.45 m membrane filters and 10 ml of the filtered solution was transferred to a 50 ml volumetric flask and diluted to volume with diluents to give test solution containing 100 g / ml ate and 20 g / ml ler .
tablet powder equivalent to the weight of one tablet was transferred to 250 ml round bottomed flask and treated under acidic , alkaline , oxidizing , thermal and photolytic stress conditions .
when degradation was complete , the solution were left to equilibrate to room temperature and diluted with diluents to furnish solutions of concentration equivalent to 100 g / ml ate and 20 g / ml ler .
was heated under reflux with 1 m hydrochloric acid for 30 min at 80 on oil bath and the drug was treated with 0.1n naoh at room temperature for 2 h in alkaline degradation . then resulting solution
the drug was treated with 2% ( v / v ) h2o2 at room temperature for 2 h in oxidative degradation .
thermal degradation was performed by exposing the solid drug to dry heat in a convection oven at 70 for 72 h and photolytic degradation was performed by exposing the drug to sunlight for 72 h.
in the system suitability standard solution of 100 g/ ml ate and 20 g / ml ler ( n=5 ) was prepared and injected
. then system suitability parameters like retention time , theoretical plates , peak asymmetry and resolution were calculated from the chromatogram .
the specificity of the method was evaluated by assessing interference from excipients in the pharmaceutical dosage form prepared as a placebo solution .
the specificity of the method for the drug was also established by checking for interference with drug quantification from degradation products formed during the forced degradation study .
test solutions for assessment of the linearity of the method where prepared at seven concentrations from 40 to 160% of the analyte concentration in the assay ( i.e. 40 , 60 , 80 , 100 , 120 , 140 and 160 g / ml for ate and 8 , 12 , 16 , 20 , 24 , 28 and 32 g / ml for ler ) .
the precision of the method , as intra - day repeatability was evaluated by performing six independent assays of the test sample preparation and calculating the rsd % .
the intermediate ( interday ) precision of the method was checked by performing same procedure on different days by another person under the same experimental conditions .
accuracy was studied by adding three different amounts ( corresponding to 50 , 100 and 150% of the test preparation concentrations ) of ate and ler to the placebo preparation and comparing the actual and measured concentrations . for each level , three solutions were prepared and each was injected in duplicate .
the robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions .
the conditions studied were flow rate ( altered by 0.1/min ) , mobile phase composition ( by using 57:43 and 53:47 v / v acetonitrile : buffer ph 3.5 ) , buffer ph ( altered by 0.2 ) and use of hplc columns from different batches .
sample solution stability was evaluated by shorting the solution at ambient temperature and at 2 - 5 and analysis after 6 , 12 , 24 , 36 and 48 h. the responses from the aged solutions were compared with those from freshly prepared standard solution .
in this work hplc method with uv detection for analysis of ate and ler in a tablet formulation was developed and validated .
the analytical conditions were selected after testing the different condition effecting hplc analysis , for example diluent and buffer composition , buffer concentration , organic solvent in the mobile phase , buffer to organic solvent ratio and other chromatographic conditions .
preliminary trials with mobile phases comprising mixtures of water with methanol or acetonitrile did not give good peak shape .
the best peak shape was obtained by use of 0.02 m potassium dihydrogen phosphate buffer , adjusted to ph 3.5 with phosphoric acid and use of mobile phase of composition 55:45 ( v / v ) acetonitrile-0.02 m potassium dihydrogen phosphate buffer .
acetonitrile chosen as a organic constituent of the mobile phase to reduce retention times and buffer was chosen to reduce peak asymmetry and achieve good peak shape .
the optimized mobile phase enabled good resolution of ate and ler and of compounds generated during forced degradation .
the newly developed analytical method was validated according to the ich guidelines and its updated international convention , usp and aoac international .
typical chromatogram of tablet extract , showing peaks of ate and ler system suitability was verified by measurement of peak asymmetry ( a<2.0 ) , resolution ( rs>3.0 ) and number of theoretical plates ( n>2000 ) after chromatography of standard solution .
the specificity of the method was determined by checking for interference with the analytes from placebo components by measuring peak purity for ate and ler during the forced degradation study .
ate showed extensive degradation in oxidative conditions while extensive degradation of ler occurred in acidic conditions as shown in ( fig .
table 1 indicated the extent of degradation results of ate and ler under various stress conditions .
chromatographic separation of ate and ler from their oxidative degradation products chromatographic separation of ate and ler from their acidic degradation products chromatographic separation of ate and ler from their alkaline degradation products forced degradation study data ate and ler showed linearity in the range of 40 - 160 g / ml and 8 - 32 g / ml , respectively .
the linear regression equations were y=16456x+11776 , correlation coefficient 0.9995 for ate and y=47672x+7888 , correlation coefficient 0.9993 for ler , where x is the concentration in g / ml and y is the peak area in absorbance units .
the limits of detection and quantification were evaluated by serial dilution of ate and ler stock solution until the signal - to - noise ratios were 3:1 for lod and 10:1 for loq .
the lod for ate and ler were 0.02 and 0.05 g / ml , respectively ; the loq were 0.05 and 0.1 g / ml , respectively .
the relative standard deviations were 0.47% for ate and 0.27% for ler , which are well within the acceptable limit of 2.0% .
the rsds for intermediate precision were found to be 0.21% for ate and 0.30% for ler .
mean recovery was 98.54 - 101.54% for ate and 98.81100.86% for ler ( table 2 ) . in all deliberately varied conditions ,
the rsd of peak areas of ate and ler were found to be well within the acceptable limit of 2.0% . the tailing factor and asymmetry for both
the results obtained from study of the stability of the test preparation , it was concluded the test solution was stable for up to 48 h at 2 - 5 and at ambient temperature , because difference between measured and original values were < 2.0% .
proposed hplc method is specific , accurate and precise for the simultaneous determination of atenolol and lercanidipine hydrochloride from pharmaceutical dosage form . the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing atenolol , lercanidipine , nifedipine , indapamide , hydrochlorothiazide and amlodipine either as such or in combination with atenolol .
the method can be used to separate these drugs from their degradation products and excipients found in the tablet dosage form .
 a simple , rapid , precise and accurate isocratic reversed phase stability indicating hplc method was developed and validated for the simultaneous determination of atenolol and lercanidipine hydrochloride in commercial tablets . the chromatographic separation was achieved on phenomenex gemini c18 ( 2504.6 mm , 5 m ) column using a mobile phase consisting of acetonitrile and buffer ( 20 mm potassium dihydrogen phosphate ph 3.5 ) in the ratio of ( 55:45 , v / v ) at a flow rate of 1.0 ml / min and uv detection at 235 nm . 
 the linearity of the proposed method was investigated in the range of 40 - 160 g / ml ( r2=0.9995 ) for atenolol and 8 - 32 g / ml ( r2=0.9993 ) for lercanidipine . 
 degradation products produced as a result of stress studies did not interfere with the detection of atenolol and lercanidipine and the assay can thus be considered stability - indicating . 
MATERIALS AND METHODS
Chromatographic conditions:
Preparation of standard solution:
Preparation of sample solution:
Forced degradation studies:
Validation procedure:
RESULTS AND DISCUSSION