Method Development and Validation for Simultaneous Estimation of Olmesartan Medoxomil and Hydrochlorothiazide by RP-HPLC SURYADEVARA VIDYADHARA*, REDDYVALAM LANkAPALLI CH SASIDHAR, BALLIPALLI VENkATESwARA RAO, kODURI TEJASwI and MARUPUDI RESHMA

A simple, fast, precise reverse phase isocratic high performance liquid chromatographic (HPLC) method has been developed for the simultaneous estimation of Olmesartan medoxomil (OM) and hydrochlorothiazide (HCTZ) in marketed formulations. Estimation of drugs in this combination was done with a C18 column [ODS UG 5 column, 250mm x 4.5mm] using mobile phase of composition acetonitrile and phosphate buffer (50:50 v/v, pH 6.8). The flow rate was 1.0 ml/min and the separation was monitored at 260nm. The retention time of HCTZ and OM were 3.6min and 4.3min respectively. The method was found to be linear over a range of 10-70μg/ml for OM and 6-42μg/ml for HCTZ. The method was validated according to the guidelines of International Conference on Harmonisation (ICH) and was successfully employed in the estimation of commercial formulations. key words: Olmesartan medoxomil, Hydrochlorothiazide, RP-HPLC, Method validation. methylethyl)-2-propyl-1-{[22 -(1Htetrazol5-yl) [1, 12 -biphenyl]-4-yl] methyl}-1Himidazole5-carboxylic acid1. Hydrochlorothiazide is one of the oldest and widely used diuretics which is also used in the treatment of hyper-tension2-3. Chemically, it is 6chloro3, 4-dihydro2H1, 2, 4-benzothiadiazine7-sulphonamide 1, 1dioxide. 196 VIDyADHARA et al., Orient. J. Chem., Vol. 30(1), 195-201 (2014) Several analytical methods have been reported for the estimation of HCTZ in marketed formulations and United States Pharmacopoeia (USP) describes a RP-HPLC method for its estimation 4-12.OM has not been described officially in any pharmacopoeia yet. Extensive literature survey revealed that very few methods were reported for the simultaneous estimation of OM and HCTZ. So, an attempt has been made to develop an accurate, precise and economically viable RP-HPLC method for the simultaneous estimation of combination of interest in the current research. MATERIALS AND METHODS Equipment used The chromatographic separation was performed on Agilent 1120 compact Liquid Chromatographic system integrated with a variable wavelength programmable UV detector and a Rheodyne injector equipped with 20 μl fixed loop. A reverse phase C18 column [Agilent ODS UG 5 column, 250mm x 4.5mm] was used. Elico SL 218 double beam UV-visible spectrophotometer and Axis AGN204–PO electronic balance were used for spectrophotometric determinations and weighing purposes respectively. Reagents and chemicals Pharmaceutical grade pure Olmesartan medoxomil and Hydrochlorothiazide were obtained as gift samples from Apotex Research Private Limited (ARPL), Bangalore and Life line Formulations, Vijayawada respectively. Marketed formulation Olsertain-H 20 with dose of 20mg of OM and 12.5mg of HCTZ were procured from local market. HPLC grade Acetonitrile and Water were procured from Merck specialities private limited, Mumbai. Chromatographic conditions C18 column [Agilent ODS UG 5 column, 250mm x 4.5mm] was used for the chromatographic separation at a detection wavelength of 260nm. Mobile phase of composition acetonitrile and phosphate buffer, pH 6.8 in a ratio of 50:50 was selected for elution and same mixture was used in the preparation of standard and sample solutions. Flow rate was adjusted to 1ml/min and the injection volume was 20μL. Preparation of mobile phase Phosphate buffer was prepared by mixing 22.4ml of 0.2M sodium hydroxide to 50ml of 0.2M potassium dihydrogen ortho phosphate and the pH was adjusted to 6.8. 450ml of buffer was added to 450ml of acetonitrile, filtered through 0.45μ membrane filter and sonicated for 20 minutes. Preparation of standard solutions 25mg each of OM and HCTZ were accurately weighed and transferred into two 25ml volumetric flasks, dissolved using mobile phase and the volume was made up with the same solvent to obtain primary stock solutions A (OM) and B (HCTZ) of concentration 1000μg/ml of each drug. From the primary stock solutions, 10ml and 6ml were pipetted out from A and B respectively, transferred to a 50ml volumetric flask and the volume was made up with the mobile phase to obtain final concentrations of 200 and 120μg/ml of OM and HCTZ respectively (working stock solution A). Preparation of sample solution Twenty tablets of Olsertain-H 20 were weighed and crushed. Content equivalent to 50mg OM and 30mg HCTZ was weighed accurately and transferred to a 50ml volumetric flask. The content was dissolved with 20ml of solvent and then sonicated for 15min. The volume was made up with the mobile phase and filtered with 0.45μ membrane filter. 1ml from the primary stock solution was pipetted out and transferred to a 10ml volumetric flask and the volume was made up with the solvent to obtain a concentration of 100 and 60 μg/ml of OM and HCTZ respectively (working stock solution B). Optimization of HPLC method The HPLC method was optimized with an aim to develop a simultaneous estimation procedure for the assay of OM and HCTZ. For the method optimization, different mobile phases were tried but acceptable retention times, theoretical plates and good resolution were observed with acetonitrile and pH 6.8 phosphate buffer (50:50, v/v) using C18 column (Agilent ODS UG column, 250mm x 4.5mm) Validation of the RP-HPLC method Validation of the optimized method was performed according to the ICH Q2 (B) guidelines. 197 VIDyADHARA et al., Orient. J. Chem., Vol. 30(1), 195-201 (2014) System suitability System suitability was carried out with five injections of solution of 100% concentration having 70μg/ml of OM and 42μg/ml of HCTZ in to the chromatographic system. Number of theoretical plates (N) obtained and calculated tailing factor (T) were reported in table 1. Linearity For the determination of linearity, appropriate aliquots were pipetted out from working stock solution A to a series of 10ml volumetric flasks and the volume was made up with the solvent to obtain concentrations ranging from 10-70 μg/ml of OM and 6-42 μg/ml of HCTZ. Each solution was injected in triplicate. Calibration curves were plotted with observed peak areas against concentration followed by the determination of regression equations and calculation of the correlation coefficients. The calibration curves for OM and HCTZ were shown in Figure 3 and their corresponding linearity parameters were given in table 2. Accuracy To ensure the reliability and accuracy of the method recovery studies were carried out by standard addition method. A known quantity of pure drug was added to pre-analysed sample and contents were reanalysed by the proposed method and the percent recovery was reported. The results were given in Table 4. Precision The repeatability of the method was verified by calculating the %RSD of six replicate injections of 100% concentration (70μg/ml of OM and 42μg/ml of HCTZ) on the same day and for intermediate precision %RSD was calculated from repeated studies on different days. The results were given in table 3. Specificity Specificity of a method was determined by testing standard substances against potential interferences. The method was found to be specific when the test solution was injected and no interferences were found because of the presence of excipients. Limit of detection (LOD) and limit of quantitation (LOQ) The LOD and LOQ were calculated from the slope(s) of the calibration plot and the standard deviation (SD) of the peak areas using the formulae LOD = 3.3 σ/S and LOQ = 10 σ/S. the values were given in table 2. Robustness Robustness of the method was verified by altering the chromatographic conditions like mobile phase composition, flow rate, detection wavelength, etc. and the % RSD should be reported. Small changes in the operational conditions were allowed and the extent to which the method was robust was determined. A deviation of ±2nm in the detection wavelength and ±0.1 mL/min in the flow rate, were tried individually. A solution of 100% test concentration with the specified changes in the operational conditions were injected to the instrument in triplicate. %RSD was reported in table 5. Assay of marketed formulation 20μl of sample solution of concentration 70μg/ml of OM and 42μg/ml of HCTZ was injected into chromatographic system and the peak responses were measured. The solution was injected three times into the column. The amount present in each tablet was calculated by comparing the areas of standards with the test samples. RESULTS AND DISCUSSION After a number of trials with mobile phases of different composition, acetonitrile and phosphate buffer, pH 6.8 50:50 v/v was selected as mobile phase because of better resolution and symmetrical peaks. OM and HCTZ were found to show appreciable absorbance at 260nm when determined spectrophotometrically and hence it was selected as the detection wavelength. An optimized chromatogram showing the separation of HCTZ and OM at different RTs was shown in figure 2. System suitability was carried out by injecting 5 replicate injections of 100% test concentration, number of theoretical plates, HETP and resolution were satisfactory. The chromatogram confirms the presence of HCTZ and OM at 3.6min and 4.3min respectively without any interferences. The parameters were given in table 1. 198 VIDyADHARA et al., Orient. J. Chem., Vol. 30(1), 195-201 (2014) Table 1: System suitability parameters Parameters Hydrochlorothiazide Olmesartan medoxomil Retention time (min) 3.6 4.3 Theoretical plates (N) 11421 9897 Tailing factor (T) 1.2 1.4 Resolution (Rs) 1.9 Table 2: Results for Linearity (n=3) Parameters Hydrochlorothiazide Olmesartan medoxomil Slope 963140 727006 Intercept 586967 17530 r2 0.9996 0.9996 Regression equation y = 963140x + 586967 y = 727006x + 17530 Linearity range 6-42μg/ml 10-70 μg/ml LOD 0.10 μg/ml 0.25 μg/ml LOQ 0.32 μg/ml 0.78 μg/ml *n= No. of determinants Table 3: Results for Precision (n=6) Drug Intraday Interday Precision (% RSD) Precision (% RSD) Hydrochlorothiazide 0.69 1.13 Olmesartan medoxomil 0.57 0.72 *n= No. of determinants Table 4: Results for Accuracy (n=3) Recovery Olmesartan medoxomil Hydrochlorothiazide level Amount Amount % Amount Amount % Added Found Recovery Added Found Recovery (μg/mL) (μg/mL) (μg/mL) (μg/mL) Std. test Std. test 80% 40 10 49.51 99.02 24 6 30.28 100.94 100% 50 10 60.93 101.55 30 6 36.21 100.59 120% 60 10 69.61 99.44 36 6 41.32 98.39 Mean Recovery 99.02-101.55% w/w 98.39-100.94%w/w *n= No. of determinants 199 VIDyADHARA et al., Orient. J. Chem., Vol. 30(1), 195-201 (2014) Table 5: Results for Robustness Parameters (n=3) % RSD Hydrochlorothiazide Olmesartan medoxomil At λ max 258 nm 0.42 0.43 At λ max 262 nm 0.71 0.59 With flow rate 0.9 ml 0.66 0.48 With flow rate 1.1 ml 0.72 0.78 *n= No. of determinants Table 6: Results for Assay (n=3) of marketed formulation Drug Label claim (mg/tab) Amount recovered % Amount found in drug OM 20 mg 20.06 mg 100.34 % w/w HCTZ 12.5 mg 12.4 mg 99.2%w/w *n= No. of determinants Concentration range of 10-70 μg/ml for OM and 6-42μg/ml for HCTZ were found to be linear with correlation coefficients 0.9996 and 0.9996 for OM and HCTZ respectively. The results were given in table 2. The proposed method was found to be precise and reproducible with %RSD of 1.13 and 0.72 for HCTZ and OM respectively. %RSD was reported in table 3. Accuracy of the method was verified by performing recovery studies by standard addition method. The percent recovery of the standard added to the pre-analysed sample was calculated and it was found to be 98.39 to 100.94% w/w and 99.02101.55%ww/w for HCTZ and OM respectively which indicates that the method was accurate. Values obtained were given in table 4. a) Olmesartan medoxomil b) Hydrochlorothiazide Fig. 1: Chemical Structures of a) OM and b)HCTZ 200 VIDyADHARA et al., Orient. J. Chem., Vol. 30(1), 195-201 (2014) The limits of detection for HCTZ and OM were found to be 0.106μg/ml and 0.259 μg/ml respectively and the limits of quantitation were 0.323 μg/ml and 0.786 μg/ml respectively. Fig. 3: Calibration Plot of Olmesartan medoxomil and Hydrochlorothiazide Fig. 4: A typical chromatogram for assay of marketed formulation containing 42 μg/ml of HCTZ and 70 μg/ml of OM The method was found to be specific for the combination of interest after verifying the chromatograms showing no interference of the excipients present. Hence, the method was well Fig. 2: Optimized chromatogram of HCTZ and OM by RP-HPLC 201 VIDyADHARA et al., Orient. J. Chem., Vol. 30(1), 195-201 (2014) suitable for the estimation of the commercial formulations of the selected combination. Values obtained were given in table 5. The method was found to be robust after changing the conditions like detection wavelength (± 2nm) and flow rate (± 0.1 ml). %RSD was calculated for each variation and reported. Values obtained were given in table 6.