Spectrophotometric and Hplc Methods for Simultaneous Estimation of Amlodipine Besilate, Losartan Potassium and Hydrochlorothiazide in Tablets

Hyderabad, for supplying gift samples of drotaverine hydrochloride and paracetamol and the Principal, Dr. A HPLC method is developed for the determination of drotaverine HCl in human plasma and urine. Two UV-spectrophotometric and one reverse phase high performance liquid chromatography methods have been developed for the simultaneous estimation of amlodipine besilate, losartan potassium and hydrochlorothiazide in tablet dosage form. The fi rst UV spectrophotometric method was a determination using the simultaneous equation method at 236.5, 254 and 271 nm over the concentration range 5-25, 10-50 and 5-25 µg/ml for amlodipine besilate, losartan potassium and hydrochlorothiazide, respectively. The second UV method was a determination using the area under curve method at 231.5-241.5, 249-259 and 266-276 nm over the concentration range of 5-25, 5-25 and 10-50 µg/ml for amlodipine besilate, hydrochlorothiazide and losartan potassium, respectively. In reverse phase high performance liquid chromatography analysis is carried out using 0.025 M phosphate buffer (pH 3.7):acetonitrile (57:43 v/v) as the mobile phase and Kromasil C18 (4.6 mm i.d×250 mm) column as stationery phase with detection wavelength of 232 nm linearity was obtained in the concentration range of 2-14, 20-140 and 5-40 µg/ml for amlodipine besilate, losartan potassium and hydrochlorothiazide, respectively. Both UV-spectrophotometric and reverse phase high performance liquid chromatography methods were statistically validated and can be used for analysis of combined dose tablet formulation containing amlodipine besilate, losartan potassium and hydrochlorothiazide.

In the UV-spectrophotometric methods, simultaneous equation method (method A), standard stock solutions of AMLO (100 µg/ml), HCTZ (100 µg/ml) and LOS (100 µg/ml) were prepared in methanol.For the selection of analytical wavelength solutions of AMLO (2 µg/ml), HCTZ (5 µg/ml) and LOS (20 µg/ml) were prepared separately by appropriate dilution of standard stock solution and scanned in the spectrum mode from 200 to 400 nm.From the overlain spectra of these drugs (fi g. 1), wavelengths 236.5 nm (λ max of AMLO), 254 nm (λ max of LOS) and 271 nm (λ max of HCTZ) were selected for analysis.The calibration curves for AMLO, HCTZ and LOS were prepared in the concentration range of 5-25 µg/ml, 5-25 µg/ml and 10-50 µg/ml, respectively at the selected wavelengths.Absorptivity values were determined for AMLO, HCTZ and LOS and were found to be 32.20/13.36/3.09,10.86/19.07/61.14 and 37.24/26.55/12.74 at 236.5/254/271 nm, respectively.Using these absorptivity values following Eqns.were developed for determining concentration of AMLO, HCTZ and LOS in tablet sample solution.A 1 = 32.20CAMLO +10.86CHCTZ +37.24CLOS (1) ,A = 13.36CAMLO +19.07CHCTZ +26.55CLOS (2) and A 3 = 3.09C AMLO +61.14CHCTZ +12.74CLOS (3), where A1, A2 and A 3 are absorbance of the tablet sample solution at 236.5, 254 and 271 nm, respectively.C AMLO is the concentration of AMLO, C HCTZ is the concentration of the HCTZ, and C LOS is the concentration of the LOS.For estimating AMLO, HCTZ and LOS in tablet formulation, twenty tablets were weighed and average weight was calculated.The tablets were crushed to obtain fi ne powder.Tablet powder equivalent to 75 mg of LOS was transferred to 50.0 ml volumetric flask added 30 ml of methanol, sonicated for 20 min and volume was made up to the mark with methanol.The solution was then filtered through Whatmann filter paper No. 41.The filtrate was appropriately diluted with the same solvent to obtain fi nal concentration within Beer Lambert's range for each drug.Absorbance of diluted sample solution was measured at selected wavelengths.The concentration of drugs was determined by using the Eqns 1, 2 and 3. Results of analysis of tablet formulation mentioned in Table 1.
In the area under curve method (method B), preparation of standard stock solution was same as mentioned in method A. From the overlain spectra of drugs (fi g. 1) wavelengths range 231.5-241.5 nm (AMLO), 266-276 nm (HCTZ) and 249-259 nm (LOS) were selected for the analysis.The calibration curves for AMLO, HCTZ and LOS were prepared in the concentration range as mentioned in method A at the selected wavelength range.Absorptivity values were determined for AMLO, HCTZ and LOS and were found to be 313.28  (5)  and A 3 = 30.96CAMLO +581.14CHCTZ +123.96CLOS (6), where A1, A2 and A 3 are area under curve of the sample at 231.5-241.5, 266-276 and 249-259 nm, respectively, C AMLO is the concentration of AMLO, C HCTZ is the concentration of the HCTZ, and C LOS is the concentration of the LOS.Preparation of sample solution for analysis of tablet formulation was same as described under method A. The concentrations of AMLO, HCTZ and LOS were determined by using the Eqns.4, 5 and 6.Result of analysis of tablet formulation are mentioned in Table 1.
In the reverse phase high performance liquid chromatography (method C), following optimum conditions were established for quantitative analysis of AMLO, HCTZ and LOS in tablet formulation by trial and error.Mobile phase: a mixture of phosphate buffer (0.025M, pH-3.7 adjusted with ortho phosphoric acid) and acetonitrile in the ratio of (57:43 v/v), column: Kromasil C18 (4.6 mm i.d×250 mm), fl ow rate: 1.0 ml/min for 6.3 min then 1.3 ml/ min from 6.3 min onwards, detection wavelength: 232 nm, temperature: room temperature.Sample solution of AMLO, HCTZ and LOS 6 µg/ml, 15 µg/ml and 60 µg/ml, respectively were prepared in mobile phase and chromatographed under optimum chromatographic conditions.The proposed chromatographic conditions were found suitable for effective separation and quantitation of AMLO (RT-5.12min), HCTZ (RT-  3.42 min) and LOS (8.02 min) with resolution of 2.32 (between HCTZ and AMLO) and 7.91 (between AMLO and LOS), tailing factor-1.5 for AMLO, 1.33 for HCTZ and 1.05 for LOS.The calibration curves (mean peak area Vs concentration) for AMLO, HCTZ and LOS were prepared in the concentration range of 2-14 µg/ml, 5-40 µg/ml and 20-140 µg/ml, respectively at 232 nm.For the estimation of these drugs in the tablet formulations, twenty tablets were weighed and average weight was calculated.The tablets were crushed to obtain fine powder.Tablet powder equivalent to 50 mg of LOS was transferred to 50.0 ml volumetric fl ask added 30 ml mobile phase and ultrasonicated for 20 min, volume was then made up to the mark with mobile phase.The solution was then filtered through a Whatmann filter paper No. 41.The filtrate was appropriately diluted with the mobile phase to obtain final concentration within linearity range for each drug in the ratio 60:15:6 μg/ ml LOS: HCTZ: AMLO, respectively.The contents were mixed thoroughly and fi ltered through a 0.2 μ fi lter.Twenty microlitres of solution was injected and chromatographed under optimum chromatographic condition.A typical chromatogram of AMLO, LOS and HCTZ is shown in (fi g. 2).The concentration of AMLO, HCTZ and LOS in tablet sample solution was determined by comparing the peak area of the sample with that of standard at 232 nm.Results of analysis tablet formulation are shown in Table 1.
Accuracy of the proposed UV-spectrophotometry and reverse phase high performance liquid chromatography method was studied by performing recovery studies by standard addition method at three different levels 80%, 100% and 120%.The results of recovery studies, expressed as % recovery, are mentioned in Table 2. Robustness of the proposed methods was studied by analyzing tablet formulation under varied conditions like different analysts, on different days and different times on same day.The standard deviation for analysis under different conditions was below 2% indicating robustness of method.Ruggedness of the reverse phase high performance liquid chromatography method was studied by deliberately changing the method parameters viz.change in mobile phase composition (±1 ml) and change in flow rate (±0.1 ml/min).Reverse phase high performance liquid chromatography method was found to withstand these variations as there was no signifi cant change in retention time, tailing factor and resolution.
Based on the results obtained, it is found that the proposed UV-Spectrophotometric and reverse phase high performance liquid chromatography methods are accurate, precise, reproducible economical and can be employed for routine analysis of AMLO, LOS and HCTZ in combined dose tablet formulation.

TABLE 1 : ANALYSIS OF TABLET FORMULATION
is amlodipine besilate, LOS is losartan potassium and HCTZ is hydrochlorothiazide *Average of six determinations, SD -Standard Deviation, CV-Coeffi cient of Variation. AMLO

TABLE 2 : RESULTS OF RECOVERY STUDIES
is amlodipine besilate, LOS is losartan potassium and HCTZ is hydrochlorothiazide, *Average of three determinations, SD -Standard Deviation, CV-Coeffi cient of Variation. AMLO