Simultaneous Estimation of Amlodipine Besilate and Olmesartan Medoxomil in Pharmaceutical Dosage Form

Petri dishes (9 cm diameter) with one sheet of Þ lter paper inside of the same size were used for the experiment. Five different concentrations of neem oil, 100, 80, 60, 40 and 20% were used for the study. One milliliter (100%), 0.8 ml (80%), 0.6 ml (60%), 0.4 ml (40%) and 0.2 ml (20%) of neem oil were evenly soaked (spread with a spatula) on different filter papers of each Petri dish. Distilled water soaked in filter paper was used as a control (0% concentration). Then different numbers of larvae were put inside each plate with the help of a soft brush and then monitored at certain interval of time. The number of larvae found dead in each plate was recorded and the results were summarized in Table 1.

weeks before use.
Petri dishes (9 cm diameter) with one sheet of Þ lter paper inside of the same size were used for the experiment.Five different concentrations of neem oil, 100, 80, 60, 40 and 20% were used for the study.One milliliter (100%), 0.8 ml (80%), 0.6 ml (60%), 0.4 ml (40%) and 0.2 ml (20%) of neem oil were evenly soaked (spread with a spatula) on different filter papers of each Petri dish.Distilled water soaked in filter paper was used as a control (0% concentration).Then different numbers of larvae were put inside each plate with the help of a soft brush and then monitored at certain interval of time.The number of larvae found dead in each plate was recorded and the results were summarized in Table 1.
From the results obtained, it is clear that the mortality of larvae was concentration and time dependent.With 0% concentration of neem oil (control), there was no mortality at any time.100% mortality was observed with 20, 40, 60, 80 and 100% concentrations after 27, 27, 27, 27 and 24 h, respectively.Thus the neem oil can be safely used for the control of ticks in animals due to its non-adverse effect in animals.Moreover, the neem oil with both fungicidal and bactericidal properties could treat these infections caused from the bite of ticks.The oil is easily available for its use as an inexpensive herbal medicine.This result will be very much useful for the farmers all over the world.Amlodipine besilate (AMLO), chemically, [3-ethyl-5-methyl(4RS)-2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-methyl-1-dihydropyridine-3,5dicarboxylate benzenesulfonate [1] , is a long acting calcium channel blocker used which is used as an antihypertensive agent [2][3][4] .Olmesartan medoxomil (OLME), chemically, 2,3-dihydroxy-2-butenyl 4-(1-hydroxy-1-methylethyl)-2-propyl-1-[p-(o-1Htetrazol-5-ylphenyl)benzyl]imidazole-5-carboxylate, cyclic-2,3-carbonate, is an angiotensin II receptor blockers used as an antihypertensive agent [5] .AMLO is official in BP [1] , whereas OLME is not official in any pharmacopoeia.Both the drugs are marketed as combined dose tablet formulation in the ratio of AMLO:OLME 05:20 mg.Literature survey revealed that a number of methods have been reported for estimation of AMLO [6][7][8][9][10][11][12][13][14][15] and OLME [16][17] individually or in combination with other drugs.However, there is no analytical method reported for the simultaneous estimation of AMLO and OLME in a combined dosage formulation.For the selection of analytical wavelength for the simultaneous equation method (method-A), solutions of AMLO and OLME (20 µg/ml, each), were prepared separately by appropriate dilution of standard stock solution and scanned in the spectrum mode from 200 nm to 400 nm.From the overlain spectra of both drugs (Þ g. 1), wavelengths 237.5 nm (λ max of AMLO) and 255.5 nm (λ max of OLME) were selected for the simultaneous equations.Calibration curves for AMLO and OLME were prepared in the concentration range of 10-50 µg/ml and 10-50 µg/ml at both the wavelengths, respectively.The absorptivity values were determined for both the drugs at both the wavelengths and following Eqns were used, A 1 =41.86CAMLO +38.99COLME (1) and A 2 =28.25CAMLO +41.39COLME (2), where A1 and A2 are absorbances of the sample at 237.5 nm and 255.5 nm, respectively, 41.86 and 28.25 are absorptivities of AMLO at 237.5 nm and 255.5 nm, respectively, 38.99 and 41.39 are the absorptivities of OLME at 237.5 nm and 255.5 nm, respectively.C AMLO is the concentration of AMLO and C OLME is the concentration of the OLME.The mixture concentration was determined by using the Eqns 1 and 2.
In the area under curve method (method-B), from the overlain spectra of both drugs (fig.1), wavelengths range 242.5-232.5 nm (for AMLO) and 260.5-250.5 nm (for OLME) were selected for the analysis.The calibration curves for AMLO and OLME were prepared in the concentration range of 10-50 µg/ml and 10-50 µg/ml at both the wavelength range, respectively.The absorptivity values were determined for both the drugs at both the wavelength range and following Eqns were used, A 1 = 414.13CAMLO +389.32COLME (3) and A 2 = 283.08CAMLO +408.97COLME (4), where A1 and A2 are area under curve of the sample at 242.5-232.5 nm and 260.5-250.5 nm, respectively, 414.13 and 283.08 are absorptivities of AMLO at 242.5-232.5 nm and 260.5-250.5 nm, respectively, 389.32 and 408.97 are the absorptivities of OLME at 242.5-232.5 nm and 260.5-250.5 nm, respectively.C AMLO is the concentration of AMLO and C OLME is the concentration of the OLME.
The mixture concentration was determined by using the Eqns 3 and 4.
In the reverse phase high performance liquid chromatography (method-C), standard stock solution of AMLO and OLME (1000 μg/ml) was prepared in mobile phase separately.The wavelength selected for analysis is 238 nm.The calibration curves for AMLO and OLME were prepared in the concentration range of 04-20 µg/ml and 10-50 µg/ml, respectively at 238 nm.Calibration curve was constructed by plotting concentration against peak area.
In the UV spectrophotometric method, for estimating AMLO and OLME in commercial formulations, twenty tablets were weighed and average weight was calculated.The tablets were crushed to obtain a fine powder.Tablet powder equivalent to 5 mg of AMLO was transferred to 25.0 ml volumetric flask containing 20.0 ml methanol and exposed to ultrasonic radiations for 20 min and then final volume was made up to the mark with methanol.The solution was then Þ ltered through a Whatmann filter paper No. 41.The filtrate was appropriately diluted with the same solvent to obtain final concentrations of 8 µg/ml for AMLO and 32 µg/ ml for OLME.Concentrations of both AMLO and OLME were determined by measuring the absorbance of the sample at 237.5 and 255.5 nm (method-A) and at 242.5-232.5 nm and 260.5-250.5 nm (method B) in the spectrum mode and values were substituted in the respective formulae to obtain concentrations.Results of the tablet analysis were analysed against the calibration curve in quantitation mode.
In the RP-HPLC method, for estimating AMLO and OLME in commercial formulation, tablet sample solution containing 8 µg/ml of AMLO and 32 µg/ ml of OLME was prepared in similar manner as described under UV spectrophotometric method using mobile phase as diluent instead of methanol.The diluted solutions were filtered through 0.20 μ Þ lter.Twenty microlitres of solutions were injected and chromatographed under above mentioned chromatographic conditions.A typical chromatogram of AMLO and OLME is shown in (fig.2).The concentration of both AMLO and OLME was determined by comparing peak area of sample with that of standard at 238 nm.The results of tablet analysis are shown in Table 1.
The proposed chromatographic system was found suitable for effective separation and quantitation of AMLO (RT-3.79min) and OLME (5.39 min).The system suitability parameters were found to be, resolution-2.22,tailing factor-1.30for AMLO and 1.17 for OLME.Recovery studies were carried out by standard addition method at three different levels

TABLE 1 : ANALYSIS OF TABLET FORMULATION
Average of six determinations, SD denotes standard deviation and CV denotes coefÞ cient of variation. *

TABLE 2 : RESULTS OF RECOVERY STUDIES
SD is the standard deviation, CV is the coefÞ cient of variation and SE is the standard error.