

ORIGINAL ARTICLE 

Year : 2011  Volume
: 2
 Issue : 3  Page : 155160 


Spectrophotometric methods for the simultaneous estimation of losartan potassium and hydrochlorothiazide in tablet dosage forms
Kareti Srinivasa Rao, Minakshi Panda, Nargesh Kumar Keshar
Departments of Pharmaceutical Analysis and Quality Assurance, Roland Institute of Pharmaceutical Sciences, Berhampur, Orissa, India
Date of Web Publication  16Dec2011 
Correspondence Address: Kareti Srinivasa Rao Department of Pharmaceutical Analysis and Quality Assurance, Roland Institute of Pharmaceutical Sciences, Berhampur, Orissa  760 010 India
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/22295186.90893
Abstract   
Aim: This work deals with the simultaneous determination of Losartan potassium (LSP) and Hydrochlorothiazide (HZ) in a binary mixture form, without prior separation, by three different techniques. Materials and Methods: The present work was carried out on Shimadzu electron UV1800 double beam UVVisible spectrophotometer. The absorption spectra of reference and test solutions were carried out in 1 cm matched quartz cell over the range of 200400 nm. Standard gift sample of LSP and HZ were obtained from Torrent pharmaceuticals Ltd, Baddi, Himachal Pradesh. Combined LSP and HZ tablets were purchased from local market. Methanol from Merck Ltd. and distilled water are used as solvent. Results: The first method is the application of simultaneous equation. Where the linearity ranges for LSP and HZ were 525 μg/ml and 120 μg/ml, respectively. The second method is the determination of ratio of absorbance at 272 nm, the maximum absorption of HZ and isosbestic wavelength 266.5nm, the linearity ranges for LSP and HZ were 580μg/ml and 125μg/ml respectively. The third method is the first order derivative method, where the linearity ranges for LSP and HZ were 130 μg/ml and 140 μg/ml respectively. The proposed procedures were successfully applied for the simultaneous determination of both the drugs in commercial tablet preparation. The validity of the proposed methods was assessed by applying the standard addition technique where the percentage recovery of the added standard was found to be 99.06±1.210 and 99.30±1.159 using the simultaneous equation method, 99.66±0.573 and 99.95±0.272 using the graphical absorbance ratio method and 99.64±0.301 and 99.91±0.614 using first derivative method, for LSP and HZ respectively. Conclusions: The proposed procedures are rapid, simple, require no preliminary separation steps and can be used for routine analysis of both drugs in quality control laboratories. Keywords: First derivative method, hydrochlorothiazide, losartan potassium, QAnalysis
How to cite this article: Rao KS, Panda M, Keshar NK. Spectrophotometric methods for the simultaneous estimation of losartan potassium and hydrochlorothiazide in tablet dosage forms. Chron Young Sci 2011;2:15560 
How to cite this URL: Rao KS, Panda M, Keshar NK. Spectrophotometric methods for the simultaneous estimation of losartan potassium and hydrochlorothiazide in tablet dosage forms. Chron Young Sci [serial online] 2011 [cited 2015 Aug 2];2:15560. Available from: http://www.cysonline.org/text.asp?2011/2/3/155/90893 
Introduction   
Losartan potassium (LSP) is an angiotensin II receptor antagonist and chemically it is 2nbutyl4chloro5hydroxymethyl1[2'(1Htetrazol5yl)(biphenyl4yl)methyl]imidazole, a strong antihypertensive agent. Losartan was developed by DuPontMerck laboratories as a potent nonpeptide angiotensin II receptor (type AT1) antagonist for hypertension treatment. ^{[1]} It is administered in its active form and is partially converted into an active metabolite, which is responsible for the drug's prolonged pharmacological effect. The therapeutic efficacy of losartan, as well as its renal and antihypertensive effects, seems to be similar to those of angiotensin converting enzyme (ACE) inhibitors. Hydrochlorothiazide (HZ) is chemically 6chloro3, 4dihydro2H1, 2, 4benzothiadiazine7sulfonamide1,1dioxide. It is the prototype of the thiazide group and antihypertensive drug. ^{[2]}
Literature survey reveals that LSP was determined by several methods including Spectrophotometric, ^{[3],[4],[5],[6]} high performance liquid chromatography (HPLC) ^{[6],[7],[8],[9]} and liquid chromatography, capillary electrophoresis and supercritical fluid chromatography. ^{[10]} HZ was determined by capillary electrophoresis ^{[11]} and electrochemical study ^{[12]} and by spectrophotometrically, ^{[13],[14],[15],[16],[17]} reverse phaseHPLC (RPHPLC). ^{[18],[19],[20]} Literature survey revealed that spectrophotometric ^{[21],[22]} and HPLC ^{[23]} methods have been reported for the estimation of LSP and HZ in combination with the other drugs. Few HPLC ^{[24],[25],[26],[27],[28],[29],[30]} methods are available for the simultaneous estimation of LSP and HZ in pharmaceutical formulations. However, there were two spectrophotometric methods for simultaneous estimation of LSP and HZ reported. First one ^{[31]} describes simultaneous estimation of LSP and HZ by simultaneous equation method using methanol as a solvent throughout the method. The second one ^{[32]} describes two methods namely simultaneous equation method and dual wavelength method for simultaneous estimation of LSP and HZ in tablets using 0.1M hydrochloric acid (HCL) for both the methods. Nevertheless, the methods we developed involve methanol for the preparation of standard stock solution and water for further dilution in all the three developed methods namely simultaneous equation method, Qanalysis method and derivative spectrophotometry method. Later two methods (Qanalysis method and derivative spectrophotometry method) were developed for the first time. The aim of this paper was to explore the possibility of using techniques of simultaneous equation, the absorbance ratio (Qanalysis) and first derivative method for quantifying LSP and HZ simultaneously in their mixture forms. The proposed methods are simple, convenient, precise, accurate and economical than the reported method.
Materials and Methods   
Instrument
The present work was carried out on Shimadzu electron UV1800 double beam UVvisible spectrophotometer. The absorption spectra of reference and test solutions were carried out in 1 cm matched quartz cell over the range of 200400 nm.
Materials
Standard gift sample of Losartan potassium and Hydrochlorothiazide were obtained from Torrent pharmaceuticals Ltd, Baddi, Himachal Pradesh. Combined LSP and HZ tablets were purchased from the local market.
Solvent used
Methanol (MERCK Ltd.), Distilled water.
Procedure
Simultaneous equation method (MethodI)
Standard stock solutions (1 mg/ml) of LSP and HZ were prepared by dissolving 100 mg of each in 20 ml methanol in a 100 ml volumetric flask and diluted to 100 ml with distilled water. From this suitable aliquots are taken and diluted with distilled water to get 20 μg/ml of LSP and 5 μg/ml of HZ. The absorption spectra of all the solutions were recorded between 200400 nm. The absorbance were measured for LSP and HZ at 218 nm (l_{1} ) (maximum absorbance of LSP), 272 nm (λ_{2} ) (maximum absorbance of HZ) and 266.5 nm (isosbestic point). Wavelengths 218 nm and 272 nm were selected for the formation of simultaneous equation [Figure 1]. The absorbances were measured at the selected wavelengths. The molar absorptivity values were 112 at λ_{1} and 418.4 at λ_{2} for LSP and 118 at λ_{1} and 419.6 at λ_{2} for HZ. The absorbance and absorptivity values were substituted in the following equation to obtain the concentrations:  Figure 1: Overlain spectra of LSP (20 μg/ml) and HZ (5 μg/ml) for simultaneous equation method
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Cx = A _{2} ay _{1}  A _{1} ay _{2} / ax _{2} ay _{1}  ax _{1} ay _{2}
Cy = A _{1} ax _{2}  A _{2} ax _{1} / ay _{1} ax _{2}  ay _{2} ax _{1} Where A _{1} and A _{2} are absorbances of the mixture at λl_{1} and λ_{2} respectively, ax _{1} and ax _{2} are absorptivity of X at λ_{1} and l_{2} respectively, ay _{1} and ay _{2} denotes absorptivity of Y at λ_{1} and λ_{2,} respectively, Cx and Cy are concentrations of LSP and HZ, respectively.
The graphical absorbance ratio method (Qanalysis method) (MethodII)
In the quantitative assay of two components by Qanalysis method, absorbances were measured at two wavelength, one being the isosbestic wavelength and the other being wavelength of maximum absorption of one of the two components. From overlain spectra of LSP and HZ, absorbances were measured at the selected wavelength, i.e., 266.5 nm (isosbestic wavelength) and 272 nm (wavelength of maximum absorption of HZ) [Figure 2]. The concentration of each component can be calculated by mathematical treatment of the following mentioned equation.  Figure 2: Overlain spectra of LSP (10 μg/ml) and HZ (10 μg/ml) for Qanalysis: Graphical absorbance ratio method
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For LSP
C _{1} = Qm  Qy / Qx  Qy. A _{1} / a
For HZ
C _{2} = Qm  Qx / Qy  Qx. A _{1} / a
Where, C _{1} = concentration of LSP
C _{2} = concentration of HZ
A _{1} = absorbance of sample at isosbestic wavelength (266.5 nm)
a = Absorptivity of LSP and HZ at isosbestic wavelength (266.5 nm)
Qx = Absorptivity of LSP at 272 nm / Absorptivity of LSP at 266.5 nm
Qy = Absorptivity of HZ at 272 nm / Absorptivity of HZ at 266.5 nm
Qm = Absorptivity of sample solution at 272 nm / Absorptivity of sample solution at 266.5 nm.
First order derivative method (Method III)
Solutions of 10μg/ml of LSP and HZ were prepared separately. Both the solutions were scanned in the spectrum mode from 200 to 400 nm. The absorption spectra thus obtained were derivatized from first to fourth order. First order derivative (n=1) was selected for analysis of both the drugs. The zero crossing wavelengths, 222 nm and 332 nm were selected for HZ and LSP, respectively [Figure 3].  Figure 3: Overlain first derivative spectra of LSP (20 μg/ml) and HZ (5 μg/ml)
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Preparation of calibration curve
Six mixed standards having concentration 1, 5, 10, 15, 20, 25, 30 μg/ml of LSP and 1, 5, 10, 20, 30, 40 μg/ml of HZ, respectively, were prepared and scanned in the spectrum mode from 200 nm to 400 nm. The absorption spectra so obtained were derivatized to obtain first derivative order spectra. The absorbances of LSP and HZ were measured at 332 nm and 222 nm, respectively, and calibration curve of both the drugs were plotted separately. The concentration of individual drug present in the mixture was determined against calibration curve in quantitation mode.
Application of the proposed procedure for the determination of Losartan potassium and Hydrochlorothiazide in tablets
Twenty tablets were weighed and average weight was calculated. The tablets were crushed to fine powder. The powder equivalent to 100 mg of LSP and 25 mg of HZ was transferred to 100 ml volumetric flask. The powder was dissolved in 20 ml of methanol by intermittent shaking followed by sonication for 15 min and then the volume was made up to 100 ml with distilled water. The solution was then filtered through a Whatmann filter paper (No. 41). The solution was diluted further with distilled water to obtain 20 μg/ml of LSP and 5 μg/ml of HZ. The concentration of both LSP and HZ were determined by measuring the absorbance of the samples at 218nm (l_{max} for LSP), 272 nm (l_{max} for HZ) and 266.5 nm (isosbestic point). The recorded data was then substituted in the equation and results obtained are summarized in [Table 1]. The analysis procedure was repeated three times. The selectivity of the proposed procedure was examined by determining the recovery of the two drugs by standard addition method [Table 2].  Table 1: Determination of LSP and HZ in tablet using the proposed methods
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 Table 2: Results of the application of the standard addition technique to the simultaneous determination of LSP and HZ in tablet by the proposed method (n=3)
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Results and Discussion   
The proposed methods were found to be simple, accurate, economic, and rapid for routine simultaneous estimation of two drugs. The values of relative standard deviation are satisfactorily low and recovery was closed to 100%, indicating reproducibility and accuracy of all methods. These methods also gave excellent result and can be employed for routine analysis of these two drugs in combined dosage form.
In simultaneous equation method, the overlay spectra of LSP and HZ shows overlap, that prevents the use of direct absorbance measurement for determination of both the drugs in their mixture. The [Figure 1] represents that the l_{max} for LSP at 218 nm and for HZ at 272 nm. The absorbance curve at the selected wavelengths were found to be proportional to the corresponding concentration of the two drugs in the range of 525 μg/ml for LSP and 120 μg/ml for HZ as shown by the small intercept and correlation coefficient approaching unity in the regression equation [Table 3]. The absorptivity values of the drugs were determined at selected wavelength. The absorptivity is the ratio of mean absorbance of the drug at selected wavelength with the concentration of component in mg/ml. These absorptivity values were the mean of six independent determinations. A set of two simultaneous equations obtained by using mean absorptivity values are given below:  Table 3: Data for calibration graph for LSP and HZ using simultaneous equation, graphical absorbance ratio method and first derivative method
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A _{1} = 112 C _{LSP} + 118 C _{HZ}  (at l_{218} )
A _{2} = 418.4 C _{LSP} + 419.6 C _{HZ}  (at l_{272} )
Where A _{1} and A _{2} are absorbances of the sample at 218 nm and 272 nm, respectively. Here, 112 and 418.4 are the absorptivity values of LSP at 218 nm and 272 nm, respectively; 118 and 419.6 are the absorptivity values of HZ at 218 nm and 272 nm, respectively. C _{LSP} is the concentration of the LSP and C _{HZ} is the concentration of HZ in mg/ml.
The proposed Q analysis method is also a simple method. In this method, the absorbances of the sample solution at the two selected wavelengths were measured and few calculations were done.
The first derivative spectrophotometry method requires spectral data processing and hence can be applied only on recording spectrophotometers with such facilities. This method was employed to totally eliminate the spectral interference from one of two drugs while eliminating the other drug. This was achieved by selecting the zero crossing point on the derivative spectra of one drug as the wavelength for the estimation of other drug. First derivative method is simple, less time consuming, no manual calculation, and gives marginally better result than Q analysis method.
Validation of methods
The methods were validated with respects to linearity, limit of detection (LOD), limit of quantification, precision, accuracy, and selectivity/sensitivity.
For linearity, the calibration plots for each method were constructed after analysis of different concentration and each concentration was measured for six times. The regression equation and correlation coefficients of the mean of six consecutive calibration curves are given in [Table 3].
LOD (k = 3.3) and Limit of quantitation (LOQ) (k = 10) of the methods were established according to ICH definitions (C _{1} = k S _{0} /s), where C _{1} is LOD or LOQ, S _{0} is the standard error of blank determination, s is the slope of the standard curve and k is the constant related to the confidence interval). The LOD, LOQ, and standard error of the methods are given in [Table 3].
Accuracy was investigated by analyzing three different concentration of binary mixture of LSP and HZ in linear range in six independent replicates. The data evaluated using equations are summarized in [Table 4]. Accuracy was expressed as bias (%). The bias values were close to zero [Table 4]. The relative standard deviation (RSD) values and also the low RSD values obtained from the analysis of pharmaceutical formulations indicated that the intermediate precision of the method was good.  Table 4: Precision and accuracy of spectrophotometric method developed for analysis of tablet (n=6)
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Conclusion   
The proposed method based on simultaneous equation, graphical absorbance ratio and first order derivative methods can be used for the simultaneous determination of LSP and HZ either in their binary mixture form or alone in their tablet preparation. The proposed methods are precise, accurate, and simple to perform. Also, no separation step is required. Hence the proposed methods can be used for the routine analysis of LSP and HZ.
Acknowledgment   
The authors are thankful to Roland institute of pharmaceutical sciences, Berhampur, Odisha, for providing necessary facilities.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]
