Simultaneous Determination of Carvedilol and Hydrochlorothiazide in Pharmaceutical Dosage Form by Second Order Derivative UV Spectrophotometry

Audumbar Digambar Mali*

Department of Pharmaceutics, Sahyadri College of Pharmacy, Methwade,

Sangola-413307, Solapur, Maharashtra, India.

*Corresponding Author E-mail: maliaudu442@gmail.com

ABSTRACT:

Derivative spectrophotometry offers a useful approach for the analysis of drugs in multi-component formulation. In this study a second order derivative spectrophotometric method is applied for the simultaneous determination of Carvedilol and Hydrochlorothiazide in tablet dosage form. The measurements were carried out at wavelengths of 292 and 272 nm for Carvedilol and Hydrochlorothiazide respectively. The method was found to be linear (r²=0.999) in the range of 5-25 μg/ml for Carvedilol in the presence of 20 μg/ml of Hydrochlorothiazide at 292 nm. The linear correlation (r²=0.999) was obtained in the range of 5-25 μg/ml for Hydrochlorothiazide in the presence of 20 μg/ml of Carvedilol at 272 nm. The method was successfully used for simultaneous determination of Carvedilol and Hydrochlorothiazide in tablet dosage form without any interference from excipients and prior separation.

KEYWORDS: Carvedilol, Hydrochlorothiazide, UV visible spectrophotometry, Method Validation, Accuracy, Pricission, Second order derivative method.

INTRODUCTION:

Carvedilol is a combined alpha- and nonselective betablocker. Carvedilol chemically, 2-Propanol, 1-(9H-carbazol- 4-yloxy)-3-[[2-(2- methoxy phenoxy) ethyl amino]-, (±)-; (±)-1-(Carbazol-4-yloxy)-3-[[2-(o-methoxy phenoxy) ethyl] amino]-2-propanol^. It is a non-selective beta blocker indicated in the treatment of mild to moderate congestive heart failure (CHF). [1] It blocks beta-1 and beta-2 adrenergic receptors as well as the alpha-1 adrenergic receptors_. Carvedilol is official drug in British Pharmacopoeia. It has been prescribed as an antihypertensive agent and an angina agent.[2]

It is first beta blocker labeled in United States especially for the treatment of heart failure of ischemic or cardiomyopathic origin with significant antioxidant activity. Relative to other beta blocker, carvedilol (CAR) has minimal inverse agonist indicating a reduced negative chronotropic and inotropic effect, which decreases its potential to worsen symptoms of heart failure. At high dosage, it exerts Calcium channel blocking activity. [3,4] The benefits of using CAR in patient with CHF in both single center and multicenter trial have been reported in the literature .It prevents vitamin E, glutathione and SH protein depletion induced by oxidation stress, the main defense mechanism against tissue injury caused by free radical. Literature survey revealed the estimation methods of Carvedilol or with other drugs by UV spectrophotometry [5], HPLC [6], calorimetric method, flow injection analysis, and HPTLC.

Hydrochlorothiazide chemically known as 6-chloro-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine-7-sulphonamide-1,1-dioxide is a moderately potent thiazide diuretic. [7, 8] It exerts its effect by reducing the reabsorption of electrolytes from the renal tubules, thereby increasing the excretion of sodium and chloride ions, and consequently of water. Hydrochlorothiazide is used in the treatment of hypertension either alone or with other antihypertensives. Literature survey revealed the estimation methods of Hydrochlorothiazide or with other drugs by UV spectrophotometry [6, 7] HPLC [8] calorimetric method, flow injection analysis and HPTLC.

Application of derivative technique of spectrophotometry offers a powerful tool for quantitative analysis of multi-component mixtures. When derivatised, the maxima and minima of the original function take zero values, and the inflections are converted into maxima or minima, respectively. The derivative curves are more structured than the original spectra, thus enabling very tiny differences between the original spectra to be identified. Derivative spectrophotometry provides selectivity and offers a solution in resolving the overlapping spectra in multi-component analysis without previous chemical separation. In the last decades, this technique has rapidly gained application in the field of pharmaceutical analysis to overcome the problem of interference, due to substances other than analytes, commonly present in pharmaceutical formulations or for combination of two or more drug substances. Lack of any published method for simultaneous spectrophotometric determination of Carvedilol and Hydrochlorothiazide, therefore, provoked us to investigate the application of derivative spectrophotometry for simultaneous determination of these compounds in pharmaceutical dosage forms using zero-crossing method.

Fig. 1: Chemical structure of Carvedilol

Fig. 2: Chemical structure of Hydrochlorothiazide

MATERIALS AND METHODS:

Apparatus and instrumentation: -

A Shimadzu 1800 UV/VIS double beam spectrophotometer with 1cm matched quartz cells was used for all spectral measurements. Single Pan Electronic balance (CONTECH, CA 223, India) was used for weighing purpose. Sonication of the solutions was carried out using an Ultrasonic Cleaning Bath (Spectra lab UCB 40, India).Calibrated volumetric glassware (Borosil®) was used for the validation study.

Materials:

Reference standard of Carvedilol and Hydrochlorothiazide API was supplied as gift sample by Lupin Laboratory park Aurangabad, Maharashtra, India. The commercial formulation Co-Dilatrol^® as purchased from the local market Solapur, Maharashtra, India.

Method development:

Preparation of standard stock solution: -

Stock solution was prepared by diluting 10 mg of each drug in sufficient quantity of methanol in separate volumetric flask and volume was made up to 100 ml to get the concentrations of 100 μg/ml for each drug. Dilutions from stock solution were prepared in the range of 5-25 μg/ml for Carvedilol and 5-25 μg/ml for Hydrochlorothiazide. Methanol was used as a blank solution.

Spectrophotometric Measurements:

Zero-order spectra of standard solutions of Carvedilol (20μg/ml) and Hydrochlorothiazide (20 μg/ml) versus their solvent blank were recorded in the range of 200-400 nm (Figure 3). The second order derivative spectra of these solutions were obtained in the same range of wavelength against their blanks (Figure 4). The values of first order derivative amplitudes for Carvedilol in the presence of Hydrochlorothiazide and vice versa were measured at 292 nm (zero-crossing of Carvedilol) and 272 nm (zero-crossing of Hydrochlorothiazide), respectively. The calibration curves for derivative spectrophotometry were constructed by plotting the drug concentration versus the absorbance values of the second order derivative spectrum, at 292 nm for Carvedilol and at 272 nm for Hydrochlorothiazide.

Analysis of commercial tablet formulation:

Contents of 20 tablets were weighed and their average weight was determined and powdered. Accurately weighed powder equivalent to fill weight of one tablet was transferred to 100 ml calibrated flask containing 50 ml of methanol and sonicated for 30 minutes. The volume was then made up to the mark with methanol. The resulting solution was then filtered through Whatman filter paper (#41). From this solution, 1 ml was transferred to another 10 ml calibrated flask and diluted up to 10 ml which gives 200 μg/ml concentration of solution. Then 1 ml of this solution was further diluted to 10 ml to get approximate concentration 20 μg/ml of Carvedilol and 20 μg/ml of Hydrochlorothiazide.

Fig. 3: Zero order spectra (overlain) of Carvedilol 20 μg/ml (A) and Hydrochlorothiazide 20 μg/ml (B)

Fig. 4: Secon order derivative spectra (overlain) of Carvedilol 20 μg/ml (A) and Hydrochlorothiazide 20 μg/ml (B)

Table 1: Assay of tablet dosage form.

Sr.No.	Sample Solution Concentration (µg/ml)	Amount found (%)*	Mean % found	%RSD
1	20	100.19
2	20	98.10	100.29	0.4692
3	20	102.58

*n=3, % RSD = % Relative Standard Deviation.

RESULTS AND DISCUSSION:

Linearity and Range:

Linearity:

Calibration curves were constructed using six replicates of Carvedilol solutions between 5-25 μg/ml in the presence of 5-25 μg/ml of Hydrochlorothiazide. The same procedure was used for solutions containing Hydrochlorothiazide 5-25 μg/ml in the presence of 5-25 μg/ml of Carvedilol. The calibration curves were constructed (Fig. 5 and Fig. 6) and statistical analysis was performed. The regression equations of calibration curves were y=0.017x+0.001 (r²=0.9997) at 292 nm for Carvedilol and y=0.016x+0.005 (r²=0.9994) at 272 nm for Hydrochlorothiazide for second order derivative spectrophotometry methods. The range was found to be 5-25 μg/ml for both drugs for second order spectrophotometry methods. [9, 10]

Fig.5 Calibration curve for Carvedilol at 301 nm

Fig.6 Calibration curve for Hydrochlorothiazide at 278 nm

Fig.7 Second order derivative overlay of Carvedilol and Hydrochlorothiazide at 5, 10, 15, 20 and 25 μg/ml Concentrations

Table 2: Stastical data for the calibration graphs for determination of Carvedilol and Hydrochlorothiazide by Proposed methods.

Parameters	Carvedilol	Hydrochlorothiazide
Linearity range (µg/ml)*	5-25	5-25
r^2± S.D*	0.999	0.999

Accuracy:

For accuracy determination, the analysed samples were spiked with extra 80%, 100% and 120% of the standard solution of both drugs and the mixtures were reanalysed by the proposed method. The experiment was conducted in triplicate. This was done to check for the recovery of the drug at different levels in the commercial tablet formulations. The mean recoveries and % RSD are illustrated in Table 3. The data indicates that the proposed derivative spectrophotometric method is highly reproducible during one run and between different runs. [9, 10]

Table 3: Results of drug content and analytical recovery of Carvedilol and Hydrochlorothiazide

Parameters	Carvedilol	% R.S.D	Hydrochlorothiazide	% R.S.D
Labelled claim	25 mg	-	12.5 mg	-
% Drug content ± S.D	100.47 ± 0.9851	0.85	100.09 ± 0.2981	0.82
Analytical recovery at 80 % ± S.D	100.62 ± 0.8720	0.58	100.82 ± 0.5234	0.53
Analytical recovery at 100 % ± S.D	101.13 ± 0.8529	0.81	99.26 ± 0.2362	0.36
Analytical recovery at 120% ± S.D	98.28 ± 0.8713	0.89	102.23 ± 0.2697	0.67

Precision:

To determine the precision of the method, Carvedilol and Hydrochlorothiazide solutions at a concentration of 20μg/ml were analysed each three times for second order spectrophotometric method. Solutions for the standard curves were prepared fresh everyday. [9, 10]

Table 4: Results of Intra and Inter Day Precision

Parameters	Intra Day Precision		Inter Day Precision
	S.D*	% RSD*	S.D*	% RSD*
Carvedilol	0.0072	0.5329	0.0083	0.5417
Hydrochlorothiazide	0.0073	0.5361	0.0087	0.5453

Sensitivity:

The limit of detection (LOD) and limit of quantification (LOQ) were calculated by using the equations LOD = 3xσ/ S and LOQ = 10xσ/S, where σ is the standard deviation of intercept, S is the slope. The LOD and LOQ were found to be 0.8753μg/ml and 2.6261μg/ml respectively of Carvedilol for second order derivative and 0.8564μg/ml and 2.5697μg/ml respectively of Hydrochlorothiazide for second order derivative. [9]

Analysis of the Marketed Formulation:

There was no interference from the excipients commonly present in the tablets. The drug content was found to be 100.29% second order spectrophotometric methods. It may therefore be inferred that degradation of Carvedilol and Hydrochlorothiazide had not occurred in the marketed formulations that were analysed by this method. The low % R.S.D. value indicated the suitability of this method for routine analysis of Carvedilol and Hydrochlorothiazide in pharmaceutical dosage form.

Table 5: Summary of validation parameters

Parameter	Carvedilol	Hydrochlorothiazide
λ range	200-400 nm	200-400nm
Regression Equation (y=mx+c)	Y=0.022x+0.006	Y=0.024x+0.006
Measured wavelength	292 nm	272 nm
Linearity range	5-25µg/ml	5-25µg/ml
Slope	0.022	0.024
Intercept	0.006	0.006
Correlation coefficient (R^2)	0.999	0.999
Limit of Detection (LOD) µg/ml	0.8753	0.8564
Limit of Quantitation (LOQ) µg/ml	2.6261	2.5697
Accuracy (Mean % Recovery)	101.47	100.09
Precission (%RSD)	0.85	0.82

CONCLUSION:

From the results of this study it can be concluded that the proposed second order derivative spectrophotometric method can be used for simultaneous determination of Carvedilol and Hydrochlorothiazide. This method is simple, rapid, practical, reliable and inexpensive and can be used for routine analysis of simultaneous determination of these compounds without any prior separation in quality control laboratories.

ACKNOWLEDGEMENT:

The authors are highly thankful to the Sahyadri College of Pharmacy, Methwade, Sangola, Solapur, Maharashtra, India for proving all the facilities to carry out the research work successfully.

REFERENCES:

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Received on 24.06.2015 Accepted on 25.08.2015

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 5(3): July- Sept. 2015; Page 133-138

DOI: 10.5958/2231-5675.2015.00021.6