Development and Validation of Novel UV Methods for Irbesartan and Hydrochlorothiazide Combination

 

A.R. Chabukswar¹*, S.N. Shinde¹, B.S. Kuchekar¹, S.C. Jagdale¹, P.D. Lokhande², V.P. Choudhari¹ and K.D. Ingale¹

¹MAEER’S Maharashtra Institute of Pharmacy, S. No. 124, MIT Campus, Paud Road, Kothrud, Pune-411038

²Department of Chemistry, University of Pune, Ganeshkhind road, Pune 411 007

*Corresponding Author E-mail: anigen18@rediffmail.com

ABSTRACT:

Simultaneous determination of Irbesartan (IRBE) and Hydrochlorothiazide (HTZ) in combined tablet dosage form has been developed by ratio spectra derivative and absorption correction spectrophotometry method.  The amplitudes at 252.6 nm and 230 nm in the first derivative of the ratio spectra method were selected to determine IRBE and HTZ respectively and the second method was based on the absorption correction method in which IRBE and HTZ exhibit λ_max at 244 nm and 275.02 nm, respectively in 0.1N HCL. HTZ has some interference due to IRBE at 275.02 nm, while HTZ do not show any absorption at 244 nm. The % assay for commercial formulations was found to be in the range 98.29-99.80 for IRBE and 98.91 – 100.10 % for HTZ by the proposed methods, for both the formulations. Recovery was found in the range of 98.11 – 100.08 % for IRBE and 98.54 – 99.29% for HTZ by ratio derivative method and 98.59 – 99.56% for IRBE and 99.33-100.12% for HTZ by absorbance correction method for both the Formulations. The methods were validated and found to be linear, precise and accurate, as per ICH guidelines. Absorbance correction method was applied for dissolution studies and drug release pattern was evaluated.

 

 

 

INTRODUCTION:

Irbesartan (IRBE) is an orally active specific angiotensin II receptor antagonist used, as a hypotensive agent and does not require biotransformation into an active form. Chemically it is 2-butyl-3-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]-1,3-diazaspiro[4.4]non-1-en-4-one. Hydrochlorothiazide (HTZ) is a diuretic. Chemically it is (6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide¹. Literature survey shows that many methods are available for spectrophotometric determination of irbesartan and hydrochlorothiazide ^2-5 and chromatographic methods have been studied ^6-10 either alone and or in combination with other drugs in formulations as well as by bioanalytical techniques.

 

Literature survey reveals that, absorption correction and ratio derivative spectroscopic methods have not been studied for the simultaneous estimation of IRBE and HTZ in combined dosage form.

 

Hence the aim of this study was the development and validation of ratio derivative and absorbance correction spectroscopic methods for the determination of IRBE and HTZ in tablet dosage form and application of absorbance correction method for Dissolution study. The proposed method was optimized and validated as per the International Conference on Harmonization (ICH) guidelines¹¹.

 

IRBESARTAN

 

HYDROCHLOROTHIAZIDE

 

MATERIAL AND METHODS:

Instrumentation:

An UV-Visible double beam spectrophotometer (Varian Cary 100) with 10 mm matched quartz cells was used. Weighing of chemicals was done on electronic balance (Model Shimadzu AUW-220D). Electrolab tablet dissolution tester (Model no:TDT-08L) used for dissolution study.

 

Reagents and chemicals:

Pure drug sample of IRBE (% purity 99.75) and HTZ(% purity 99.93) was kindly supplied as a gift sample by Cipla Pvt. Ltd. Mumbai. These samples were used without further purification. Spectroscopy grade methanol and analytical reagent grade HCl was used throughout the study. Tablets each containing 150 mg of IRBE and 12.5 mg HTZ used for analysis were IROVEL-H (Formulation I) and XARB-H (Formulation II) manufactured by Sun Pharma Sikkim, India and Piramal Health care, India , respectively.

 

Preparation of Standard Stock Solutions and calibration Curve:

Standard stock solutions of pure drug containing 1000 μg mL^-1 of IRBE and HTZ were prepared separately in methanol and 0.1 N HCL for ratio derivative method              (Method A) and absorption correction method ( Method B)  respectively. The working standard solutions of these drugs were obtained by dilution of the respective stock solution in methanol and 0.1 N HCL respectively.

Derivative amplitudes of ratio spectra and concentrations were used to construct calibration curve for method A.

 

Preparation of Sample Stock Solution and Formulation analysis:

Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 100  mg of IRBE (8.33 mg of HTZ) was weighed and dissolved in the 80 mL of methanol (0.1 N HCL for method B) with the aid of ultrasonication for 15 min and solution was filtered through Whatman paper No. 41 into a 100 mL volumetric flask. Filter paper was washed with methanol, adding washings to the volumetric flask and volume was made up to the mark with methanol (0.1 N HCL for method B). The solution was suitably diluted further to get required final concentration of IRBE (100μg mL^-1) and HTZ (8.33 µg mL^-1).

 

Method A: Ratio Derivative:

The method involves dividing the spectrum of mixture by the standardized spectra of each of the analyte and deriving the ratio to obtain spectrum that is dependent of concentration of analyte used as a divisor (Fig. 1). Using appropriate dilutions of standard stock solution, the two solutions were scanned separately. The ratio spectra of different IRBE standards at increasing concentrations were obtained by dividing each with the stored spectrum of the standard solution of HTZ (2.49 μg mL^-1) as shown in (Fig 2).Wavelength 252.6 nm was selected for the quantification of IRBE in IRBE + HTZ mixture. The ratio and ratio derivative spectra of the solutions of HTZ at different concentrations were obtained by dividing each with the stored standard spectrum of the IRBE (30 μg mL^-1) as shown in (Fig 3).Wavelength 230.0 nm was selected for the quantification of HTZ in IRBE  +  HTZ  mixture. Measured analytical signals at these wavelengths were proportional to the concentrations of the drugs. Calibration curves were prepared from the measured signals at the selected wavelength and concentration of the standard solutions. The amount of IRBE and HTZ in tablets was calculated by using following equations-

At 252.6 nm:         C_IRBE = (Ratio derivative amplitude for IRBE- 2.545)/ 2.830.... (1)

At 230.0 nm: C_HTZ = (Ratio derivative amplitude for HTZ - 0.0224)/ 0.9006.… (2)

 

Fig 1: Spectrum of mixture of IRBE and HTZ for ratio derivative method.

 

Fig 2:  First derivative of Ratio spectra of (a) 10,(b) 20, (c) 30, (d) 40, (e) 50 µg mL^-1 of IRBE when 2.49 µg mL^-1 of HTZ is used as divisor.

 

Fig 3:  First derivative of Ratio spectra of (1) 0.83, (2) 1.66, (3) 2.49, (4) 3.32, (5) 4.16 µg mL^-1of  HTZ when 30 µg mL^-1 of IRBE is used as divisor

 

Method B: Absorption Correction:

λmax of IRBE and HTZ was determined by scanning the drug solution in the solvent was found to be at 244 nm and 275.02 nm respectively. IRBE also showed absorbance at 275.02 nm, while HTZ did not show any interference at 244 nm. To construct Beer’s plot for IRBE and HTZ dilutions were made in the solvent using stock solution of 100 µg mL^-1.

Table 1: Optical characteristics of the proposed methods

Parameter				Irbesartan		Hydrochlorothiazide
				Method A	Method B	Method A	Method B
λ (nm)				252.6	244	230	275.02
Beer’s law limit (μg mL-1)				10-50	10-50	0.83-4.16	0.83-4.16
Regression Equation (y = mx + c)			Slope (m)	4.896	0.04869	0.9006	0.0646
			Intercept (c)	8.879	0.07483	0.0224	0.01381
Correlation coefficient				0.9998	0.9997	0.9995	0.9991
Precision (%R.S.D.)	Repeatability (n=5)			0.64	0.54	0.79	0.92
	Intraday(3x3Times)			1.21	0.69	0.78	1.23
	Interday(3x3Days)			1.15	1.03	0.93	1.42
Formulation Analysis(% Assay, %RSD) n=6		Formulation-1		99.21,  0.46	99.80,0.32	98.91,0.51	100.10,0.30
		Formulation-2		98.29,0.84	99.34,0.67	99.25,1.21	99.56,0.63

1. RSD : Relative Standard Deviation.

2. y = mx + c, where y is the Absorbance and x the Concentration in micrograms per milliliter.

 

Also Beer’s plot was constructed for IRBE and HTZ in solution mixture at different concentration (10:0.83, 20:1.66, 30:2.49, 40:3.33, 50:4.16µg mL^-1) levels. Both the drugs followed linearity individually and in mixture within the concentration range 10-50µg ml^-1 and 0.83-4.16µg mL^-1 for IRBE and HTZ respectively (Fig .4).

 

Determination of Absorption Factor at Selected Wavelengths:

IRBE and HTZ solution in the solvent of known concentrations were scanned against blank on spectrophotometer. The value of absorption factor was found to be 1.152. Quantitative estimation of IRBE and HTZ was carried out using following equation:

Corrected Absorbance of HTZ at 275.02nm =

Abs_275.02 (IRBE+HTZ) – [(abs_275.02 (HTZ)/ abs₂₄₄ (IRBE)] *abs₂₄₄ (IRBE)

Where; abs: Absorption value at given wavelengths.

 

Fig  4: Overlain spectra of IRBE: (1) 10µg/mL;(2) 20 µg/mL; (3) 30 µg/mL;(4) 40 µg/mL; (5) 50 µg/mL; and HTZ (A) 0.83 µg/mL;(B)1.66 µg/mL; (C) 2.49 µg/mL;(D)3.33 µg/mL;(E)4.16 µg/mL in methanol.

 

Recovery studies

The accuracy of the proposed method was checked by recovery study, by addition of standard drug solution to preanalysed sample solution at three different concentration levels (50 %, 100 % and 150 %) within the range of linearity for both the drugs. The basic concentration level of sample solution selected for spiking of the drugs standard solution was 15 μg mL^-1 of IRBE and 1.245 μg mL^-1 of HTZ.

 

Solution Stability

Method stability was checked by analyzing solution kept in fridge and at room temperature by both methods. Solution at room temperature was stable for 12 and 24 hours as tested by method A and method B, respectively (% RSD < 2). Solution in fridge was stable for 10 days and 15 days as tested by method A and method B, respectively (% RSD < 2).

 

Precision of the Method:

Method repeatability was determined by six times repetition of assay procedure. For intra-day precision method was repeated 5 times in a day and the average % RSD was determined. Similarly the method was repeated on five different days for inter-day precision and average % RSD was determined (Table 1).

 

Dissolution study

The dissolution study was carried out for irbesartan and hydrochlorothiazide and was validated. A calibrated dissolution apparatus (USP II) paddle 50rpm & bath temp at 37±1^oc 900ml. Nine hundred millilitres freshly prepared and degassed 0.1N HCL solution was used as the dissolution medium. Six tablets were evaluated and dissolution sample were collected at 5, 10, 15, 25, 35, 40 and 45 min interval. At each time point, a 5 mL sample was removed from each vessel sample, filtered through a nylon filter (0.45μm, 25 mm), 2.0 mL of filtrate was diluted to5 mL with 0.1 N HCL and analyzed by Absorbance correction method, percentage release of IRBE and HTZ was calculated by using equations 5 and 6, respectively.

 

IRBE % release = (C_IRBE × 900×2.5×100)/ (1000×150) …. (5)

HTZ % release = (CHTZ × 900×2.5×100)/ (1000×12.5) …. (6)

 

RESULTS AND DISCUSSION:

Irbesartan and Hydrochlorothiazide combination is useful for the patients suffering from blood pressure related complications. The methods developed in the present work have been found to be significant due to its simplicity and novelty. Since λmax of both the drugs differ more than 20 nm, absorption corrected method was selected for their simultaneous estimation in formulation.

Table 2. Recovery Studies of IRBE and HTZ by Ratio Derivative (Method A) and Absorbance Correction Method (Method B).

Formulation studied	Recovery Level	Recovery of	Amount Spiked (µg/mL)	% Mean  Recovery,  % RSD by  n=6
				Method A	Method B
Formulation I	50%	IRBE	7.5	99.39,  1.03	99.71    0.83
		HTZ	0.62	98.83,  0.56	99.49,   0.47
	100%	IRBE	15	100.08, 0.64	99.24,   1.34
		HTZ	1.245	98.80,  0.41	100.12,  1.28
	150%	IRBE	22.5	99.41,  0.51	98.59,    0.52
		HTZ	1.86	98.54,  0.95	99.33,    0.34
Formulation II	50%	IRBE	7.5	98.53,  1.28	99.22,    0.65
		HTZ	0.62	99.29,  0.49	100.02,  1.05
	100%	IRBE	15	99.46,  0.89	99.56,    0.93
		HTZ	1.245	99.13,  0.72	100.10,  1.16
	150%	IRBE	22.5	98.11,  0.51	99.36,    0.44
		HTZ	1.86	99.04,  1.15	99.38,    0.84

 

 

Fig 5:  Dissolution profile of IRBE and HTZ tablet formulation by Absorption Correction  method.

 

Quantitative estimation of HTZ was carried out by subtracting interference of IRBE using experimentally calculated absorption factor. The values of % RSD and correlation of coefficient were satisfactory and results of the formulation analysis are shown in Table 1 and result of the recovery study in Table 2. Fig 1 represents the UV spectrum of IRBE and HTZ for ratio derivative method. Fig 2 and 3 represents the first derivative ratio spectra of both the drugs at different concentrations. Fig 4 shows the overlain spectra of the drugs at the given wavelengths. The data indicates that there is no interference due to excipients present in the formulation. Beer’s law is obeyed in the concentration range of 10-50 μg mL^-1and 0.83-4.16 μg mL^-1 for IRBE and HTZ for both the ratio derivative and absorbance correction method. The correlation of coefficient was found in the range of 0.9991 to 0.9998 for both the drugs. Statistical analysis shows that the data obtained is significant and meaningful to analyze the drugs in combination. The developed method can be easily and conveniently adopted for routine quality control analysis. Absorbance ratio method has been used for studying dissolution profile of the drug release pattern and method has been found to be useful as 92.03% drug release has been determined for HTZ and 91.49% for IRBE as shown in Fig 5.

 

CONCLUSION:

The developed method can be used as IPQC test and for routine simultaneous determination of IRBE and HTZ in tablet dosage form. Ratio derivative method can be extended to determine the analytes in biological samples. The absorption correction method is also successfully applied for Dissolution Study and drug release can be determined accurately. Both the methods are accurate, simple, rapid, precise, reliable, sensitive, reproducible and economical as per ICH guidelines.

 

ACKNOWLEDGEMENT:

The authors wish to express their gratitude Cipla Ltd. Mumbai, India, for the sample of pure Irbesartan and Hydrochlorothiazide. The authors are also thankful to the Management of MAEER’s Maharashtra Institute of Pharmacy for providing necessary facilities.

 

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Received on 31.03.2010        Modified on 12.04.2010

Accepted on 22.05.2010        © AJRC All right reserved