INTRODUCTION:

Amlodipine (a dihydropyridine) is a calcium-channel blocker. It is given as besylate and used in the management of hypertension and angina pectoris. It is chemically 3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methyl pyridine-3,5-dicarboxylate mono benzene sulphonate. It is official in BP¹. Bisoprolol is a cardio selective beta-blocker. It is given as the fumarate in the management of hypertension and angina pectoris. Chemically it is (±)-1-[4-[[2-(1-methylethoxy) ethoxy] methyl] phenoxyl-3-[(1-methyl ethyl) amino]-2-propanol. It is official in USP².

Literature survey reveals the availability of several methods for estimation of amlodipine besylate (AMLO) includes UV^3-5, HPLC^6-8, HPTLC⁹ as alone or in combination with other drugs. Bisoprolol fumarate (BISO) can be determined by UV¹⁰, HPLC¹¹, HPTLC¹² in combination with hydrochlorothiazide and by RP-HPLC¹³as alone is reported. No method has been reported for the estimation of AMLO and BISO in combined dosage form. Present work emphasizes on the quantitative estimation of AMLO and BISO in their combined dosage form by UV visible spectrophotometric method.

MATERIAL AND METHODS:

Instrument

UV visible double beam spectrophotometer, Shimadzu UV- 2401PC with matched pair quartz cells corresponding to 1 cm path length.

Chemicals

All chemicals and reagents used were of analytical grade and purchased from Merck Chemicals, India.

Preparation of stock solution

AMLO (Amlodipine besylate equivalent to 50 mg of amlodipine) and BISO (50 mg) were accurately weighed and transferred to two separate 50 ml volumetric flasks, dissolved in methanol to obtain stock solution of concentration 1 mg/ml each.

Selection of λ_max

From these stock solutions, working standard solutions were prepared by appropriate dilution of aliquot portions with distilled water to get final concentration of 10 µg/mleach and were scanned in the wavelength range of 200-400 nm to determine the λ_max. AMLO shows λ_maxat 365 nm while BISO at 222 nm, respectively (Figure 1).

Preparation of standard solution

From the standard stock solution 5 ml each of AMLO and BISO was taken in 100 ml volumetric flask. Volume was made upto the mark with distilled water. Aliquot portion was appropriately diluted with distilled water to get final concentration of 10 µg/ml.

Figure 1: Overlain spectra of AMLO and BISO

Preparation sample solution

Twenty tablets were weighed and finely powdered. An accurately weighed tablet powder equivalent to 5 mg of amlodipine was transferred to 100 ml volumetric flask. Ten milliliter of methanol was then added and contents were sonicated for 15 min. The volume was made upto the mark with distilled water. The resulting solution was filtered through Whatmann filter paper No. 1. Aliquot portion was appropriately diluted with distilled water to get final concentration of 10 µg/ml.

The absorbances of standard and sample solutions were measured at 222 and 365 nm using solvent blank. The results were calculated by the formula¹⁴,

Where, A₁ and A₂ are absorbance of diluted mixture at 222 and 365 nm respectively, c_xand c_y are the concentration of AMLO and BISO respectively (g/100 ml), ax₁ and ax₂ are absorptivities of BISO at 222 and 365 nm respectively, ay₁ and ay₂ are absorptivities of AMLO at 222 and 365 nm respectively.

Validation of Method¹⁵

The method was validated in terms of linearity, accuracy, precision and specificity of the sample applications. The linearity of the method was investigated by serially diluting the stock solutions of AMLO, BISO and measured the absorbance at 222 and 365 nm. Calibration curves were constructed by plotting the absorbance against the concentration. Both the drugs show linearity in the concentration range from 5-100 µg/ml with correlation coefficient of 0.999.

Recovery studies were carried out to study the accuracy of the proposed method and ascertained by standard addition method. A known amount of drug was added to preanalysed tablet powder, at three levels and the percentage recoveries were calculated. Precision was found to be lower than 2 %. Ruggedness of the proposed method is determined by analysis of aliquots from homogenous slot by different analysts using similar operational and environmental conditions. Specificity study was performed by keeping the sample under various stressed conditions as at 60^oC and at 50^oC by adding 1 ml of 0.1N HCl, 0.1N NaOH, 3% H₂O₂, exposing with UV light at 265 nm.

RESULTS AND DISCUSSION:

Tablets were analyzed and amount of drug determined by proposed method was in good agreement with the labeled claim. The results of the marketed formulations were found to be 99.45±0.142 and 100.58±0.269 for AMLO and BISO respectively. The proposed method was validated as per the ICH guidelines. Linearity was determined at different concentration, AMLO and BISO both shows linearity in the concentration range of 5-100 µg/ml with correlation coefficient of 0.999. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by standard deviation of response and slope of calibration curve. LOD and LOQ were found to be 4.31, 13.07 for AMLO and 1.45, 4.42 for BISO respectively. System reproducibility was determined by five replicate applications and five times measurement of a laboratory mixture at the analytical concentration. The reproducibility of sample was expressed in terms of S.D. and % R.S.D. There was no interference from the common excipients present in tablets. The recovery of drug was determined at 80, 100 and 120 % levels. The percent recovery was from 99.33 to 99.61 for AMLO and 100.28 to 100.80 for BISO indicating that method has required accuracy. Specificity studies results reveal that both the drugs degraded in presence of peroxide. Ruggedness was performed under three different conditions different days, different analysts and intraday. The results show the % RSD values < 2.0% signifies the precision of the method. The proposed method for simultaneous estimation of AMLO and BISO in combined dosage form was found to be simple, accurate and rapid and can be employed for estimation of pharmaceutical formulations in quality control departments.

ACKNOWLEDGEMENT:

The authors are thankful to the Head of Department, Department of Pharmaceutical Sciences, R.T.M. Nagpur University, Nagpur for providing laboratory facilities. The authors are also thankful to Ranbaxy Laboratories Ltd. and Merck Ltd. for gift samples of amlodipine besylate and bisoprolol fumarate respectively.

Table 1: Results of marketed formulation, recovery and intermediate precision

Drugs

Parameters

% Labeled claim

% Recovery*

Intermediate Precision**

Interday

Intraday

Different analyst

AMLO

Mean

± SD

% RSD

99.39

1.210

1.217

99.45

0.142

99.20

0.485

0.488

99.37

0.525

0.528

99.43

0.500

0.502

BISO

Mean

± SD

% RSD

101.55

0.227

0.223

100.58

0.269

0.267

101.02

0.813

0.804

99.34

0.717

0.721

100.10

0.493.

0.492

*Mean

REFERENCES:

1. British Pharmacopoeia Vol. 1, Department of Health Social Services and Public Safety, H.M. Stationary Office London, 2004;126.

2. United States Pharmacopoeia, 24^th edition, United States Pharmacopoeial Convention, Inc., Rockville, MD., 2000;268.

3. Gohil K. et al. Spectrophotometric analysis of amlodipine besylate in bulk and in tablet dosage forms. Indian J. Pharm. Sci., 2005; 67(3):376.

4. Rahman N. and Md. Nurul Hoda. Validated spectrophotometric methods for the determination of amlodipine besylate in drug formulations using 2,3-dichloro 5,6-dicyano 1,4-benzoquinone and ascorbic acid J.Pharm. Biomed. Anal., 2003; 31: 381-392.

5. Rahman N. and Azmi S. N. H. Spectrophotometric method for the determination of amlodipine besylate with ninhydrin in drug formulations Farmaco, 2001; 56: 731-735.

6. Zarapkar S.S. and Kanyawar N.S. Simultaneous estimation of amlodipine and losartan potassium in pharmaceutical dosage by reverse phase high performance liquid chromatography. Indian Drugs, 2002; 39(6):338-341.

7. Rao J.R. et al. Methods of estimation of multi-component formulations: a review. Indian Drugs, 2002; 39(7): 378-381.

8. Zarghi A. et al. Validated HPLC method for determination of amlodipine in human plasma and its application to pharmacokinetic studies. Farmaco, 2005; 60: 789-792.

9. Josefsson M. et al. Sensitive high-performance liquid chromatographic analysis of amlodipine in human plasma with amperometric detection and a single-step solid-phase sample preparation. J. Chromatogr B: Biomed Appl., 1995; 672: 310-313.

10. Patel L.J. et al. HPTLC method for the simultaneous estimation of bisoprolol fumarate and hydrochlorothiazide in tablet dosage form. Indian Drugs, 2006; 43(8): 630-635.

11. Patel L.J. et al. Simultaneous estimation of bisoprolol fumarate and hydrochlorothiazide in tablet dosage form by RP-HPLC method. Indian J. Pharma. Sci., 2006; 68(5): 635-638.

12. Sahu R. and Patel V.B. Simultaneous spectrophotometric estimation of hydrochlorothiazide and bisoprolol fumarate in combined dosage forms.. Indian J. Pharma. Sci, 2006; 68(6):764-767.

13. Clarice M.B.R. et al. Development and validation of an RP-HPLC method for the dissolution studies of bisoprolol in pharmaceutical dosage form. J. Liq. Chromatogr. and Rel.Tech., 2005; 28: 477-486.

14. Beckett A. H. and Stenlake. T. B., Practical pharmaceutical chemistry, 4^thedition, part II, CBS Publisher and Distributor, New Delhi, 1997; 277-280.

15. PART II: Validation of analytical procedure: methodology Q2B, ICH Harmonized Tripartite Guidelines, 1996; 6-12.

Received on 19.12.2008 Modified on 22.12.2008

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008; Page 513-515