INTRODUCTION:

Amlodipine besylate (AMB), 2 ‐ [(2 ‐ amino ethoxy) ‐ methyl] ‐ 4 ‐ (2– chloro phenyl) ‐1, 4 ‐dihydro ‐ 6 ‐ methyl ‐ 3, 5 ‐ pyridine dicarboxylic acid 3 ‐ ethyl ‐ 5 ‐ methyl ester, benzene sulfonate is a potent dihydro calcium channel blocker¹. Various analytical methods have been reported for the assay of AMB alone or in combination with other anti ‐ hypertensive agents in pharmaceutical formulations. They include UV spectroscopy²^‐⁴, high performance liquid chromatography⁵^‐⁸, high performance thin layer chromatography^9-10, LC ‐ MS¹¹ and LC ‐ MS/ MS¹².AMB is a calcium antagonist that inhibits the trans membrane influx of calcium ions into vascular smooth muscles and cardiac muscles, which in turn affects their contractile process and results in reduced blood pressure. It is used in the treatment of hypertension and angina¹³.

Olmesartan medoxomil (OM) (5-methyl-2-oxo-1, 3-dioxolen-4-yl) methoxy-4-(1-hydroxy-1-methylethyl)-2-propyl-1-{4-[2-(tetrazol-5-yl)-phenyl] phenyl} methylimidazol-5-carboxylate, is a potent and selective angiotensin AT1 receptor blocker¹⁴ (Fig. 2). It has been approved for the treatment of hypertension in the United States, Japan and European countries.

The drug contains a medoxomil ester moiety and is cleaved rapidly by an endogenous esterase to release the active metaboliteolmesartan¹⁵. The tablet manufacturer claims that the combined oral administration of amlodipine with olmesartan has been found to be more effective than either drug alone in the treatment of hypertension.¹⁶

Literature survey reveals that there are many methods for determination of either AMB or OM and in combination with other drug in combination dosage forms. None of the reported analytical method describes a simultaneous determination of AMB and OM in tablet dosage form by UV absorption correction method. Hence it was proposed to attempt the development of simple, accurate, rapid and economic method for simultaneous determination of AMB and OM in pharmaceutical formulation.

MATERIAL AND METHODS:

Instrumentation:

A UV/Visible double beam spectrophotometer, model Shimadzu UV-1601 with 1cm UV matched quartz cells was used. One Pan Balance (K-14 super By K Roy) was used for experimental purpose.

Reagent and chemicals:

Pharmaceutically pure sample of Amlodipine besylate and olmesartan medoxomil were obtained as a gift sample from Lupin labaratories. All solvents were of AR grade obtained from Qualigens and Loba Chemie Laboratory.

Experimental condition:

Amlodipine Besylate and Olmesartan Medoxomil were dissolved separately in methanol so as to get 1000μg/ml concentration of each drug. These solutions were diluted to a conc. 10μg/ml in methanol and the solutions were scanned in the wavelength range of 200-400 nm. Two wavelengths were selected at 363nm and 258nm, λmax of Amlodipine and Olmesartan. The UV-Spectra of Amlodipine besylate and Olmesartan standard and sample are shown in Figure: 1 and 2.

Fig-1 Overlay Spectrum of Amlodipine Besylate and Olmesartan Medoxomil Standard.

Fig-2 Overlay Spectrum of Amlodipine Besylate and Olmesartan Medoxomil Sample.

Preparation of standard stock solution:

An accurately weighed 50mg of AMB and OM was transferred into a 50ml volumetric flask and dissolved separately in methanol and was made up to volume with Methanol. From this pipetted out 5ml of the solution and made up to the volume to 50ml with methanol (100µg/ml). From this pipetted out 12.5ml of AMB was made up to 100ml with methanol (12.5 µg/ml) and 25ml of OM was made up to 50ml with methanol (50µg/ml). The drug obey Beer’s law in the concentration range of 0 to 25 μg/ml. Quantitative estimation of these drugs was carried out by using following formulae’s.

C_AM = A₁/ax₁

C_OM = A₂ – (ax₂* C_AM ) /ay₁

C_AM = A₃₆₃/_126.6

C_OM = A₂₅₈ – (128.32* C_AM ) /456.2

Where,

C_AM=Concentration of Amlodipine Besylate

C_OM= Concentration of Olmesartan Medoxomil

A₁ = Absorbance of sample solution at 363nm

A₂ = Absorbance of sample solution at 258nm

ax₁ = Absorptivity value of standard Amlodipine at 363nm

ax₂ = Absorptivity value of standard Amlodipine at 258nm

ay₁ = Absorptivity value of standard Olmesartan at 258nm

For the estimation of the drug in tablet formulation twenty tablets were weighed and their average weight was determined. The tablets were then finely powdered. An accurately weighed 50mg of AMB and OM was transferred into a 50ml volumetric flask and dissolved separately in methanol and was made up to volume with Methanol. From this pipetted out 5ml of the solution and made up to the volume to 50ml with methanol (100µg/ml). From this pipetted out 12.5ml of AMB was made up to 100ml with methanol (12.5 µg/ml) and 25ml of OM was made up to 50ml with methanol (50µg/ml).The absorbance’s values were read at 363.00 and 258.0 nm and concentration was obtained by solving the absorption correction equations. Results of analysis of tablet formulation are shown in Table No. 2.The proposed method was validated on the basis of parameters namely accuracy, precision, specificity, ruggedness and linearity and range. The accuracy of the proposed method was ascertained by carrying out recovery studies using standard addition method. The recovery study was performed to determine if there was any positive or negative interference from excipients present in the formulation. Precision of an analytical method is expressed as SD or RSD of a series of measurements. It was ascertained by replicate estimation of drug by the proposed method. The specificity is the ability to assess unequivocally the analyte for the presence of interesting components that may be expected to be present, such as impurities, degradation product and matrix components and stored for 24 hours under different conditions, like room temperature (normal), at 50º after addition of 1.0 ml of 0.1 N of hydrochloric acid, at 50º after the addition of 1.0 ml of 0.1 N of sodium hydroxide, at 50º after the addition of 3 % of hydrogen peroxide and at 60º. After 24 hours, the contents of the flask were subjected to same procedures as previously described. Test for ruggedness was carried out by repeating the procedure under different conditions, i.e., on different days, at different time and by different analysts. Linearity and range study was done by preparing concentration in the range of 80 -120 % of test concentration and absorbance values were recorded at 258.0 nm and at 363.0 nm.

RESULT AND DISCUSSION:

An attempt has been made to develop a fast, sensitive, precise, reproducible and economical analytical method for simultaneous estimation of AMB and OM in their combined dosage form. In this method drugs obey Beer’s law in the concentration range of 0 to 25 μg/ml. The absorption additivity study was carried out to see whether the drugs in mixture showed additivity or not. It was observed that both the drugs showed additivity of absorbance at selected wavelengths indicating that both the drugs do not interact with each other in the solvent system used. The method was validated as per the ICH and USP guidelines. The results of recovery study were found to be within the prescribed limit of 98 - 102 %, proving the accuracy and showing that the method is free from interference from excipients. The results are shown in Table No. 2. For precision, replicate estimation of both AMB and OM in the same batch of tablets was done by proposed method, which yielded quite concurrent results, indicating reliability of the method. The values of SD or RSD are within the prescribed limit of 2 %, showing high precision of the method, as shown in Table No. 1. In the specificity study, the sample was exposed to different stress conditions like acid, alkali, Peroxide and heat. The study showed degradation of AMB under peroxide, but the exact nature of degradation of drug could not be found out. During the linearity study it was observed that absorbance values of AMB and OM in the marketed formulation were linear in the range of 80 % to 120 % of the test concentration with R2 close to one for this method of analysis. From the study of validation parameters namely accuracy, precision (SD and RSD), ruggedness (interday, intraday and different analyst), specificity, linearity and range, it was observed that the method is specific, accurate, precise, reproducible and rugged. Hence both the methods can be employed for routine analysis of tablet dosage form.

Table No 1: Spectral and linearity characteristic data

Parameters	AMB	OM
λmax(nm)	363.0	258.00
Beers’s law limit (μg/ ml)	1.25-6.25	5-25
Regression Equation(y =mx +c) Slop(m)	0.012	0.004
Interccept(c)	0.0001	0.009
Correlation coefficient	0.999	0.998
LOD (μg/ ml)	0.4072	1.669
LOQ (μg/ ml)	1.234	5.056
Precision (% R.S.D.)	0.76	0.82
Repeatability (n=5) Intra-day(3x3 times)	0.78	1.40
Inter-day(3x5 days)	0.95	0.82
SD	0.00158	0.0224

Table No 2: Assay results of AMB and OM in tablets.

Drug	Labeled (mg)	Found (Mean± SD, n=6)	% Label claim (%Found ± SD, n=6)
Amlodipine Besylate	5	4.94±0.04	98.80±0.95
Olmesartan medoximil	20	19.94±0.20	99.70±1.13

CONCLUSION:

The validated spectrophotometric methods employed here proved to be simple, economical, rapid, precise, and accurate. Thus these can be used for routine simultaneous estimation of AMB and OM in tablet dosage form.

REFERENCES

1. Budavari S. The Merck Index. 14th ed. Whitehouse Station, NJ,USA: Merck Research Lab, Division of Merck & Co., Inc., 2006.

2. Permender Rathee, Sushila Rathee, Shyama Thakur and Vikash Kumar. Simultaneous Estimation of Amlodipine Besylate and Atenolol as A.P.I. and in Tablet Dosage Forms by Vierodt’sMethod using UV Spectrophotometry. Int J Chem Tech Res 2010; 2 (1): 62 ‐ 68.

3. Neela Manish Bhatia, Snehal Jawaharlal Deshmane, Harinath Nivrutti More and Prafulla Balkrishna Choudhari. Simultaneous Spectrophotometric Estimation of the Amlodipine Besylate and Hydrochlorothiazide. Asian J Res Chem 2009; 2 (4): 393 ‐ 397.

4. Pallavi Salve, Deepali Gharge, Rupali Kirtawade, Pandurang Dhabale and Kishor Burade. Simple Validated Spectroscopic Method for Estimation of Amlodipine Besylate from Tablet Formulation. Asian J Res Chem 2009; 2 (4): 553 ‐ 555.

5. Priyanka R Patil, Sachin U Rakesh, Dhabale PN and Burade KB.RP ‐ HPLC Method for Simultaneous Estimation of Losartan Potassium and Amlodipine Besylate in Tablet Formulation. Int J Chem Tech Res 2009; 1 (3): 464 ‐ 469.

6. European Pharmacopoeia. 3rd edition, Strasbourg: Council of Europe: 2001.

7. Dhorda VJ and Shetkar NB. Reversed phase liquid chromatographic determination of Ramipril and Amlodipine in tablets. Indian Drugs 1999; 36: 638.

8. Vaijanath G Dongre, Sweta B Shah, Pravin P Karmuse, Manisha Phadke and Vivek K Jadhav. Simultaneous determination of metoprolol succinate and amlodipine besylate in pharmaceutical dosage form by HPLC. J Pharm Biomed Anal 2008; 46 (3): 583 ‐ 586.

9. Ilango K, Kumar PB and Prasad VRV. Simple and rapid highperformance thin layer chromatographic determination of amlodipine in pharmaceutical dosage forms. Indian J Pharm Sci 1997; 59 (6): 171 ‐173.

10. Agrekar AP and Powar SG. Simultaneous determination ofatenolol and amlodipine in tablets by high performance thin layer chromatography. J Pharm Biomed Anal 2000; 21: 1137 ‐ 1142.

11. Feng Y, Zhang L, Shen Z, Pan F and Zhang Z. Analysis of AML in human plasma by Liquid chromatography ‐ mass spectrometry.JChromatogr Sci 2002; 40 (1): 49 ‐ 53.

12. Bhatt J, Singh S, Subbaiah G, Shah B, Kambli S and Ameta S. A rapid and sensitive Liquid Chromatography Tandem Mass Spectrometry (LCMS/ MS) for the estimation of AML in human plasma. J Biomed Chromatogr 2007; 21: 169 ‐ 175.

13. Norvasc, Online Drug Description and Clinical Pharmacology of amlodipine, The Internet Drug Index, Rx List Inc., 2008. Website: http://www.rxlist.com/cgi/generic/amlod2.htm.

14. K. Koga, S. Yamagishi, M. Takeuchi, Y. Inagaki, S.Amano Mol. Med. 2002; 8: 591-598.

15. L.R. Schwocho, H.N. Masonson, Pharmacokinetics of CS-866 a, new angiotensin II receptor blocker in healthy subjects. J. Clin. Pharmacol. 2001; 41:515-520.

16. Amlodipine and Olmesartan Study Results Released; Late Breaker Presented At American Society of Hypertension Twenty-Second Annual Scientific Meeting, Hypertension news 22 May 2007.

Received on 19.04.2011 Modified on 02.05.2011

Asian J. Research Chem. 4(7): July, 2011; Page 1100-1102