INTRODUCTION

Metoprolol is a selective beta 1-adrenoceptor blocking agent. It is given as succinate and used for treatment of hypertension. It is chemically 1-[4-(2-methoxyethyl) phenoxy]-3-(propan-2-yl amino) propan-2-ol. It is official in USP. Atorvastatin is a lipid lowering agent and is given as calcium salt. It is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This enzyme catalyzes the conversion of HMG-CoA to mevalonate, an early and rate-limiting step in cholesterol biosynthesis^1-2. It is chemically [R-(R*, R*)]-2-(4-fluorophenyl)-β, δ- dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1Hpyrrole-1-ptanoic acid, calcium salt (2:1) trihydrate. Combination of these two is used for the treatment of hypertension. Literature survey reveals availability of UV, HPLC and HPTLC methods for estimation of METO and ATOR individually with many drugs other than this particular combination^3-9. However no method has been reported till date for the estimation of METO and ATOR in combined dosage form. Present work deals with the quantitative estimation of METO and ATOR in their combined dosage form by various UV spectrophotometric methods.

EXPERIMENTAL:

Instrumentation:

All spectral measurements were made on shimadzu UV-VIS spectrophotometer – 1650 with 1 cm matched quartz cells.

Preparation of standard stock solution:

An accurately weighed quantity of 50mg of metroprolol and atorvastatin were separately taken in a 50ml volumetric flask, dissolved in methanol and made up to volume using the same to get 1mg /mL.

Preparation of sample solution:

The average net content of 20 capsules were determined and the powder equivalent to 23.75mg of meto was taken in 50 mL volumetric flask and dissolved in methanol and made up to the volume with the same. The solution was then filtered using whatman filter paper, the first few mL of the filtrate was discarded and the remaining solution was used for further analysis.

ASSAY PROCEDURE:

Method A - Simultaneous equation method:^8-9

Aliquots of the standard stock solutions were transferred to a series of 50mL volumetric flask and suitably diluted with methanol to give varying concentrations ranging from 5 - 50 µg/mL for both METO and ATOR. The solutions of METO and ATOR were scanned in the range of 200 to 400 nm against distilled water as blank.Two wavelengths selected for simultaneous equation method were 223 nm and 241nm. Absorbance and absorptivities of series of standard solutions were recorded at selected wavelengths λ₁ and λ₂. Two simultaneous equations were formed using these absorptivity coefficient values

A₁ = 367.25 C₁ + 9.044 C₂--------------------------- (1)

A₂ = 331.87 C₁ + 288.01 C₂-------------------------- (2)

Where, A₁ and A₂ were absorbances of sample solution at 223 nm and 241 nm respectively. C₁ and C₂are the concentrations of ator and meto respectively in sample solution in g / L. Aliquots of sample solution were diluted suitably and absorbances of the final dilution were measured at 223 nm and 241 nm and concentration of two drugs in sample was calculated using above framed simultaneous equations – (1) & (2).

Results of analysis are reported in Table-2. Validity of above framed equation was checked by repeating the procedure with different standards of METO and ATOR, measuring their absorbance at respective wavelengths and calculating concentration of two components. The results of validation studies was found satisfactory. The analysis procedure was repeated five times with the formulation.

Method B - Absorbance Ratio method:^10-12

Absorbance ratio method (Q-anaysis) uses the ratio of absorbances at two selected wavelengths, one at isoabsorptive point and other being the λmax of one of the two components. Aliquots of the standard stock solutions were transferred to a series of 50mL volumetric flask and suitably diluted to give varying concentrations ranging from 5 - 50 µg/mL for METO and ATOR.. The solutions of meto and ATOR were scanned in the range of 200 to 400 nm against distilled water as blank. The overlain spectra of metoprolol and atorvastatin was taken and isoabsorptive wavelength was found to be at 231 nm (fig-1). From the overlain spectra, the wavelengths 231 nm ( isoabsorptive point ) and 241nm ( λmax of atorvastatin ) were selected for formation of Q – absorbance equation. The absorptivity values of each drug at isoabsorptive point were determined. The concentration of each drug in the combination was determined by substituting the absorbance and absorptivity values in the following equations:

C_X = ( Qm-Qy / Qx-Qy ) x ( A / ax₁ )

C_Y = ( Qm-Qx / Qy-Qx ) x ( A / ay₁ )

Where, Cx = concentration of ATOR, Cy= concentration of METO, Qm= ratio of absorbance of sample at selected wavelengths, Qx= ratio of absorptivity of ATOR, Qy= ratio of absorptivity of METO, ay₁= absorptivity of METO at 231 nm, ax₁= absorptivity of ATOR at 231 nm.

Results of analysis of capsule formulation are reported in Table-2.

Method C - Area under curve method:¹³

The mixed standard solutions of meto and ator were scanned between 200-400 nm using distilled water as blank. The area under curve between 215-235 nm for meto and 240-295 nm for atorvastatin were measured by using the inbuilt software. The inbuilt software calculates the area bound by the curve and the horizontal axis. The horizontal axis is selected by entering the wavelength range over which the area has to be calculated .The wavelength range is selected on the basis of repeated observations so as to get the linearity between area under curve and concentration. The amount of meto and ator in marketed sample was computed from the calibration curve obtained by plotting the area versus concentration. Results of analysis are reported .

Fig(1)

RECOVERY STUDIES:

To study the accuracy and reproducibility of the proposed methods, recovery experiments were carried out by adding a known amount of standard drug solution of METO and ATOR to preanalysed sample and the percentage recovery is calculated. The results are furnished in table-2.

RESULTS AND DISCUSSION:

The optical characteristics such as absorption maxima, Beer's law limits, molar absorptivity and Sandell's sensitivity are furnished in Table-1. The regression characteristics like slope, intercept, and correlation co-efficient (r), obtained from different concentrations were calculated and the results are summarized in Table-1.

The results indicate that there is no interference of other ingredients present in the formulation. Thus, the proposed methods are simple, sensitive, precise, accurate, rapid, economic, reproducible and useful for the simultaneous determination of meto and ator in pharmaceutical formulation. The value of standard deviation & correlation coefficient were satisfactory and recovery studies were indicative of the accuracy of the proposed methods in method A, two wavelengths of respective absorbance maxima ie 223nm for METO and 241nm for ATOR were used for the analysis of the durgs. The criteria for obtaining maximum Precision¹⁴ by this method were calculated and found to be outside the range of 0.1-2. In method B, the primary requirement for developing a method for analysis is that the entire spectra should follow the Beer’s law at all wavelengths which was fulfilled in case of both these drugs.

Table 1: Optical & statistical parameters

Parameters	Simultaneous equation		Absorbance ratio method				Area under curve
Parameters	METO	ATOR	METO-223	ATOR-241	METO-231	ATOR-231	METO	ATOR
λmax (nm)	223	241	223	241	231	231	215-235	240-295
Beer’s law limits (µg/mL)	5-50	5-50	5-50	5-50	5-50	5-50	5-50	5-50
Molar absorptivity(L mol^-1 cm^-1)	18801	44415	18801	44415	8286.93	41408	-	-
Sandell’s sensitivity (µg cm^-2 /0.001 abs unit)	0.034776	0.0272	0.03477	0.0272	0.07899	0.0292	-	-
Slope (m)	0.027663	0.0363	0.02766	0.0363	0.01199	0.0337	0.11805	0.31599
Intercept(c)	0.013067	0.0051	0.01306	0.0051	0.00826	0.0065	0.0702	-0.0424
Regression equation*	0.02766X +0.013	0.0363X +0.0051	0.02766X +0.013	0.0363X +0.0051	0.01199X +0.008	0.0337X +0.0065	0.011805X+0.07	0.31599X-0.042
Correlation coefficient (r)	0.9997	0.9999	0.9997	0.9999	0.9993	0.9999	0.9996	0.9994
Standard deviation	0.026133	0.01033	0.02613	0.0103	0.01653	0.0129	0.1404	-0.0848
% RSD**	0.1099	0.1036	0.1099	0.1036	0.06962	0.12991	0.29231	-0.84378
LOD	3.1175	0.92143	3.1175	0.9214	4.54883	1.2666	3.92492	-0.88561
LOQ	9.4471	2.7922	9.4471	2.7922	13.7843	3.8381	11.8937	-2.68367

* (Y=mx+c), ** mean of three readings

Table – 2: Assay and recovery of METO and ATOR in dosage forms

Method	Drug	Label claim (mg)	Amount obtained(mg)*		% Label claim	Percentage recovery **
Simultaneous equation	METO	23.75	23.77		100.1	99.00
Simultaneous equation	ATOR	10	9.97		99.7	100.17
Absorbance ratio method	METO	23.75		23.74	99.95	99.73
Absorbance ratio method	ATOR	10		9.93	99.3	99.65
Area under curve	METO	23.75		23.82	100.29	99.82
Area under curve	ATOR	10		10.05	100.50	100.95

* Average of three determinations, ** Average of three determinations

The two wavelengths used for the analysis of both drugs were 231nm (isoabsorptive point) and 241nm (λmax of ATOR) The validation parameters were studied at all the three wavelengths for both the methods. Accuracy was determined by calculating the % recovery and precision was calculated as repeatability (standard deviation & RSD) and inter & intra day variation for both the drugs. Both the methods were successfully used to determine the amounts of METO and ATOR present in the formulation. The results obtained are in well agreement with the corresponding labeled amount.

CONCLUSION:

The methods described in this paper for the simultaneous estimation of METO and ATOR are found to be simple, sensitive, economical, accurate, rapid and precise. Hence these three methods could be successfully employed for the routine analysis of METO and ATOR in their combined dosage form.

REFERENCES:

1. Budavari S , Editor. The Merck index.12^th ed. Whitehouse station (NJ): Merck and Co. Inc; 1996. p-897.

2. Gennaro AE, editor. Remington’s-the science and practice of pharmacy.20^th ed. Vol 2. Eaaston (PA): Mack publishing Co; 2000, p-1294.

3. D.A. Shah, K.K Bhatt, et al. Stability indicating RP-HPLC estimation of atorvastatin calcium and amlodipine besylate in pharmaceutical formulations. Indian J of pharmaceutical sciences. Nov-Dec 2008; 70(6): 754-760.

4. M.R Khan, Deepti Jain, et al. Simultaneous spectrophotometric determination of atorvastatin calcium and amlodipine besylate in tablets . Indian J of pharmaceutical sciences. Jul-Aug 2006; 68(4): 546-548.

5. K. Raja Rajeswari, G.G. Sankar, et al. RP-HPLC method for the simultaneous determination of atrovastation and amlodipine in tablets dosage form. Indian J of pharmaceutical sciences. Mar-Apr 2006;

6. Sivakumar T, Manavalan R, et al. An improved HPLC method with theaid of a chemometric protocol:Simultaneous analysis of amlodipine and atorvastatin in pharmaceutical formulations. J Sep Sci 2007;30:3143-53.

7. K.R. Gupta, M.R. Tajne, S.G. Wadodkar. New spectrophotometric method for simultaneous determination of metoprolol tartarate & hydrochlothiazide in tablets . Indian J of pharmaceutical sciences. July-Aug 2008; 70(4): 511-513.

8. Sonali Paramane, Lata Kothapalli, et al. Simultaneous spectrophotometric estimation of gatifloxacin and ornidazole in tablet dosage form. Indian J of Pharmaceutical Sciences;Nov-dec 2009:p819-821.

9. Permender Rathee, Sushila Rathee. Stability indicating UV-spectrophotometric methods for simultaneous determination of losartan potassium and hydrochlorthiazide in pharmaceuticals. Eurasian J. Anal. Chem 2009;4(1):98-109.

10. Anandakumar K, Vetrichelvan T. Simultaneous estimation of aspirin and clopidogrel bisulphate in solid dosageform by uv spectrophotometry. Indian drugs;44(5):may-2007;p342-345.

11. Shveta Chandra, A.V.Kasture. Simultaneous spectrophotometric determination of pioglitazone HCl and glimepride in tablets. Indian J of Pharmaceutical Sciences; Sep-Oct 2005:67(5); p627-629.

12. S.J.Daharwal, Swarnlata Saraf, et al. Spectrophotometric methods for the simultaneous estimation of doxycycline and tinidazole in tablet dosage form. Indian J of Pharm Education and Research; Jan-Mar 2008, 42(1): p27-31.

13. Thomas A B, et al. Simultaneous spectrophotometric method for assay and dissolution of tramadol and ibuprofen in combined dosage form. Indian drugs:Aug 2008;45(8): p643-648.

14. Beckett A H and Stenlake J B; Practical pharmaceutical chemistry: CBS publisher; New Delhi; fourth edition;part two;1997; p285.

15. Part II; Validation of analytical procedures; methodologyQ2B, ICH harmonized tripartite guidelines, 1996;6-12.

Received on 22.12.2009 Modified on 17.02.2010

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 586-588