Simultaneous Determination of Amlodipine Besylate and Atorvastatin Calcium in Pharmaceutical Tablet Formulation by Derivative Spectrophotometric Method

 

Vandana B. Patel1*, Rajnikanta Sahu2 and Bhumika M. Patel1

1Baroda College of Pharmacy, Limda, Vadodara, Gujarat, India.

2Department of Pharmacy, The Maharaja Sayajirao University of Baroda, Gujarat, India.

*Corresponding Author E-mail: vbpatel04@yahoo.com

 

ABSTRACT:

Two simple, accurate and precise methods viz. first derivative zero crossing spectrophotometry (method 1) and derivative ratio spectra zero crossing method (method 2) have been developed for simultaneous determination of amlodipine besylate and atorvastatine calcium in pure and commercial formulation without any prior separation or purification. The linearity range was found to be 5- 40 g ml-1 for both the drugs. The value of limit of detection and limit of quantification was 0.397 g ml-1 and 1.323 g ml-1 for amlodipine and 0.220g ml-1 and 0.735 g ml-1 for atorvastatine respectively for method 1 and 0.600 g ml-1 and 0.262 g ml-1 for amlodipine and 0.262g ml-1 and 0.874 g ml-1 for atorvastatine respectively for method 2. The method was satisfactorily validated in terms of accuracy and precision. The results of the study showed that the proposed spectrophotometric methods are useful for the routine determination of amlodipine and atorvastatin in its combined pharmaceutical tablet dosage form.

 

KEYWORDS: First derivative zero crossing spectrophotometry; Derivative ratio spectra zero crossing method; Amlodipine besylate; Atorvastatin calcium.

 


 

INTRODUCTION:

Amlodipine besylate (AMLB), 3-ethyl-5-methyl ()-2-[(2-aminoethoxy)methyl ]-4-(o-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate, monobenzenesulphonate, is a calcium channel blocker. It inhibits the trans-membrane influx of calcium ions into vascular smooth muscles and cardiac muscle. Atorvastatin calcium (ATVC) is chemically [R-(R*,R*)]-2-(4-flurophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoic acid, calcium salt trihydrate. It is a selective, competitive inhibitor of HMG-CoA reductase, the rate-limiting enzyme that converts 3-hydroxy-3-methyl-glutaryl-coenzyme A to mevalonate, a precursor of sterols, including cholesterol. The combination dosage forms of atorvastatin calcium and amlodipine besylate are available in the market for the treatment of hypertension, chronic stable angina, vasospastic angina, elevated serum triglyceride levels, and primary dysbetalipoproteinemia. The literature survey reveals many analytical methods for the quantitative determination of either AMLB or ATVC alone1,2,3.

 

Also, some spectrophotometric and chromatographic methods have also been developed for simultaneous determination of AMLB and ATVC4,5,6,7,8. In this communication, two new spectrophotometric methods viz. first derivative zero crossing spectrophotometry (method 1) and derivative ratio spectra zero crossing method (method 2) are proposed for simultaneous determination of AMLB and ATVC.

 

MATERIALS AND METHODS:

Spectrophotometric measurements were made on Shimadzu 1601 double beam UV-spectrophotometer with a fix slit width of 1 nm coupled HP7540 computer loaded software of version 2.0 and EPSON-300 printer.

 

Reagents:

Methanol (AR grade) was obtained from Allied Chemical Corporation (Vadodara, India). Standard bulk drug sample of AMLB and ATVC was obtained as gift sample by Torrent Pharmaceuticals Ltd. (Ahmedabad, India) and M/s. Alembic Ltd (Vadodara, India) respectively. The pharmaceutical dosage form used in this study, tablets with 5 mg AMLB and 10 mg ATVC per tablet, was procured from local market.

 

Procedure:

First Derivative Zero Crossing Spectrophotometry (Method 1):

Preparation of calibration curve:

Standard stock solution of AMLB and ATVC were made by dissolving 50 mg of each drug in 50 ml of methanol individually. Suitable aliquots of these stock solution were taken to prepare binary mixture containing varying amount of ATVC and AMLB (AMLB: ATVC = 5: 5, 10: 10 15: 15, 20: 20, 25: 25, 30: 30 and 40:40 g ml-1) in 10 ml volumetric flask and the volume was made with methanol. The absorption spectra of the binary mixture solutions of AMLB and ATVC were recorded in the range of 200 nm to 300 nm and were stored in the memory of computer. The 1st derivatives of stored spectra of the binary mixtures were stressed after smoothing these at ∆λ = 4 interval and multiplying the entire spectra with a constant factor 10. The amplitudes at 246.5 nm were plotted against the respective concentrations of AMLB. Similarly the amplitudes at 290 nm were plotted against the respective concentrations of ATVC.

 

Analysis of tablet formulation:

Twenty tablets were weighted accurately and ground to fine powder. A quantity of powder equivalent to 5 mg AMLB and 10 mg ATVC was weighed and transferred to 100 ml volumetric flask containing approximately 20 ml methanol. The mixture was ultrasonicated for 5 min. The solution was filtered using Whatman no. 41 paper and volume was made upto the mark with methanol. After suitable dilution the solution was tested by the procedure as described in preparation of calibration curve.

 

Ratio Spectra First Derivative Spectrophotometry (Method 2):

Preparation of calibration curve:

Standard stock solution of AMLB and ATVC were made by dissolving 50 mg of each drug into methanol. Suitable aliquots of the standard stock solution were diluted with methanol to produce standard solutions of 15 g ml-1 of ATVC and 17 g ml-1 of AMLB for obtaining divisor spectra. The absorption spectra of 15 g ml-1 of ATVC and 17 g ml-1 of AMLB were recorded in the range of 200 nm to 250 nm and stored in the memory of the instrument after smoothing it at ∆λ = 4 interval and multiplying with constant 10 as divisor spectra. The binary mixture containing AMLB and ATVC in 1:2 ratio were prepared by mixing suitable aliquots of the standard solution and diluting it with methanol. The absorption spectra of binary mixture solution of AMLB and ATVC were recorded in range of 200 nm to 250 nm and were stored in the memory of instrument. The stored spectra of binary mixture solutions were divided by previously stored divisor spectrum of 15 g ml-1 of ATVC to get the ratio spectra of AMLB. The first derivative of ratiospectra of AMLB were traced with Δ l = 4 interval, multiplication factor was 10 and the amplitude at 244.6 nm was plotted against the respective concentrations of AMLB. Similarly, the absorption spectra of binary mixture were divided by a previously stored standard divisor spectrum of 17 g ml-1 of AMLB to get the ratiospectra of ATVC. The first derivative of ratiospectra of ATVC were traced with Δ l = 4 interval, multiplication factor was 10 and the amplitude at 222.7 nm was plotted against the respective concentrations of ATVC.

 

Analysis of tablet formulation:

Twenty tablets were weighted accurately and ground to fine powder. A quantity of powder equivalent to 5 mg AMLB and 10 mg ATVC was weighed and transferred to 100 ml volumetric flask containing approximately 20 ml methanol. The mixture was ultrasonicated for 5 min. The solution was filtered using Whatman no. 41 paper and volume was made upto the mark with methanol. After suitable dilution the solution was tested by the procedure as described in preparation of calibration curve.

 

METHOD VALIDATION:

Accuracy:

For studying the accuracy of the proposed methods and for checking the interference from excipients used in the dosage forms, recovery experiments were carried out by the standard addition method. This was performed at three different levels (80, 100 and 120%) by addition of known amount of AMLB and ATVC to a pre-analyzed sample of commercial tablets. The amount of standard recovered were calculated in terms of mean recovery with the upper and lower limits of percentage standard deviation.

 

Precision:

Intra-day precision and inter-day precision for the developed methods were measured in terms of % RSD. The experiments were repeated five times in a day for intra-day precision and on five different days for inter day precision. The concentration value for both intra-day and inter-day precision were calculated five times separately and percentage relative standard deviation were calculated. Finally, the mean of % R.S.D (% R.S.D= [S/X]100, where S is standard deviation and X is mean of the sample analyzed) was calculated.

 

Limit of detection and limit of quantification:

Limit of detection (LOD) and limit of quantification (LOQ) were calculated according to 3 s/m and 10 s/m criterions, respectively, where s is the standard deviation of the absorbance (n=10) of the sample and m is the slope of the corresponding calibration curve.

 

RESULTS AND DISCUSSION:

The absorption spectra of the two drug compounds, AMLB and ATVC overlapped closely as shown in Fig 1. For this reason, the determination of the above compounds was not possible by direct measurement of absorbance in zero-order spectra. On the other hand, derivative spectroscopy shows more resolution and makes it possible to analyze each drug in presence of one another as well as in presence of other excipients without any pretreatment.

 


Table 1.results of recovery study of AMLB and ATVC by all three methods

% Amount added to preanalyzed sample

*% Recovery SD for method 1

*% Recovery SD for method 2

 

AMLB

ATVC

AMLB

ATVC

80%

99.52 1.038

98.96 1.736

99.79 1.302

100.59 0.744

100%

99.76 0.415

100.00 1.389

100.08 1.332

100.11 0.573

120%

100.92 1.248

99.92 0.668

99.79 1.611

99.97 0.65

Mean recovery

100.06

99.626

99.88

100.2233

 

Table 2.Results of validation parameters

Parameters

Method 1

Method 2

AMLB

ATVC

AMLB

ATVC

Range

5-40

g ml-1

5-40

g ml-1

5-40

g ml-1

5-40

g ml-1

Slope

0.0139

0.0072

0.0258

0.0276

Intercept

0.0073

0.0001

0.0013

0.0103

Correlation coefficient ( R2)

0.9988

0.9997

0.9999

0.9999

Accuracy

100.06

99.626

99.88

100.2233

Precision (% RSD)

1.484

1.284

1.049

1.554

LOD

0.397 g ml-1

0.220 g ml-1

0.600 g ml-1

0.262 g ml-1

LOQ

1.323 g ml-1

0.735 g ml-1

1.999 g ml-1

0.874 g ml-1

 

Table 3 Assay results of AMLB and ATVC in combined commercial formulation by zero crossing spectrophotometry ( method 1) and derivative ratio spectra zero crossing ( method 2)

Method

% labeled claim obtained for AMLB

% labeled claim obtained for ATVC

Method 1

98.216 0.465

99.022 0.598

Method 2

99.424 0.565

98.750 0.787

 


 

Fig 1. Overlay spectra of AMLB and ATVC

`

Fig 2. 1st derivative spectra of 15 g ml-1 each of AMLB and ATVC

 

Fig 3. 1st derivative of Ratiospectra obtained when spectra of mixture solutions containing 5, 10, 20, 30 and 40 g ml-1 of AMLB was divided by 15 g ml-1 of ATVC

 

Fig 4. 1st derivative of Ratiospectra obtained when spectra of mixture solutions containing 5, 10, 20, 30 and 40 g ml-1 of ATVC was divided by 15 g ml-1 of AMLB

 

First derivative zero crossing spectrophotometry:

In contrast to zero-order spectra, first derivative spectra showed more resolution in terms of zero crossing point shown in Fig 2. There is no contribution of ATVC at 246.5 nm and there is no contribution of AMLB at 290 nm, hence 246.5 nm was considered to be the first derivative wavelength for AMLB determination and 290 nm for ATVC determination. Thus one drug can be determined at the zero crossing point of another drug.

 

In this method, the standard and sample preparation required less time and no tedious extractions were involved. The results of recovery studies, 100.06 % of amlodipine and 99.62% of atorvastatin, indicated high accuracy of the method (Table 1). Results of validation parameters are depicted in Table 2. A good linear relationship (r =0.9988 for amlodipine and r = 0.9997 for atorvastatin) was observed within the concentration range of 5-40 mg/ml for both the drugs. Low values of standard deviation are indicative of the high precision of the method. The satisfactory assay results of amlodipine and atorvastatin combined tablet formulation, 98.216 0.465 % for amlodipine and 99.022 0.598 % for atorvastatin (Table 3), revealed the potential utility of the developed method in the tablet dosage form.

 

Ratio spectra first derivative spectrophotometry:

An accurate choice standard divisor is fundamental for this method. Several tests were made in a preliminary investigation by using standard divisors in the concentration range from 5 to 40 g ml-1 for each drug. The best result in terms of signal-to-noise ratio, sensitivity, repeatability and range of validity of Beers law were found by using 15 g ml-1 of ATVC as divisor for AMLB determination and 17 g ml-1 of AMLB as divisor for ATVC determination. The first derivative wavelength was considered 244.6 nm for AMLB determination because there is no contribution of ATVC at this wavelength as shown in Fig 3. Similarly, 222.7 nm was used as first derivative wavelength for ATVC determination because there is no contribution of AMLB at this wavelength (Fig.4).

 

Accuracy of the developed method was checked by recovery studies. It was found that 99.88 % of amlodipine and 100.22% of atorvastatin were recovered, which indicated high accuracy of the method (Table 1). A good linear relationship with the regression coefficient r = 0.9999 was observed within the concentration range of 5-40 g/ml for both the drugs. Low values of standard deviation are indicative of the high precision of the method. The limit of detection (LOD) for amlodipine and atorvastatin were found to be 0.600 g /ml and 0.262 g /ml respectively and the limit of quantification (LOQ) for amlodipine and atorvastatin were found to be 1.999g/ ml and0.874 g/ ml respectively. Summary of various validation parameters are listed in Table 2. The assay of amlodipine and atorvastatin combined tablet formulation was found to be 99.424 0.565% for amlodipine and 98.750 0.787 % for atorvastatin respectively (Table 3)

CONCLUSION:

Both the newly developed methods viz., First derivative zero crossing spectrophotometry and Ratio spectra first derivative spectrophotometry, for simultaneous estimation of AMLB and ATVC are simple, specific, accurate and precise which indicates its suitability for routine pharmaceutical analysis without prior separation of individual drugs.

 

REFERENCES:

1.       Kamble AY, Mahadik MV, Khatal LD and Dhaneshwar SR. Validated HPLC and HPTLC Method for Simultaneous Quantitation of Amlodipine Besylate and Olmesartan Medoxomil in Bulk Drug and Formulation. Analytical Letters. 2010; 43; 251 258.

2.       Dongre VG, Shah SB, Karmuse PP, Phadke M and Jadhav VK. Simultaneous determination of metoprolol succinate and amlodipine besylate in pharmaceutical dosage form by HPLC. Journal of Pharmaceutical and Biomedical Analysis. 2008; 46; 583-586.

3.       Nakarani NV, Bhatt KK, Patel RD and Bhatt HS. Simultaneous determination of amlodipine besylate and benazepril hydrochloride in pharmaceutical dosage form by LC. Journal of AOAC International. 2007; 90; 700-705.

4.       Sahu R and Patel VB. Simultaneous spectrophotometric determination of amlodipine besylate and atorvastatin calcium from their binary mixture by dual wavelength and zero absorbane measurement. Indian Drugs. 2006; 43; 160-161.

5.       Sahu R and Patel VB. Simultaneous Spectrophotometric determination of amlodipine besylate and atorvastatin calcium in binary mixture. Indian Journal of Pharmaceutical Science. 2007; 69; 110-111.

6.       Khan MR and Jain Deepti. Simultaneous spectrophotometric determination of atorvastatin calcium and amlodipine besylate in tablets. Indian Journal of Pharmaceutical Science. 2006; 68; 546-548.

7.       Mishra P, Gupta A and Shah K. Simultaneous estimation of atorvastatin calcium and amlodipine besylate from tablets. Indian Journal of Pharmaceutical Science. 2007; 69; 831-833.

8.       Shah DA, Bhatt KK, Mehta RS, Baldania SL and Gandhi TR. Stability Indicating RP-HPLC Estimation of Atorvastatin Calcium and Amlodipine Besylate in Pharmaceutical Formulations. Indian Journal of Pharmaceutical Science. 2008; 70; 754-760.

 

 

 

Received on 26.08.2010 Modified on 02.09.2010

Accepted on 06.09.2010 AJRC All right reserved

Asian J. Research Chem. 4(1): January 2011; Page 128-131