Ratio Derivative Spectrophotometry Method for Simultaneous Estimation of Metoprolol and Amlodipine in their Combined Dosage Form

 

Anuruddha R. Chabukswar*, Shital D. Tambe, Vishnu P. Choudhari, Shailesh N. Sharma, Mayuri N. Mohokar, Subhash Chate.

MAEER’s Maharashtra Institute of Pharmacy, Pune-411038, Maharashtra, India

*Corresponding Author E-mail: anichem18@gmail.com

 

ABSTRACT:

Simple, precise and economical spectrophotometric method have been developed for the simultaneous estimation of Metoprolol Succinate (METO) and Amlodipine(AMLO) in combined tablet dosage form. Present study describes the development and validation for the simultaneous estimation of metoprolol(METO) and Amlodipine(AMLO)  by the Ratio derivative UV spectroscopy method. Methanol is used as solvent.  The amplitudes at 277.017nm and 235.62nm of the first derivative of ratio spectra were selected to determine METO and AMLO, respectively by ratio derivative method. Beer’s law is obeyed in the concentration range of 50-250 μg/mL and 5-25 μg/mL for Metoprolol Succinate and Amlodipine, respectively by both the methods. The % assay in commercial formulation was found to be 99.56% for METO and 98.96% for AMLO by the proposed method. The method was validated with respect to linearity, precision and accuracy. Recovery was found in the range of 99.51-99.92% for METO and 99.69-100.6% for AMLO by ratio derivative method. The present result shows that the proposed method can be successfully employed for routine analysis of the marketed formulations.

 

KEYWORDS: Metoprolol Succinate, Amlodipine, Ratio Spectra, Derivative Spectrophotometry.

 


INTRODUCTION:

Metoprolol succinate (METO) is a selective β-adrenergic antagonist, which is used in the treatment of cardiovascular disorders such as hypertension, angina pectoris, cardiac arrhythmias, congestive heart failure and myocardial infarction. Metoprolol is administered orally as tablet. Chemically Metoprolol succinate is (RS)-1-(Isopropylamino)-3-[p-(2-methoxyethyl) phenoxy]propan-2-ol succinate  with molecular formula C34H54N2O10 [1,2] (Fig. 1). Amlodipine(AMLO),  is calcium channel blockers, used for patients with angina pectoris and hypertension. Chemically it is 2-[(2-Aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridine dicarboxylic acid- 3-ethyl-5-methyl ester[3] having the molecular formula C20H25ClN2O5 [4](Fig.2).

 

A combination of METO (25) and AMLO (2.5) are marketed as tablet formulation (Metpure-AM) in the ratio 10:1. This combination is indicated for treatment of angina pectoris, cardiac arrhythmia and hypertension.

 

Literature survey shows that few analytical techniques such as spectrophotometry [5], HPLC [6] have been reported for the combination as well as the individual drugs. Simultaneous determination of METO with other CVS acting drugs has been reported[7-11]. As far as Amlodipine is concerned, few reports are available for its estimation in bulk and formulation such as spectrophotometry[12-16], HPLC and bioanalytical methods[17]. The method was validated for linearity, accuracy, precision, sensitivity, robustness, etc. in accordance with International Conference on Harmonization (ICH) guidelines [18].

 

Fig.1Structure of Metoprolol Succinate

 

Fig.2 Structure of Amlodipine

 

In the present work an attempt has been made to develop an analytical method development for the simultaneous estimation of METO and AMLO in the combine dosage form by using ratio derivative method by UV spectroscopy.

 

MATERIALS AND METHODS:

Instrumentation

An UV-Visible double beam spectrophotometer (Varian Cary 100) with 10 mm matched quartz cells was used. Electronic balance (Model Shimadzu AUW-220D) was used for weighing.

 

Reagents and Chemicals

Pure drug sample of both METO, percentage purity 99.86% and AMLO, percentage purity 99.92% was kindly supplied as a gift sample by Cipla Ltd. These samples were used without further purification. Spectroscopy grade methanol was used throughout the study. Tablets each containing 25 mg of METO and 2.5 mg of AMLO used for analysis was Metpure-AM manufactured by Emcure Ltd.

 

Analytical Method Development

Preparation of Standard Stock Solutions

Standard  stock  solutions  of pure  drug  containing 1000  μg/mL  of  METO and  AMLO were  prepared separately  in methanol. Standard stock solutions were further diluted with methanol to get working standard solutions of analytes in the concentration range of 50-250 μg/mL and 5-25 μg/mL of Metoprolol Succinate (METO) and Amlodipine (AMLO), respectively and scanned  in the range of 200-400nm. For ratio derivative amplitudes (at interval 1.2 and filter size 9) of ratio spectra were measured at 277.017nm and 235.62 nm for METO and AMLO, respectively.  First derivative amplitudes of ratio spectra and concentrations were used to construct calibration curve.

 

Preparation of Sample Solution and Formulation analysis

Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 25mg of METO and AMLO (2.5 mg) was weighed and dissolved in the 30 mL of methanol  with the aid of  ultrasonication  for 7 min and solution was filtered through Whatman paper No. 41 into a 100 mL volumetric flask.  Filter  paper  was  washed  with  same solvent,  adding  washings  to  the  volumetric  flask and volume was made up to the mark with methanol. The solution was suitably diluted further with methanol to get required final concentration of METO (150 μg/mL) and AMLO (15µg/mL).(fig 3)

 

Fig 3: Overlay spectra of AMLO, METO and Formulation

 

Theoretical aspects of Ratio Derivative

The  method  involves  dividing  the  spectrum  of  mixture  by  the  standardized  spectra  of each  of  the  analyte  to get ratio spectra and first derivative of ratio spectrum was obtained which was independent of concentration of divisor .Using appropriate dilutions of standard stock solution, the two solutions were scanned separately. The ratio spectra of different METO standards at increasing concentrations were obtained by dividing each with the stored spectrum of the standard solution of  AMLO (2.5 μg/mL) as shown in (Fig 4A).Wavelength277.017nm was  selected  for  the  quantification  of  METO  in  METO + AMLO  mixture. The ratio derivative spectra of the solutions of AMLO at different concentrations were obtained by dividing each with the stored standard spectrum of the METO (6μg/mL) as shown in (Fig 4B). Wavelength 235.62nm was selected for the quantification of AMLO in METO + AMLO mixture. Measured analytical signals at these wavelengths were proportional to the concentrations of the drugs over the selected concentration range. Calibration curves were prepared from the measured signals at the selected wavelength and concentration of the standard solutions. The concentrations of METO (CMETO) and AMLO (CAMLO) in solution of tablets was calculated by using equations (1) and (2), respectively.

 

At 277.017nm nm: CMETO =          ....(1)

 

 

At 235.62 nm: CAMLO =           … (2)

 

Fig. 4A: First Derivative of ratio spectra of  50, 100, 150, 200, 250 µg/ml of METO when 2.5µg/ml of AMLO is used as divisor

 

Fig.4B: First Derivative of ratio spectra of 5, 10, 15.20, 25 µg/ml of AMLO when 25µg/ml of METO is used as divisor

 

Validation of Analytical method

Validation is the process of establishing documented evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes. Present method has been validated as per ICH guidelines as follows.

 

1) Accuracy

Accuracy of the method was determined in terms of % recovery of standard. Recovery studies were carried out by addition of standard drug solution at the level of 80%, 100% and 120% to the pre analyzed sample. Results of the recovery study were found to be within the acceptance criteria 100±10 %, indicating a good degree of sensitivity of the method towards detection of analytes in sample. Accuracy results for METO & AMLO are shown in Table no 1.1 & 1.2 respective.

 

2) Solution Stability

Method stability was checked by analyzing solution kept in refrigerator and at room temperature by both methods. Solution at room temperature was stable for 12 hours and solution in refrigerator was stable for 30 days (% RSD < 2).

 

3) Precision of the Method

Method repeatability was determined by six times repetitions 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 2).

 

4) Linearity

The linearity of the proposed method was evaluated for each drug by analyzing a series of different concentrations of each of METO; AMLO within the range stated in Table3. The assay was performed according to the experimental conditions previously established. The absorbance values for METO and AMLO were measured, at the specified wavelengths (Table 3), and plotted against its concentration. A straight line was obtained in each case. The statistical analysis of these graphs using least squares method was made for the slope, intercept and correlation coefficients. The results obtained show that the linearity of calibration graphs and the compliance with Beer`s law for the both drugs i.e METO and AMLO fig no. (5A & 5B).The correlation coefficients of calibration plots for METO and AMLO were 0.998 and 0.999 as indicated in Table 3


 

Table 1.1 Accuracy result for METO

INGREDIENT

RECOVERY

DRUG

AMOUNT SPIKED

AMOUNT RECOVERED

% MEAN  RECOVERY

 

LEVEL %

AMOUNT (µg/ml)

(µg/ml) 

(µg/ml)

% RSD (N=3)

 

80%

75

60

134.68

99.762,0.96296

METO

100%

75

75

149.89

99.926,0.66667

 

120%

75

90

164.2

99.515,0.915152

 

Table 1.2 Accuracy result for AMLO

INGREDIENT

RECOVERY

DRUG

AMOUNT SPIKED

(µg/ml))

AMOUNT RECOVERED (µg/ml)

% MEAN  RECOVERY

LEVEL %

AMOUNT (µg/ml)

% RSD (N=3)

 

80%

7.5

6

13.49

99.925,0.92593

AMLO

100%

7.5

7.5

15.09

100.6,0.850

 

120%

7.5

9

16.45

99.696,0.9697

 

 


Table 2: Determination of precision

SR.NO.

REPRODUCI-BILITY

(%)

(INTRA-DAY PRECISION)

(n=4)(%)

(INTER-DAY PRECISION)

(n=3)(%)

1

99.02716

99.02716

99.2057

2

99.55

100.4089

99.3763

3

100.0667

100.2095

99.02716

4

100.45

99.97441

98.02734

5

100.15

100.266

99.31679

6

100.58

100.0756

99.31679

Mean

99.97064333

99.993595

99.045013

Std. deviation

0.584738518

0.496867626

0.513668

RSD

0.005849102

0.004968995

0.0051862

%RSD

0.584910228

0.496899453

0.5186207

1

99.02716

99.02716

99.2057

 

 (A)

(B)

Fig 5: calibration curve of A) Metoprolol and B) Amlodipine

5) Specificity

Specificity is a procedure to detect quantitatively the analyte in the presence of component that may be expected to be present in the sample matrix. Commonly used excipients in tablet preparation were spiked in a pre-weighed quantity of drugs and then absorbance was measured and calculations done to determine the quantity of the drugs.

 

6) Limit of Quantification (LOQ) and Limit of Detection (LOD)

LOD and LOQ were calculated statistically from formula shown in equation No 3 and 4 respectively:

 

                                                   ….. (3)

 

                                                    ….. (4)

 

Where, SD: Standard Deviation of y- intercepts of regression lines,

 

The LOD and LOQ of METO and AMLO by statistical and visualization methods were mentioned in Table 3.

 

Table 3: Optical characteristics of the proposed methods and result of linearity, sensitivity and formulation analysis

SR. NO.

PARAMETERS

METOPROLOL

(METO)

AMLODIPINE

(AMLO)

1

Wavelength

Nm

277.017

235.62

2

Beer’s law limit

(μg/mL)

50-250

5-25

3

 

Linearity

 

 

(μg/mL

50-250

5-25

Regression Equation*

Y=0.004X+

0.024

y = 0.012x - 0.050

R2

0.998

R˛ = 0.999

5

Formulati-on Analysis (%Assay, %RSD), n=6

Metpure-AM

99.56% ,0.5864

98.96%,0.6549

6

LOD

(μg/mL)

0.07508

-0.3154

7

LOQ

(μg/mL)

0.2275

-0.9559

Y* = mX + c, where Y is the absorbance and X the concentration in micrograms per millilitre

 

 

7) Robustness

Robustness of the method was determined by repeating the proposed method by other analyst in the same laboratory and calculating % RSD. The results are indicated in Table 4.

 

Table 4: Results of Robustness

SR.NO.

ANALYST I

ANALYST II

1

99.02716

99.2057

2

100.51

99.3763

3

100.0667

99.02716

4

100.1053

98.02734

5

100.1172

99.31679

6

100.397

99.31679

Mean

100.037227

99.0450133

Std. deviation

0.52632396

0.51366797

RSD

0.00526128

0.00518621

%RSD

0.5261281

0.51862073

 

RESULT AND DISCUSSION:

Under experimental conditions described, calibration curve, assay of tablets and recovery studies were performed. Using appropriate dilutions of standard stock solution the two solutions were scanned separately. A critical evaluation of proposed method was performed by statistical analysis of data where slope, intercept, correlation coefficient are shown in Table 3. As per the ICH guidelines, the method validation parameters checked were linearity, accuracy and precision, stability, LOD& LOQ. Beer’s law is obeyed in the concentration range of 50-250 µg/mL and 5-25µg/mL for METO and AMLO, respectively. Correlation coefficient was greater than 0.999 for both the drugs. The proposed methods were also evaluated by the assay of commercially available tablets containing METO and AMLO. The results of formulation analysis are presented in Table 3. Recovery was found in the range of  99.51-99.92% for METO (Table no 1.1) and 99.69-100.6-% for AMLO  (Table  1.2) by ratio derivative method . The present method has been found to be suitable for routine analytical work of the marketed formulation.

 

CONCLUSION:

The validated spectrophotometric method employed here proved to be simple, economical, precise and accurate. Thus it can be used as IPQC test and for routine simultaneous determination of METO and AMLO in tablet dosage form.

 

ACKNOWLEDGEMENT:

The authors wish to express their gratitude to Cipla Ltd. India for gift samples of pure Metoprolol Succinate and amlodipine respectively. The authors are also thankful to the Principal and Management of MAEER’s Maharashtra Institute of Pharmacy, Pune-411038 for providing necessary facilities.

 

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Received on 09.05.2012       Modified on 31.05.2012

Accepted on 09.06.2012      © RJPT All right reserved

Research J. Pharm. and Tech. 5(7): July 2012; Page 950-954