Development and Validation of UV Spectroscopic Method for Simultaneous Estimation of Metoprolol Succinate and Cilostazol

 

Abhishek Chauhan, Ankit Sharma, Nikhil Sharma, Amar Deep Ankalgi,

Mahendra Singh Ashawat

Department of Pharmaceutical Analysis, Laureate Institute of Pharmacy, Kathog, Jawalamukhi 176031, Himachal Pradesh, India.

*Corresponding Author E-mail: ankitsharma.0506@gmail.com

 

ABSTRACT:

In the present work, A Simple, rapid, sensitive, precise, and reproducible, specific UV spectrophotometric method for the determination of Metoprolol succinate and Cilostazol in bulk drug form were developed and validated. The estimated wavelength for Metoprolol succinate was 223nm, while Cilostazol was 257nm. Beer's law applies to Metoprolol succinate and Cilostazol at concentrations of 15-40μg/ml and 6-16μg/ml, respectively with correlation coefficients of 0.9971 and 0.9995. The recovery rates for Metoprolol succinate and Cilostazol were found to be 99.24 - 100.6% and 99.35 - 100.4%, respectively. Intraday precision of Metoprolol succinate and Cilostazol were found to be 0.167 - 0.258 and 0.657 - 0.847 %RSD, while interday precision were found to be 0.118 - 0.287 and 0.426 - 0.747 %RSD respectively. The LOD and LOQ of Metoprolol succinate were found to be 5.971μg/ml and 18.09μg/ml, Cilostazol was found to be 1.075μg/ml and 3.258μg/ml, respectively. Validation of proposed methods was carried out according to ICH Q2R1Guidelines. The proposed methods were found accurate and reproducible for routine analysis of both the drugs.

 

KEYWORDS: Metoprolol succinate, Cilostazol, Simultaneous equation method, UV Spectroscopy, Validation.

 

 

 

INTRODUCTION:

Metoprolol Succinate and Cilostazol are two widely used bulk drugs in the pharmaceutical industry. Metoprolol Succinate, a beta-blocker, is primarily used to treat hypertension, angina pectoris, and heart failure1. Cilostazol, a phosphodiesterase inhibitor, is used to treat intermittent claudication and for secondary prevention of strokes and transient ischemic attacks2. Metoprolol Succinate and Cilostazol combination with both drugs help to treatment of post-operative cardiac diseases. This research paper focuses on the analysis of Metoprolol Succinate and Cilostazol drug combination, with an emphasis on the development and validation of chromatographic methods. The study aims to contribute to the existing knowledge on the analysis of these bulk drugs, providing insights into their analytical methodologies. The findings of this research will be useful for pharmaceutical manufacturers, regulatory agencies, and researchers in the field of pharmaceutical analysis. Hence, there is a scope to develop analytical methods for Metoprolol Succinate and Cilostazol in combination3-5.

 

Figure 1: Structure of Metoprolol succinate and Cilostazol

 

The literature review indicates that a number of analytical techniques, including UV spectrophotometric, High-performance liquid chromatography (HPLC), Stability indicating RP-HPLC, HPTLC, TLC, LC/MS/MS, and UPLC, have been reported for the estimation of Metoprolol Succinate and Cilostazol in pharmaceutical formulations, and bulk drugs6-8. literature review reveals that there isn't a documented technique for estimating both drug together at the same time. Thus, it is deemed worthwhile to create quick, easy, accurate, and exact procedures for estimating Metoprolol Succinate and Cilostazol simultaneously9-13.

 

MATERIAL AND METHOD:

Instrument:

Shimadzu model 1900i double beam UV-visible spectrophotometer two matched quartz cells with 1cm light path was used to measure absorbance of the resulting solutions.

 

Reagents and Materials:

The bulk drug Metoprolol Succinate and Cilostazol was both obtained from (Yarrow Chem products Pvt. Ltd, Mumbai). Methanol was used as a solvent throughout the experimentation.

 

PREPARATION OF SOLUTIONS:

Standard Stock Solution of Metoprolol succinate:

An accurately weighed quantity of Metoprolol succinate (10mg) was transferred into 100ml volumetric flask and dissolved with 25ml with methanol, sonicated for 15 min. diluted up to the mark with Methanol to obtain standard solution having concentration of METO (100μg/ml).

 

 

Standard Stock Solution of Cilostazol:

An accurately weighed quantity of Cilostazol (10mg) was transferred into 100ml volumetric flask and dissolved with 25ml with methanol, Sonicated for 15 min. diluted up to the mark with Methanol to obtain standard solution having concentration of CIL (100μg/ml).

 

Working standard solution of Metoprolol succinate:

From 100μg/ml solution of Metoprolol succinate 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0ml was transferred into 10ml volumetric flask and adjust with methanol up to mark to get the final concentration of 15, 20, 25, 30, 35 and 40μg/ml.

 

Working standard solution of Cilostazol:

From 100μg/ml solution of Cilostazol 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6ml was transferred into 10ml volumetric flask and adjust with methanol up to mark to get the final concentration of 6, 8, 10, 12, 14 and 16μg/ml.

 

SIMULTANEOUS EQUATION METHOD:14

To determine wavelength for measurement, standard spectra Metoprolol succinate and Cilostazol was scanned between 200-400nm against Methanol. The method was based on the measurement of absorbance of Metoprolol succinate and Cilostazol at 223nm and 257nm respectively. This method obeyed Beer’s law in the concentration range of 15-40μg/ml for Metoprolol succinate and 6-16μg/ml for Cilostazol.

 

Cx = A2ay1 - A1ay2 / ay1ax2 - ay2ax1

Cy= A1ax2 - A2ax1 / ay1ax2 - ay2ax1

 

Where,

Cx = Concentration of Cilostazol

Cy = Concentration of Metoprolol succinate

A1 = Absorbance of test at λ1 (λmax of CIL= 257 nm)

A2 = Absorbance of test at λ2 (λmax of METO= 223nm)

ax1 = Absorptivity of x drug (METO) at λ1

ax2= Absorptivity of x drug (METO) at λ2

ay1 = Absorptivity of y drug (CIL) at λ1

ay2= Absorptivity of y drug (CIL) at λ2

 

SPECTROPHOTOMETRIC CONDITION:

Table 1: Spectrophotometric conditions for Spectroscopic Method

Mode

Spectrum

Scan Speed

Medium

Wavelength Range

400-200 nm

Initial base line correction

Methanol

 

PREPARATION OF CALIBRATION CURVE:

For Metoprolol succinate:

An aliquot of stock solution of Metoprolol succinate 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0ml were pipette out in five different 10ml volumetric flasks and further diluted to attain concentration of about 15, 20, 25, 30, 35 and 40μg/ml respectively. Graph of Absorbance Vs Concentration was plotted.

 

For Cilostazol:

An aliquot of stock solution of Cilostazol l 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6 ml were pipette out in five different 10ml volumetric flasks and further diluted to attain concentration of about 6, 8, 10, 12, 14 and 16μg/ml. respectively. Graph of Absorbance Vs Concentration was plotted.

 

METHOD VALIDATION:15

Linearity and Range:

The linearity of Metoprolol succinate and Cilostazol was found to be in the range of 15-35μg/ml and 6-14μg/ml respectively. Calibration curve of Metoprolol succinate and Cilostazol are shown in Figure 3, 4 at 223nm (λmax of Metoprolol succinate), 257nm (λmax of Cilostazol), 231nm (Isoabsorptive point).

 

Accuracy:

To assess the accuracy of the proposed method, recovery studies were carried out three different levels i.e. 80%, 100% and 120%. To the pre-analyzed sample solution, a known amount standard drug solution was added at three different levels, absorbance was recorded. The % recovery was then calculated and results are given in Table 3.

 

Precision:

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions and results are given in Table: 4, 5. Precision may be considered at three levels:  

 

Intraday Precision:

Solutions containing 15, 20, 25μg/ml of METO and 6, 8, 10μg/ml of CILO were analyzed three times on the same day and %R.S. D was calculated.

 

Interday Precision:

Solutions containing 15, 20, 25μg/ml of METO and 6, 8, 10μg/ml of CILO were analyzed on three different successive days and %R.S. D was calculated.

 

Repeatability:

Solutions containing 25μg/ml of METO and 10μg/ml of CILO were analyzed for six times and %R.S.D. was calculated. R.S.D was not more than 2%.

 

LOD and LOQ:

Limit of detection (LOD) and Limit of quantization (LOQ) were determined by using the formula based on the standard deviation of the response and the slope. The LOD and LOQ were calculated by mathematical equation and results are given in Table: 6.

 

LOD= 3.3 σ/s

LOQ= 10 σ/s

 

Where,

S= the slope of calibration curve

σ == the standard deviation of the response

 

Robustness:

The robustness of the method was tested as a function of change in wavelength. Experimental results showed that the change in %RSD value was 0.8% (less than the acceptance limit of <2% RSD). The UV strategy appeared to be durable. The results obtained are presented in Table 7.

 

RESULTS AND DISCUSSION:

The methods were validated with respect to linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy, robustness as per the ICH guidelines.

 

 

Figure 2: Overlain spectra of Metoprolol succinate at 223nm (10 µg/ml) and Cilostazol at 257nm (10µg/ml)

 

Table 2: Linearity

Metoprolol succinate (µg/ml)

Absorbance

Cilostazol (µg/ml)

Absorbance

223nm

257nm

223nm

257nm

15

0.347

0.031

6

0.079

0.234

20

0.486

0.036

8

0.091

0.297

25

0.592

0.041

10

0.103

0.354

30

0.697

0.045

12

0.114

0.409

35

0.810

0.049

14

0.123

0.472

 

 

 

Figure 3: Calibration curve of Metoprolol succinate at 223 nm and 257nm

 

Figure 4: Calibration curve of Cilostazol at 223 nm and 257nm

Table 3: Accuracy and Recovery study data

Name of Drug

% Level of recovery

Amount of drug Taken (µg/ml)

Amount of drug Added (µg/ml)

Total amount Taken (µg/ml)

Total amount found (µg/ml)

% Recovery (n=3)

METO

(223nm)

80

20

15

35

34.87

99.64

100

20

20

40

40.24

100.6

120

20

25

45

44.87

99.72

METO

(257nm)

80

20

15

35

35.15

100.4

100

20

20

40

39.69

99.24

120

20

25

45

45.15

100.3

CILO

(257nm)

80

8

6

14

13.96

99.71

100

8

8

16

16.07

100.4

120

8

10

18

17.96

99.77

CILO

(223nm)

80

8

6

14

14.05

100.3

100

8

8

16

15.89

99.35

120

8

10

18

18.05

100.2

 

Table 4: Precision study of Metoprolol succinate

Intraday precision of Metoprolol succinate (n=3)

Metoprolol succinate(223nm)

Metoprolol succinate(257nm)

Conc. (µg/ml)

Mean Absorbance ±SD

% RSD

Conc. (µg/ml)

Mean Absorbance ±SD

% RSD

15

0.346±0.0005

0.167

15

0.030±0.0005

1.883

20

0.487±0.0011

0.237

20

0.035±0.0005

1.634

25

0.591±0.0015

0.258

25

0.040±0.0005

1.420

Interday precision of Metoprolol succinate (n=3)

15

0.348±0.010

0.287

15

0.031±0.0057

1.823

20

0.487±0.0005

0.118

20

0.037±0.0005

1.546

25

0.593±0.0011

0.195

25

0.041±0.0005

1.386

Repeatability of Metoprolol succinate (n=6)

25

0.591±0.0018

0.316

25

0.041±0.0007

1.829


Table 5: Precision study of Cilostazol

Intraday precision of Cilostazol (n=3)

Cilostazol(223nm)

 Cilostazol(257nm)

Conc. (µg/ml)

Mean Absorbance ±SD

% RSD

Conc. (µg/ml)

Mean Absorbance ±SD

% RSD

6

0.078±0.0005

0.734

6

0.232±0.0015

0.657

8

0.091±0.0015

1.672

8

0.296±0.0020

0.702

10

0.102±0.0010

0.980

10

0.354±0.0030

0.847

Interday precision of Cilostazol (n=3)

6

0.080±0.0011

1.437

6

0.235±0.0010

0.426

8

0.092±0.0015

1.648

8

0.299±0.0020

0.695

10

0.103±0.0005

0.559

10

0.354±0.0026

0.747

Repeatability of Cilostazol (n=6)

10

0.103±0.0014

1.373

10

0.353±0.0023

0.655

 

Table 6: Limit of Detection and Limit of Quantification

Parameter

Metoprolol succinate

Cilostazol

223nm

257nm

LOD (µg/ml)

5.971

1.075

LOQ (µg/ml)

18.09

3.258

 

Table 7: Robustness

Name of Drugs

Wavelength

(nm)

Mean Absorbance ±SD

% RSD

METO

221

0.580±0.0011

0.149

223

0.591±0.0015

0.258

225

0.605±0.0010

0.165

CILO

255

0.349±0.0020

0.573

257

0.354±0.0030

0.847

259

0.357±0.0005

0.162

Table 8: Summary of Validation Parameter

Sr. No

Parameters

Metoprolol succinate

Cilostazol

Acceptance Criteria

1

Wavelength (nm)

223

257

-

2

Beer’s Law Limit (μg/ml)

15-40

6-16

-

3

Regression equation (y = mx +c)

0.0227x+0.0179

0.0293x+0.0604

-

4

Correlation Coefficient (r˛)

0.9971

0.9995

NLT 1

5

Intraday Precision (%RSD, n=3)

0.167 - 0.258

0.657 - 0.847

% RSD NMT 2.0 %

6

Interday Precision (% RSD, n=3)

0.118 - 0.287

0.426 - 0.747

7

Repeatability (% RSD, n=6)

0.316

0.655

8

Accuracy (% Recovery) (n=3)

99.24 - 100.6

99.35 - 100.4

NLT 98.0 % to NMT 102.0 %

9

LOD (μg/ml)

5.971

1.075

-

10

LOQ (μg/ml)

18.09

3.258

-

11

Robustness (n=3)

0.149 - 0.258

0.162 - 0.847

% RSD NMT 2.0 %

 

CONCLUSION:

The developed UV spectrophotometric method in that linearity, precision, accuracy, LOD, LOQ, and robustness were found to be more accurate, precise, and reproducible. The method were found to be simple and time-saving could be applied for routine analysis in quality control laboratories for simultaneous estimation of Metoprolol succinate and Cilostazol.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGEMENT:

The authors are thankful Dr. Amar Deep Ankalgi,       Dr. M.S. Ashawat Director and Principal of Laureate Institute of Pharmacy, for giving all facilities for my research work, as well as Yarrow Chem Products Pvt. Ltd, Mumbai, for providing drug samples Metoprolol succinate and Cilostazol to carry out this work.

 

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Received on 27.05.2025      Revised on 23.07.2025

Accepted on 05.09.2025      Published on 08.10.2025

Available online from October 15, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(4):273-278.

DOI: 10.52711/2231-5675.2025.00043

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