Development and Validation of Q-Absorbance UV Spectrophotometric Method for Simultaneous estimation of Amlodipine Besylate and Candesartan Cilexetil In bulk drugs

 

Prof. Sushil D. Patil*, Rohit B. Badhan*, Dr. Sanjay J. Kshirsagar.

MET’s Institute of  Pharmacy, MET League of Colleges, Bhujbal Knowledge City, Adgaon, Nashik, Maharashtra State 422003, India

*Corresponding Author E-mail: sushilpharma@rediffmail.com

 

ABSTRACT:

A new, simple, accurate and sensitive UV-spectrophotometric absorption correction method has been developed for simultaneous determination of Candesartan Cilexetil and Amlodipine Besylate in bulk utilizing concept of standard addition. The method is based upon determination of Amlodipine Besylate at 237 nm & Candesartan Cilexetil at 254 nm methanol as a solvent. Overlay spectra of both drugs shows absorbance at 244 nm. Linearity was observed in range of 10-60 µg/ml and 2-10 µg/ml for Candesartan Cilexetil and Amlodipin Besylate respectively. The correlation coefficient value was found to near to 1. All methods were statistically validated as per ICH guidelines.

 

KEYWORDS: Candesartan Cilexetil, Amlodipine Besylate, UV-spectrophotometric, Simultaneous estimation, Method Development and Validation, ICH Guideline.

 

 


1. INTRODUCTION:

Candesartan Cilexetil:

Candesartan is a angiotensin (II) receptor blocker is alone or with other anti hypertensive agent to treat hypertension. 2-ethoxy-1-({4-[2-(2H-1,2,3,4-tetrazol-5 yl)phenyl]phenyl}methyl)-1H-1,3-benzodiazole-7-carboxylic acid. The chemical structure of Candesartan Cilexetil shown in Fig.1.[1]

 

Fig 1: Structure of Candesartan Cilexetil.

 

Amlodipine Besylate:

Amlodipine is a calcium channek blocker, it act as anti angina agent. chemically is 3-ethyl 5-methyl (4RS)-2-[(2-aminoethoxy) methyl]-4-(2chlorophenyl)-6-methyl-1, 4dihydropyridine-3, 5-dicarboxylate benzene sulphonate, The chemical structure of Amldipine Besylate shown in Fig.2.[1]

 

Fig 2: Structure of Amlodipine Besylate

 

As per literature review, several method was developed in individual /plain drug but this combination one or two method was developed. Candesartan Cilexetil and Amlodipine Besylate was estimated by only few method UV spectroscopy [2,3, 5], HPLC [4]. Only one stability indicating RP-HPLC method. The aim of present work was to develop and validate a accurate, cost effective and precise UV spectroscopy by simultenious equation method,Q-absorbance ratio method , RP-HPLC method for determination Candesartan Cilexetil and Amlodipine Besylate

 

2. MATERIALS AND METHODS:

2.1    Apparatus and Equipments:

A Shimadzu UV–visible spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) was used for all absorbance measurements with 1 cm paired quartz cell.

 

2.2    Reagents and Chemicals:

Pharmaceutical grade Candesartan Cilexetil and Amlodipine Besylate was supplied as a gift sample from Glenmark Pharmaceutical Pvt. Ltd.Mumbai, India. Methanol used in analysis was of Analytical grade and all other chemicals and reagents were of analytical grade and were purchased from Thomas Baker (Chemicals), Mumbai, India.

 

2.3 Preparation of standard stock solution :( 100µg/mL):

An accurately weighed quantity of 10 mg Candesartan Cilexetil was transferred to 100 mL volumetric flask, dissolved with 10 mL of methanol and sonicated for 15 min, volume was then made up to the mark with methanol.

 

An accurately weighed quantity of 10 mg Amlodipine Besylate was transferred to 100 mL volumetric flask, dissolved with 10 mL of methanol and sonicated for 15 min, volume was then  made up to the mark with methanol.

 

2.4 Selection of wavelength for analysis:

By appropriate dilution of standard stock solution, solution containing 10 μg/ml of CAND and 10 μg/ml AMLO separately, these diluted solutions were scanned in range 200-400 nm separately. CAND showed λmax at 254 nm in Methanol. (figure 3) and AMLO shows λmax at  237 nm (figure 4).

 

 

Fig 3. UV Spectra of Candesartan Cilexetil

 

 

Fig 4. UV Spectra of Amlodipine Besylate

 

 

Figure 5 : Overlay UV spectra of AML and CAN

2.6 Method validation:

The UV Spectrophotometric method was validated as per ICH guidelines for method validation. The performance parameters like linearity, precision and accuracy were evaluated. [9]

 

2.6.1.1 Limit of Detection (LOD):

LOD is the lowest level of concentration of analyte in the sample that can be detected, though not necessarily quantitated. It was calculated by using the formula, [8, 9]               

 

LOD= 3.3σ/S

Where,

σ = Standard deviation of the response,

S = Slope of calibration curve.

 

2.6.1.2 Limit of Quantitation (LOQ):

LOQ is the lowest concentration of analyte in a sample that may be determined with acceptable accuracy and precision when the required procedure is applied. It was calculated by using the formula, [9]  

 

LOQ=10σ/S

Where,

σ = Standard deviation of the response,

S = Slope of calibration curve.

 

Linearity:

Linearity was studied by diluting standard stock solution of Candesartan Cilexetil 10-50μg/ml and Amlodipine Besylate 2-10μg/ml concentrations (n=3). Calibration curves with concentration verses absorbance were plotted at their respective wavelengths and the obtained data was subjected to regression analysis using the least square method. The standard curves for Candesartan Cilexetil and Amlodipine Besylate are shown in (Fig. 6, 7) respectively and data is presented in Table 2. [8]

 

Table 1: Calibration study data CAND

Sr. No

Concentration (µg/mL)

Absorbance at 227 nm

1

10

0.291

2

20

0.552

3

30

0.799

4

40

1.046

5

50

1.321

 

Table 2: Calibration study data CAND

Sr.No

Concentration (µg/mL)

Absorbance at 227 nm

1

2

0.067

2

4

0.144

3

6

0.215

4

8

0.291

5

10

0.37

 

 

Fig 6: Calibration curve of Candesartan (10-50 µg/mL)

 

 

Fig 7: Calibration curve of Amlodipine Besylate (2-10 µg/mL)

 

2.7.1 Specificity:

Specificity is the ability to measure unequivocally the desired analyte in the presence of components such as excipients and impurities.[6-9]

 

Here, Lactose Monohydrate was used as excipient and which is prepare a stock solution (B) (100µg/mL). Spiking Lactose Monohydrate in 3 different levels 80,100,120% respectively from stock solution B and stock solution (A) which is spike 100% and mix with 3 different level of Lactose Monohydrate solution to determine the amount of  ℅ recovery at 244nm. 

 

Accuracy:

To check the accuracy of the developed methods and to study interference of formulation additives, analytical recovery experiments were carried out by using standard addition method. Reference standard solution of each drug was added to tablet samples at three different concentrations level (80, 100 and 120%). At each level, samples were prepared in triplicate and the mean percentage recoveries and % RSD value were calculated. Table .6 shows the result for accuracy of the method.[6-9]


 

Table 3: % Recovery studies for specificity of CAND

Sr. No.

Conc. Of API(µg/mL)

Add % level

Spike stock sol.(µg/mL)

Abs. at 244 nm

Drug found (µg/mL)

% Recovery

1

20

80

16

0.479

17.76

88.80

2

20

100

20

0.475

17.60

88.00

3

20

120

24

0.478

17.72

88.60

 

Table 4: % Recovery studies for specificity of AMLO

Sr. No.

Conc. Of API(µg/mL)

Add % level

Spike stock sol.(µg/mL)

Abs. at 244nm

Drug found (µg/mL)

% Recovery

1

6

80

4.8

0.203

5.702

95.04

2

6

100

6

0.213

5.972

99.54

3

6

120

7.2

0.209

5.864

97.74


 

 


Table 5: Accuracy study data (Plane drug + Product) of CAND

Sr. No.

API(A) (µg/mL)

Add % level

API(B) (µg/mL)

Absorbance at 244nm

Conc. found for both (A+B)

% Recovery

Avg. % Recovery

Standard Deviation

1

20

 

80

16

0.850

32.6

90.55

90.29

0.4474

2

20

16

0.842

32.28

89.66

3

20

16

0.851

32.64

90.66

4

20

 

100

20

0.920

35.4

88.50

88.80

0.2160

5

20

20

0.925

35.6

89.00

6

20

20

0.924

35.56

88.90

7

20

 

120

     24

0.988

38.12

86.63

87.09

0.4131

8

20

24

0.992

38.28

87.00

9

20

24

0.999

38.56

87.63

 

Table 6: Accuracy study data (Plane drug + Product) of AMLO

Sr. No.

API(A) (µg/mL)

Add % level

API(B) (µg/mL)

Absorbance at 244nm

Conc. found for both (A+B)

% Recovery

Avg. % Recovery

Standard Deviation

1

6

 

80

4.8

0.360

9.94

92.09

93.76

0.9737

2

6

4.8

0.366

10.10

93.59

3

6

4.8

0.374

10.32

95.59

4

6

 

100

6

0.399

11.00

91.66

92.41

0.5617

5

6

6

0.403

11.10

92.56

6

6

6

0.405

11.16

93.01

7

6

 

120

7.2

0.460

12.64

95.82

96.46

0.8245

8

6

7.2

0.469

12.89

97.66

9

6

7.2

0.468

12.86

97.46

 


Precision:

It is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings. It was determined by studying repeatability, intra-day and inter-day precision of method. The average recovery of the analyte of 80%, 100% and 120% solution.

 

For intraday, the analysis was carried out at different intervals on the same day and for inter day, the analysis was carried on different days. Table 8 and 9 give the results for intraday and inter-day studies respectively.[8,9]

 

Table 7: Precision study data CAND

Sample No.

% Assay

Intraday

Interday

1

98.94

97.21

2

97.75

97.68

3

97.60

97.88

Mean

98.09

97.59

SD

0.5993

0.2808

%RSD

0.611

0.2877

 

 

 

Table 8: Precision study data AMLO

Sample No.

% Assay

Intraday

Interday

1

90.83

90.21

2

91.14

90.58

3

91.75

93.88

Mean

91.57

91.55

SD

0.8415

1.6491

%RSD

0.9189

1.8018

 

Ruggedness:

Ruggedness of the proposed method is determined by analysis of aliquots from homogenous slots by different analysts using similar operational and environmental conditions. The results are shown in Table.10 and Table.11.[8]

 

Table 9: Robustness study data at different wavelength of CAND

Sample No.

Wavelength (nm)

243

245

248

1

0.522

0.520

0.519

2

0.519

0.521

0.516

3

0.523

0.518

0.515

Avg. %Recovery

97.26

93.93

96.33

SD

0.3398

0.2492

0.3398

%RSD

0.3493

0.2571

0.3527

Table 10: Robustness study data at different wavelength of AMLO

Sample No.

Wavelength (nm)

240

245

250

1

0.173

0.180

0.190

2

0.174

0.182

0.188

3

0.177

0.183

0.187

Avg. %Recovery

82.28

85.43

88.43

SD

0.7655

0.5617

0.5617

%RSD

0.9303

0.6575

0.6351

 

3.RESULTS AND DISCUSSION:

Development and optimization of the spectrophotometric method:

Proper wavelength selection of the methods depends upon the nature of the sample and its solubility. To develop a rugged and suitable spectrophotometric method for the quantitative determination of Candesartan Cilexetil, the analytical condition were selected after testing the different parameters such as diluents, buffer, buffer concentration, and other chromatographic conditions.

 

Our preliminary trials were by using different compositions of diluents consisting of methanol best result was obtained and degassed in an ultrasonic bath. Below figure represent the spectrum (Figure 1).

 

3.1  Selection of wavelength:

Scan standard stock solution in UV spectrophotometer between 200 nm to 400 nm on spectrum mode, using Methanol as a blank. Candesartan Cilexetile shows λmax at 254nm and Amlodipine Besylate shows λmax at 237nm.The combine overlay of both drugs shows λmax at 244nm. The proposed analytical method is simple, accurate and reproducible (Figure 3, 4, 5).

 

3.2 Method validation:

The set parameter the method is validated, the Linearity range was found to be 10 to 50 μg/ml for CAND(Table 1, and Fig. 6)  and 2 to 10 μg/ml for AMLO.(Table 2, and Fig. 7).Method was found to be specific for the CAND (Table 3) and AMLO (Table 4). For accuracy data supported by ANNOVA test with at P ≤ 0.05 and other data indicates that the results lie between 97.79-99.71% for both the drugs (Table 5 and 6).The precision confidence interval of 99% was considered precise and the %RSD values for the repeatability and intermediate precision studies were <0.611%,<0.287% and <0.918%,<1.801%, for CAND and AMLO respectively shown in (Table 7 and 8). The evaluation of robustness at different wavelength (Table 9 and 10), the value of the test preparation solution of robustness was not affected and it was in accordance with that of actual. Hence the analytical method would be concluded as robust.

 

 

 

Table 11: Result summary of various validation parameter

Parameter

Values for EMPA

Values for MET

Linearity and Range (µg/mL)

1-3 µg/mL

2-10 µg/mL

Accuracy (%Recovery)

99.44%

93.27%

ANOVA

P≤ 0.05

P≤ 0.05

Precision (%RSD)

RSD< 2%

RSD< 2%

 

4.CONCLUSION:

The developed UV methods were found to be more accurate, precise and reproducible. These statistical parameter of these methods shows good results. The recovery studies revealed excellent accuracy and high precision than previous reported method. The method was found to be simple & time saving. Two proposed methods such as simultaneous equation and Q-absorbance ratio method could be applied for routine analysis in quality control laboratories.

 

5. ACKNOWLEDGMENT:

The authors are thankful to the management and trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Nashik, for providing necessary chemicals and analytical facilities and to Glenmark Pharmaceutical Pvt. Ltd. Mumbai, India, for providing pharmaceutical grade Candesartan and Amlodipine Besylate as gift sample.

 

6. REFERENCE:

1.     M.Bindu and G.Kumaraswamy Method Development and Validation of simultaneous estimation of Amlodipine and Candesartan by RP-HPLC in Tablet dosage forms.Indo American J Pharm Reach .2014:4(10):3922-3928

2.     N. Padmaja and G. Veerabhadram. Development and validation of analytical method for simultaneous estimation of Empagliflozin and linagliptin in bulk drugs and combined dosage form using UV-visible spectroscopy. Scholar Research Library. 2015; 7(12):306-312

3.     B.Kotecha, M.Pambhar .Q-absorbance ratio spectrophotometer method for the Simulteneous estimation of Amlodipine Besylate and Candesartan Cilexetil in synthetic mixture. Pharmatutor Magazine, 2014;2(5);167-178

4.     R.Khaire and J.Landge.Method Development and Validation of Candesartan by RP-HPLC Int J Pharm P’ceutical Reach.2016:6(3):345-360

5.     A.Kumar Sen and Denish N.Hinsu .Analytical method development and validationfor simultaneous estimation of Teneligliptine hydrobromide hydrate and Metformin hydrochloride from it’s pharmaceutical dosage form by three different UV spectrophotometric method Journal of Applide P’ceutical Scie.2016:6(09),157-165

6.     Siladity Bahera. UV visible Spectrophotometric method development and validation of assay of paracetamol and tablet formulation. Analytical and Bioanalytical Technique. 2012; 3(6):3-6.

7.     ICH Harmonized Triplicate Guidelines, “Validation of analytical procedures: text and methodology, Q2 (R1),” in International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2005.

8.     International Conference of Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of Analytical Procedures: Methodology, Adopted in Geneva (1996).

 

 

 

 

 

 

 

 

 

 

 

Received on 12.12.2017          Accepted on 28.01.2018        

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(1): 53-57.

DOI:  10.5958/2231-5675.2018.00010.8