RP-HPLC Analysis for Quantitation of Candesartan Cilexetil in Solid Dosage Forms

 

Revathi R.1*, Saravanan V.S.1, Ethiraj T.2, Jhansi Lakshmi Marreddy1

1Department of Pharmaceutical Analysis, The Erode College of Pharmacy and Research, Erode, Tamil Nadu, India.

2Department of Pharmaceutics, The Erode College of Pharmacy and Research,  Erode, Tamil Nadu, India.

*Corresponding Author E-mail revathethiraj@gmail.com

 

 

ABSTRACT:

A simple reverse phase liquid chromatographic method was developed and validated as per the ICH guidelines for the quantitative determination of candesartan cilexetil in pharmaceutical dosage forms. The mobile phase consisted of 0.02 M mono basic potassium phosphate buffer: acetonitrile: triethyl amine (40:60:0.2) and adjust pH to 6.0 with ortho phosphoric acid. The eluent was monitored at 254 nm, at a flow rate of 2 mL/min and retention time was observed at 9.153 min. The linearity was observed from 0.08071-0.24213 mg/mL with R2= 0.998. The LOD and LOQ were found to be 0.00005 mg/mL and 0.00017 mg/mL respectively. This method was also validated and can be successfully applied for routine analysis.

 

KEYWORDS: Candesartan cilexetil, RP-HPLC, Validation.

 

 

INTRODUCTION:

Candesartan cilexetil (Fig 1) is chemically, 2-ethoxy-3-[21-(1H-tetrazol-5-yl)-4-yl methyl]-3H- benzoimidazole-4-carboxylic acid 1-cyclohexyloxy carbonyl oxy ethyl ester1. It has a molecular formula of C33 H34 N6 O6 and a molecular weight of 610.67. It acts by blocking the vasoconstrictor aldosterone secreting effects of angiotensin II by selectively blocking the binding of angiotensin II to the AT1 receptor in many tissues such as vascular smooth muscle and the adrenal gland. A stability indicating LC method was developed for candesartan cilexetil2. Simple and sensitive HPLC method for simultaneous analysis of candesartan cilexetil and hydrochlorthiazide was developed3,4. HPTLC densitometric method and Q-absorbance ratio method for analysis of candesartan cilexetil and hydrochlorothiazide were developed5,6. First derivative UV spectroscopic method for determination of candesartan cilexetil and dissolution testing were also prescribed7.

 

Few literatures revealed about its pharmacological action8,9. The objective of this work is to develop simple, accurate, precise and economic RP-HPLC method with less cost to estimate the candesartan cilexetil in pharmaceutical dosage forms.

 

Fig 1: Chemical Structure of Candesartan Cilexetil.

 

MATERIALS AND METHODS:

HPLC Instrumentation and conditions

The Experiments were conducted on Agilent Technology (1100 series) HPLC system with G1329 A Auto injector and G1314 A multi wavelength detector. The chromatographic studies were performed using Inertsil ODS-3 C18 column (250 × 4.6 mm) with 5 µm particle size at ambient temperature and eluted with mobile phase consisting of 0.02 M mono basic potassium phosphate buffer: Acetonitrile: Triethyl amine (40:60:0.2) and adjusted pH to 6.0 with phosphoric acid at a flow rate of 2 mL/min. The run time was 14 mins.  The mobile phase was filtered through 0.45 µm nylon filter and degassed in ultra Sonicator prior to use10.

 

An absorption maximum of pure drug was scanned over the range 200 - 400 nm by using Shimadzu UV-2456 UV-Visible spectrophotometer11. Chromatograms were made with injection volume of 20 µL and a wavelength of 254 nm.

 

Preparation of buffer solution

Accurately 2.72 gms of mono basic potassium phosphate was dissolved in 1000 mL of Mille-Q water, filtered through 0.45 µm nylon membrane filter and degassed.

 

Preparation of working standard solution

The stock solution of candesartan cilexetil was prepared by weighing 32 mg of working standard which is transferred in to a 200 mL volumetric flask and dissolved in 100 mL of diluent. The solution was sonicated for 15 min and then the volume was made up to 200 mL with diluent and mixed.

 

Preparation of sample solution

Exactly 20 tablets were weighed and grinded to fine powder. A quantity of powder equivalent to 16 mg of candesartan cilexetil was transferred into a 100 mL volumetric flask and dissolved in 50 mL of mobile phase. The solution was sonicated for 15 min and shaken for 30 min. Then diluted to volume with mobile phase and mixed. The solution was filtered through 0.45 µ membrane filter by discarding the first 5 mL. The filtrate was injected for the further analysis.

 

Validation of proposed method

Once the chromatographic and the experimental conditions were established, the method was validated by the determination of the following parameters: specificity, system suitability, linearity, precision, accuracy, robustness, limit of detection (LOD), limit of quantitation (LOQ), solutions stability,  following the ICH guidelines (Q2 (R1)12.

 

Specificity

Specificity of an analytical method is its ability to measure accurately and specifically the analyte of interest without interference from placebo and degradation products. The specificity of the method was established by injecting blank, placebo and standard solution in triplicate and recording the chromatograms.

 

System suitability

To ensure the validity of the analytical procedure, a system suitability test was established. Data from five injections of 20 µL of the working standard solution containing 0.160 mg/mL were used for the evaluation of the system suitability parameters like tailing factor, number of theoretical plates and retention time by chemstation software.

 

Linearity

Linearity is the ability of the method to respond proportionally to the changes in the concentration of the analyte in a sample. Linearity studies were conducted by using series of standard solutions of candesartan cilexetil at concentration levels from 50- 150 % of the target concentration (50 %, 75 %, 100 %, 125 % and 150 %). The calibration curve was obtained by plotting the concentration against peak area of the each standard solution. The five concentration levels were subjected to regression analysis to calculate calibration equation and correlation coefficient.

 

Precision

The precision of the method was determined by repeatability (intra-day) and intermediate precision (inter-day). Repeatability was determined by performing six repeated analysis of the same working solution of candesartan cilexetil, on the same day, under the same experimental conditions. The intermediate precision of the method was assessed by carrying out the analysis on different days and also by another analyst performing the analysis in the same laboratory (between-analysts).

 

Accuracy

Accuracy of a method is defined as the closeness of a measured value to the true value. The recovery studies were carried out at 50 %, 100 %, and 150 % of the target level in the tablet in triplicate each in the presence of placebo. The recovery was calculated with respect to the standard deviation.

 

Limit of detection and limit of quantitation

Limit of detection (LOD) and limit of quantitation (LOQ) were calculated based on the ICH guidelines.

 

Robustness

Robustness was determined by minor changes in the analytical method parameters such as by changing the flow rate from 1.8 mL/min to 2.2 mL/min, the mobile phase composition with buffer pH 6.0: acetonitrile: triethylamine (46:54:0.2, 40:60:0.2, 34:66:0.2), and pH from 5.8 to 6.2.

 

RESULTS AND DISCUSSION:

The reverse phase liquid chromatography method was developed and validated for candesartan cilexetil in uncoated tablets. The best chromatographic conditions were adequately selected (Table 1). A typical chromatogram of candesartan cilexetil (Fig 2) standard solution shows a symmetrical peak, well separated from the solvent front. The retention time observed at 9.153 min and percentage content of candesartan cilexetil in formulation was found to be 96.93 %.

 

 

Fig 2: Typical RP-HPLC chromatogram of candesartan cilexetil

 

 

Table 1: Optimized chromatographic conditions for the proposed method

S.N

Parameters

Optimized condition

1

Column

Inertsil ODS C-18 (250×4.6 mm, 5 µ)

2

Mobile phase

 

0.02 M mono basic potassium phosphate buffer: acetonitrile: triethylamine (40:60:0.2) and adjust pH to 6.0 with phosphoric acid.

3

Flow rate(mL/min)

2

4

Injection volume(µL)

20

5

Detection wavelength (nm)

254

6

Temperature

Ambient

7

Retention time(min)

9.153

8

Runtime(min)

14

 

A system suitability test was applied to representative chromatograms for various parameters. The results obtained were within acceptable limits (Table 2). Thus, the system meets suitable criteria. Specificity was observed that there was no interference from the placebo with the analyte peak.

 

Table 2: System suitability test parameters for the proposed method

S.N

Parameters

values

1

Theoretical plates

3459

2

Tailing factor

1.48

3

Retention time(min)

9.153

 

The calibration curve was obtained for a series of concentration in the range of 0.080 – 0.240 mg/mL and it was found to be linear (Fig 3) with regression equation of Y=22023X+10.45. LOD and LOQ were obtained from the slope and the standard deviation of the intercept from three calibration curves determined by a linear regression line as defined by ICH. The limit of detection and limit of quantitation were found to be 0.00005 mg/mL and 0.00017 mg/mL respectively.

 

Fig 3: Calibration curve of candesartan cilexetil

 

The precision was measured in terms of intraday precision and interday precision, which were determined by sufficient number of aliquots of a homogeneous sample. The % RSD values not more than 2 indicated that the method was precise (Table 3). This showed that the precision of the method was satisfactory. The recovery was calculated with respect to the standard deviation (Table 4). The mean % recovery of candesartan cilexetil at each level was not less than 98 % and not more than 102 %. These results indicated that the method had an acceptable level of accuracy.


 

Table 3: Precision study data

Drug

Intraday precision

Interday precision

Amount  found* (mg)

% Content

% RSD

Amount found* (mg)

% Content

% RSD

Candesartan cilexetil

7.76

96.93

1.63

7.79

97.39

1.55

*(n=5)

 

Table 4: Recovery for the proposed RP-HPLC method

Concentration of spiked level

Amount added (mg/mL)

Amount found (mg/mL)

% Recovery*

% RSD

50 %

0.08121

0.08034

98.98

0.0716

0.08045

0.08038

100 %

0.16243

0.16193

99.69

0.0252

0.16190

0.16199

150 %

0.24364

0.24628

101.08

0.0114

0.24633

0.24629

*(n=3)

 

 

The robustness was determined by analyzing the same sample under a variety of conditions. The factors considered were: variations in the flow rate, organic ratio of mobile phase and pH. The results and the experimental range of the selected variables were given in Table 5, together with the optimized values. There were no significant changes in the chromatographic pattern when the above modifications were made in the experimental conditions, showing thus that the method is robust.

 

Table 5: Robustness

Parameters

% RSD of Retention time*

Ratio of organic mobile phase

10 % Less

0.820

10 % More

0.2760

 

Flow rate

1.8 ml/min

0.1122

2.2 ml/min

1.3485

 

pH

5.8

0.6638

6.2

0.1082

*(n=3)

 

CONCLUSION:

The proposed high performance liquid chromatographic method has been evaluated and validated for linearity, precision, accuracy, specificity and proved to be simple, rapid, convenient and effective for the routine analysis of candesartan cilexetil in tablet dosage forms.

 

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3.        Quatab S S,  Razzaq S N, Ashfaq M, Shuja Z A  and Khan I U.(2007). Simple and sensitive LC-UV method for simultaneous analysis of hydrochlorothiazide and candesartan cilexetil in pharmaceutical formulations, Acta chromatographica.19; 2007: 119-129.

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8.        Akira Matsumori. Efficacy and safety of oral candesartan cilexetil in patients with congestive heart failure, The European Journal of Heart Failure.5; 2003: 669–677.

9.        Nagisa Y, Shintani A and Nakagawa S. The angiotension II receptor antagonist candesartan cilexetil (TCV-116) ameliorates retinal disorders in rats, Diabetologia. 44; 2001: 883-888.

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11.     Hobart H Willard, Lynne L Merritt, Jr. John A Dean, Frank A Settle. Instrumental methods of analysis. CBS publishers and Distributors, New Dehli. 1986. pp.580-655.

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Received on 24.10.2013          Accepted on 01.12.2013        

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Asian J. Pharm. Ana. 3(4): Oct. - Dec. 2013; Page 115-118