STANDADADIZATION AND STABILITY INDICATING STUDIES BY RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF CANDESARTAN CILEXETIL & HYDROCHLORTHIAZIDE IN TABLET DOSAGE FORM

STANDADADIZATION AND STABILITY INDICATING STUDIES  BY RP-HPLC  METHOD  FOR  THE  SIMULTANEOUS  ESTIMATION   OF CANDESARTAN CILEXETIL & HYDROCHLORTHIAZIDE  IN  TABLET DOSAGE FORM

G. Mani Kumar* ¹ and J.V.L.N. Seshagiri Rao ²

School of Pharmaceutical Sciences & Technologies ¹, J.N.T.U Kakinada, Andhra Pradesh, India.

University College of Pharmaceutical Sciences ², Andhra University, Visakhapatnam, Andhra Pradesh India.

ABSTRACT: A rapid, selective, precise, accurate, rugged and robust high performance liquid chrmatgraphic (HPLC) method for the simultaneous determination of Candesartan Cilexetil & Hydrochlorthiazide in tablet dosage form .The method was validated according to ICH and FDA guidelines. The chromatography is performed on a Kromasil C8, 150 x 4.6 mm, 5µm, in a gradient mode with a mobile phase of Water, acetonitrile and Trifluracetic acid in different ratios. UV-Visible detector at 285 nm was found to be suitable for detection. Linearity was observed in the range of 70 -130μg/ml with correlation coefficient of 0.9999. Sensitivity, accuracy, range, precision, robustness, ruggedness, stability, specificity, limit of detection, limit of quantification and system suitability parameters were validated for the developed method. The developed method was successfully applied to estimate the amounts in pharmaceutical formulations and for the stability indicating studies.

Candesartan Cilexetil & Hydrochlorthiazide , HPLC, Chromatography

INTRODUCTION: Candesar – H tablets (Ranbaxy Laboratories Ltd  India) ^1-3, which contain candesartan cilexetil ^1-6 and hydrochlorothiazide ^2-10, are one of the most commonly used formulations for treatment of high blood pressure when one medicine is not sufficiently effective. Hydrochlorothiazide: 6-chloro - 1, 1 - dioxo- 3, 4-dihydro-2H-1$l ^ {6}, 2, 4-benzothiadiazine-7-sulfonamide (Fig.1) with molecular Formula and Molecular weight of C₇H₈ClN₃O₄S₂ &297.741; is a diuretic.

Several analytical methods, including LC–MS ¹¹ Voltametry ¹², Spectrophotometry ^{13-16, 25} and HPLC ^{17-21, 24-26}, have already been reported for its determination, either alone or in combination with other drugs. Candesartan cilexetil: 2-ethoxy-1-({4-[2-(2H-1, 2, 3, 4-tetrazol-5-yl) phenyl] phenyl} methyl)-1H-1, 3-benzodiazole-7-carboxylic acid (Fig.2).  With molecular weight formula and weight of C₃₃H₃₄N₆O₆ &610.67 is a non-peptide angiotensin II receptor.

The literature contains very few methods for analysis of candesartan cilexetil; those reported include HPLC with fluorimetric detection ²² and spectrofluorimetry. HPLC ²⁵ and ratio derivative Spectrophotometry methods have been used for simultaneous determination of the two compounds. The HPLC method used UV – VIS detection for simultaneous quantification of hydrochlorothiazide and candesartan cilexetil. The retention time was 4.5 mins and 10.6 mins. And stability indicating studies proves here that this method is stabile. The main objective of this study is to produce a quick, reproducible and stable method for the routine, simultaneous analysis of candesartan cilexetil and hydrochlorothiazide.

FIG 1. MOLECULAR STRUCTURE OF HYDROCHLOROTHIAZIDE

FIG 2. MOLECULAR STRUCTURE CANDESARTAN CILEXETIL

Mobile phase B: Prepare a mixture of Water, acetonitrile and Triflouroacetic acid in the ratio of 20:80:0.1%v/v/v and sonicated to degas.

THE GRADIENT PROGRAMMING IS AS FOLLOWS:

 Injection volume: 10 µL

Column: Kromasil C8, 150 x 4.6 mm, 5µm

Column temperature: 30°C

Flow rate: 1.0 ml/min

Diluent: Methanol: water (80:20)

Detector wave length: 285nm for Hydrochlorothiazide and candesartan

Run time: 20mins

Observation:

Hydrochlorothiazide: 4.5 mins

Candesartan cilexetil: 10.6mins

Candesartan cilexetil and Hydrochlorothiazide peaks are found sharp and they are symmetrical.

 Standard Preparation: Accurately weigh and transfer about 30mg of Candensartan Cilexetil working standard and 12.5mg of Hydrochlorothiazide workig standard in to a 50 ml volumetric flask. Add 30ml of diluent. Sonicate to dissolve and make up to the volume with diluent. Filter the solution through 0.45 µm nylon filter

 Test Preparation: Transfer 10 tablets into a 500 ml volumetric flask. Add 150ml of diluent and sonicate for 25 minutes with intermediate shaking cool to room temperature and dilute to volume with Filter the solution through 0.45 µm nylon filter

 System Suitability: From the Chromatogragram of standard solution USP Tailing for candensartan Cilexetil and Hydrochlorothiazide. Peak is not more than 2.0USP Tangent for candensartan Cilexetil peak is not less than 5000 and Hydrochlorothiazide. Peak is not less than 1500. The %RSD of the peak area of candensartan Cilexetil and Hydrochlorothiazide peak from six replicate injections should be not more then 2.0.

 Procedure:  Equilibrate the system with mobile phase A and mobile phase B at least 30 minutes.

Inject Blank, Stadard solution six times and samples solution in duplicate.

 Calculation: Calculate the amount of and candensartan Cilexetil Hydrochlorothiazide. present in % of tablet using the formula          

% Assay of candensartan Cilexetil. =

Where,

AT1= Average area of candensartan Cilexetil peak in Sample Solution.

AS1 = Average area of candensartan Cilexetil peak in Standard Solution.

DS= Stanard Solution dilution,

DT=Test solution dilution

P = Potency of candensartan Cilexetil Working Standard on as is basis

C =Claim

Calculation: Calculate the amount of Hydrochlorothiazide present in % of tablet using the formula          

Where,

AT2   = Average area of Hydrochlorothiazide peak in Sample Solution.

AS2 = Average area of Hydrochlorothiazide peak in Standard Solution.

DS= Stanard Solution dilution,

DT=Test solution dilution

P = Potency of Hydrochlorothiazide Working Standard on as is basis

C=Claim

 METHOD VALIDATION SUMMARY ²³

System Suitability and System Precision:

A Standard solution was prepared by using Candensartan Cilexetil and Hydrochlorothiazide working standard as per test method and was injected six times into the HPLC system.

The system suitability parameters were evaluated from standard chromatograms and found to be within the limits and results are shown in Table 1 and Table 2. Calculated the % RSD from six replicate injections for Candensartan Cilexetil and Hydrochlorothiazide peak area.

 SPECIFICITY

Placebo Interference:

A study to establish the interference of placebo was conducted. Samples were prepared in triplicate by taking the placebo equivalent to about the weight in portion of test preparation as per the test method. Chromatogram of placebo did not show any extra peaks. This indicates that the excipients used in the formulation do not interfere in estimation of Candensartan Cilexetil and Hydrochlorothiazide. No interference at the retention times of Candensartan Cilexetil and Hydrochlorothiazide and the results are shown in Table 3.

FIG 3: HPLC CHROMATOGRAM OF STANDARD CANDESARTAN CILEXETIL AND HYDROCHLOROTHIAZIDE

 Precision of Test Method:

The precision of test method was evaluated by analysing six samples prepared as per test method. The assay and relative standard deviation of the individual Candensartan Cilexetil and Hydrochlorothiazide were calculated the results are shown in Table 4, Table 5 and Table 6 and they are within the limits.

TABLE 1 : RESULTS OF SYSTEM PRESION

TABLE 2: RESULTS OF SYSTEM SUITABILITY

TABLE 3: RESULTS OF SYSTEM SUITABILITY

Accuracy:

A study of Accuracy was conducted.  Drug Assay was performed in triplicate as per test method with equivalent amount of Candensartan Cilexetil  and Hydrochlorothiazide into each volumetric flask by spikeing API into the placebo each  level to get the concentration of Candensartan Cilexetil  and Hydrochlorothiazide equivalent to 75,100%,150% of the sample conc of Candensartan Cilexetil  and Hydrochlorothiazide as per the test method. The average % recovery of Candensartan Cilexetil and Hydrochlorothiazide was found to be within the limits and the results are shown in Table 7 and Table 8.

 Linearity of Test Method:

A graph is plotted to “mg/ml of Candensartan Cilexetil and Hydrochlorothiazide added” versus mg/ml of Candensartan Cilexetil and hydrochlorothiazide found” in Accuracy section. The correlation coefficient was found to be 0.999. And the results are shown in Table 4, Table 9 and Figure 4.

FIG 4.  LINEARITY GRAPH OF   HYDROCHLOROTHIAZIDE

FIG 5.  LINEARITY GRAPH OF CANDESARTAN CILEXETIL

From the above study it was established that the linearity of test method is from 70% to 130% of the labeled amount of Candensartan Cilexetil and Hydrochlorothiazide. The Correlation Coefficient shall be not less than 0.99. And the results are shown in Table 4, Table 9 and Figure 5.

RUGGEDNESS OF TEST METHOD:

System to system /Analyst to Analyst/column to Column variability:

System to system /Analyst to Analyst/column to Column variability study was conducted on different HPLC system, different column and different analyst under similar conditions at different times. Six samples were prepared and each were analysed as per test method.

The relative standard deviation for Candensartan Cilexetil and Hydrochlorothiazide were found to be below 2 % on the columns, systems and Analysts. Comparison of both the results obtained on two different HPLC systems, different column and different analysts shows that the related substances test method is rugged for System to system /Analyst to Analyst/column to Column variability.

 TABLE 4: RESULTS OF PRECISION

 TABLE 5: RESULTS OF METHOD PRECISION

Robustness:

Effect of variation in mobile phase composition:

A study was conducted to determine the effect of variation in composition of mobile phase. Standard solution prepared as per the test method was injected into the HPLC system using mobile phases. The system suitability parameters were evaluated and found to be within the limits

 Effect of variation of flow rate:

A study was conducted to determine the effect of variation in flow rate. Standard solution prepared as per the test method was injected into the HPLC system using flow rates 0.8ml/min and 1.2ml/min. The system suitability parameters were evaluated and found to be within the limits

Effect of variation of temperature:

A study was conducted to determine the effect of variation in temperature. Standard solution prepared as per the test method was injected into the HPLC system using temperatures 35ºC. The system suitability parameters were evaluated and found to be within the limits for temperatures 35ºC and the results are shown in Table 10.

 Stability Indicating Studies

Forced Degradation

Degradation were carried out by attempting degradation of the sample with exposure to stressconditions like Acidic (1M HCL), Alkaline (1M NaOH), Peroxide, water and UV light.

With 0.1M NaOH

Crush the ten Tablets of both Candesartan Cilexetil and Hydrochlorothiazide, accurately weigh and transfer about 60mg of Candesartan Cilexetil, 25 mg of in to a 100 ml volumetric flask. Then add 10 ml of 0.1 NaOH and reflex for 30 min at 60⁰C and cool to room temperature and add 10 ml of 0.1 N HCL to neutralize. Sonicate to dissolve and make up to the volume with diluent. Filter the solution through 0.45 µm nylon filter. Then it was injected once into the chromatographic system obtainthe chromatograms. Retention times were found.

With 0.1M HCL

Crush the ten Tablets of both Candensartan Cilexetil and Hydrochlorothiazide, accurately weigh and transfer about 60mg of Candensartan Cilexetil, 25 mg of in to a 100 ml volumetric flask. Then add 10 ml of 0.1 HCL and reflex for 30 min at 60⁰C and cool to room temperature and add 10 ml of 0.1 N NaOH to neutralize. Sonicate to dissolve and make up to the volume with diluent. Filter the solution through 0.45 µm nylon filter. Then it was injected once into the chromatographic system obtain the chromatograms. Retention times were found.

 With 1 % M H₂O₂

Crush the ten Tablets of both Candensartan Cilexetil and Hydrochlorothiazide, accurately weigh and transfer about 60mg of Candensartan Cilexetil, 25 mg of in to a 100 ml volumetric flask. Then add 10 ml of 1 % M H₂O₂and reflex for 30 min at 60⁰C and cool to room temperature. Sonicate to dissolve and make up to the volume with diluent. Filter the solution through 0.45 µm nylon filter. Then it was injected once into the chromatographic system obtain the chromatograms. Retention times were found.

 With M H₂O

Crush the ten Tablets of both Candensartan Cilexetil and Hydrochlorothiazide, accurately weigh and transfer about 60mg of Candensartan Cilexetil, 25 mg of in to a 100 ml volumetric flask. Then add 10 ml of H₂Oand reflex for 30 min at 60⁰C and cool to room temperature. Sonicate to dissolve and make up to the volume with diluent. Filter the solution through 0.45 µm nylon filter. Then it was injected once into the chromatographic system obtainthe chromatograms. Retention times were found.

 With Heat

Take both Candensartan Cilexetil and Hydrochlorothiazide, accurately weigh and transfer about 60mg of Candensartan Cilexetil, 25 mg of in to a 100 ml volumetric flask and exposed to 105⁰C for 6 hours. Add 20 ml of diluents and Sonicate to dissolve and make up to the volume with diluent. Filter the solution through 0.45 µm nylon filter. Then it was injected once into the chromatographic system obtainthe chromatograms. Retention times were found.

 Bench top stability of standard and Test preparation:

A study to establish stability of Candesartan Cilexetil and Hydrochlorothiazide standard and test preparation on bench top was conducted over period of two days. Candesartan Cilexetil and Hydrochlorothiazide test preparation with target concentration spiking are injected initial, 1 day and 2 days. The difference in % of Candesartan Cilexetil and Hydrochlorothiazide from initial to 24 hours is within the limits. Candesartan Cilexetil and Hydrochlorothiazide standard was injected initial, 1 day and 2 days and the difference of the standard over period of one day was found stable.

From the above study, it was established that the Forced degradation studies with Acid, Base, Peroxide, Heat and Light The percentage assay is within the limits Were shown in the Table 11. And got the Retention time similar with standard sample.

 TABLE 6: RESULTS OF PRECISION

  TABLE 7: RESULTS OF ACCURACY

 TABLE 8: RESULTS OF ACCURACY

 TABLE 9: RESULTS OF LINEARITY OF DETECTOR RESPONSE

 TABLE 10: RESULTS OF ROBUSTNESS SHOWING EFFECT OF VARIATION IN COMPOSITION OF ORGANIC PHASE IN MOBILE PHASE

Set I        =              Control (Proposed method)

Set II      =              Variation in flow rate (-10%)

Set III    =              Variation in flow rate (+10%)

Set IV     =              Column oven temperature (35°C)

Set V      =              Variation in wavelength (l = 280m)

Set VI     =              Variation in wavelength (l = 290m)

Set VII   =              Variation in organic content in mobile phase (-2%)

Set VIII =              Variation in organic content in mobile phase (+2%)

TABLE 11: RESULTS OF FORCED DEGRADATION STUDIES

 CONCLUSIONS: It is concluded from the RP-HPLC method development for the simultaneous qualitative determination of Candesartan Cilexetil and Hydrochlorothiazide is fast, reproducible, and simple. The proposed method is found to be specific, accurate, precise, linear, robust, and rugged. The developed method is stability indicating and can be used by a quality control department to determine assay of regular Candesartan Cilexetil and Hydrochlorothiazide commercial samples and also stability samples

 ACKNOWLEDGEMENTS: I would like to acknowledge the support of the management of the Pharmacy department, JNTU Kakinada, Andhra Pradesh, India. I would also like to thank the University authorities, for provided the necessary facilities to carry out this work.

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    How to cite this article:

    Kumar GM and Seshagiri Rao JVLN: Standadadization and Stability Indicating Studies by RP-HPLC Method for the Simultaneous Estimation   of Candesartan Cilexetil & Hydrochlorthiazide in Tablet Dosage Form. Int J Pharm Sci Res2014; 5(12): 5438-46.doi: 10.13040/IJPSR.0975-8232.5 (12).5438-46.

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