International Journal of Pharma Sciences Vol. 4, No. 5 (2014): 725-729 Research Article Open Access

ISSN: 2320-6810

Method Development and Validation for the Simultaneous Estimation of Azilsartan and Chlorthalidone by RP-HPLC in Pharmaceutical Dosage Form P. Sravani*, S. Rubesh Kumar, N. Duganath and N. Devanna Department of Pharmaceutical Analysis, Jawaharlal Nehru Technological University Anantapur, Oil Technological Research Institute Ananthapuram-515001, A.P, India

* Corresponding author: P. Sravani; e-mail: [email protected]

Received: 8 August 2014

Accepted: 21 August 2014

Online: 01 September 2014

ABSTRACT A simple, precise, accurate method was developed for the simultaneous estimation of azilsartan and chlorthalidone in pharmaceutical dosage form by RP-HPLC technique. 0.1% Ortho phosphoric acid buffer and acetonitrile in the ratio of (30:70) was used as mobile phase run through ODS (250mm: 4.6mm, 5µ) column with a flow rate of 1ml/min. The temperature of the column oven was maintained at 30°C. Wavelength was optimized to 230nm. Stock and working solutions were prepared by using the diluents water and acetonitrile in the ratio of 50:50. Run time was fixed to 9min. chlorthalidone and azilsartan were eluted at 2.266 and 4.551 with good resolution of 11. The plate count, tailing factor and all system suitability parameters are within ICH range. The developed method was validated as per ICH guidelines. %RSD of chlorthalidone and azilsartan was obtained to be 0.72 and 0.68 respectively. % Recovery found was 99.92% and 99.845, Calibration plot was done for both chlorthalidone, azilsartan and linearity equation were y = 20261x + 2072 and y = 13573x + 1593 with correlation coefficient 0.999. Assay of the tablet was performed and found as 100.15%. All the parameters were within the ICH guidelines and the method was economical and simple as retention times were less than in literature and decreased run time.

Keywords: Azilsartan, Chlorthalidone, ICH guidelines, RP-HPLC. INTRODUCTION Azilsartan is an anti hypertensive drug used in the treatment of hypertention. It is a angiotensin II receptor antagonist. It blocks the action of angiotensin receptor by vasopressor harmone that prevents vasoconstriction and decreases the blood pressure. Its IUPAC name was (5-methyl-2-oxo-1,3-dioxol-4yl)methyl 2-ethoxy-1-([2'-(5-oxo-4,5-dihydro-1,2,4oxadiazol-3-yl)biphenyl-4-yl]methyl)-1H-benzimidazol -7-carboxylate and molecular formula C30H23KN4O8. Azilsartan was practicaly insoluble in aqueous solutions soluble in DMSO and methanol. Pka of the drug was 9.21 [1]. Chlorthalidone is a diuretic drug of thiazide diuretics category used in the treatment of hypertension. Chlorthalidone inhibits Na+ and Cl- ions re-absorption in the distal convoluted tubule by blocking the Na+ /Cl- Symporter. IUPAC name was (RS)2-Chloro-5-(1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol1-yl)benzene-1-sulfonamide with molecular formula C14H11ClN2O4S. Chlorthalidone was soluble in water, http://ijps.aizeonpublishers.net/content/2014/5/ijps725-729.pdf

methanol, alcohol, methanol and DMSO. Pka was 9.57 [2]. According to literature two methods were available in which madhu et al., the retention time for Chlortalidone and Azilsartan Medoxomil were 3.923min and 7.208 min respectively [3]. Naazneen et al., the retention time for Chlortalidone and Azilsartan Medoxomil were 2.36±0.1 mins and 5.54±0.5 mins respectively [4].

MATERIALS AND METHODS Materials and reagents: Azilsartan and chlorthalidone API were gifted samples by spectrum pharma research solutions, HPLC grade water and Acetonitrile from Merk, and the combination tablet Edarbyclor with lable claim 40mg and 12.5mg of Azilsartan and Chlorthalidone from Takeda. Instruments High performance liquid chromatography of waters 2695 with quaternary pumps, Auto sampler and PDA 725

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detector. Software integrated with the HPLC was Waters software Empower 2. Double beam Labindia UV spectrophotometer integrated with UV Win 5 software, Labman ultra sonic cleaner and Denver Digital balance. Preparation of Buffer: (0.1%OPA) Accurately measured and transferred 1ml of Conc. Orthophosphoric acid in a 1000ml of volumetric flask, about 900ml of milli-Q water added, sonicate to degass and finally made up the volume with water. Preparation of standard working solution: Accurately Weighed and transferred 40mg and 12.5mg of Azilsartan and Chlorthalidone API into two 10 ml clean dry volumetric flasks, add 7ml of diluent(Water and acetonitrile 50:50), sonicated for 30 minutes and made up to the final volume with diluent. From the above stock solution, 1 ml was pipette out in to a 10ml volumetric flask and then made up to the final volume with diluents results in standard working solution containing 400ppm of azilsartan and 125ppm of chlorthalidone. Preparation of sample working solution: 5 tablets were weighed and powdered, tablet powder weight equivalent to 62.5mg of chlorthalidone and 200mg of azilsartan was weighed and transferred into a 50ml volumetric flask, 30ml of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. From the filtered solution 1ml was pipette out into a 10 ml volumetric flask and made up to 10ml with diluents results in sample working solution containing 400ppm of azilsartan and 125ppm of chlorthalidone. Chromatographic conditions: 10µl of Sample was injected in to mobile phase line composed of buffer and acetonitrile in the ratio of 30:70 pumped with a flow rate of 1ml/min through ODS 250 Column at 30°C oven temperature. Wavelength optimized for these two drugs was 230nm. Drugs were eluted at their best retention times of possible with good resolution. Method Validation: The developed method was validated as per ICH guidelines including the parameters specificity, linearity, precision, accuracy, Limit of detection, Limit of quantification and Robustness. Specificity: Specificity determines the placebo interference of the related substances or the excipients like diluents, glidants, lubricants and binders in the process of determination of the drug. The excipients were spiked to the drug concentrations and interference was estimated. Linearity: Six linear dilutions were prepared by transferring 0.25ml, 0.50ml, 0.75ml, 0.1ml, 1.25ml, 1.5ml from the standard stock solution in to six 10ml volumetric flasks http://ijps.aizeonpublishers.net/content/2014/5/ijps725-729.pdf

and made up with diluents results in solutions with 100ppm, 200ppm, 300ppm, 400ppm, 500ppm, 600ppm azilsartan and 31.25ppm, 62.5ppm, 93.75ppm, 125ppm, 156.25ppm, 187.5ppm of chlorthalidone respectively in six volumetric flasks. Precision: Intraday precision: It is also called repeatability, sample working solution was prepared by multiple sampling from a homogeneous mixture six samples were prepared, injected and reported as %Relative standard deviation. Inter day precision: It is also called intermediate precision, day-day precison, analyst-analyst precision. Sample working solution was prepared by multiple sampling from a homogeneous mixture six samples were prepared and injected on the next day. It was expressed as % Relative standard deviation. Accuracy: Three levels of sample solution were prepared 50%(200ppm of azilsartan and 62.5ppm of chlorthalidone), 100%(400ppm of azilsartan and 125ppm of chlorthalidone), 150%(600ppm of azilsartan and 187.5ppm of chlorthalidone) and injected. %recoverey was calculated and reported. LOD: Limit of detection is the lowest concentration of the drug that can be detected at the detector level without necessary quantification. S/n ratio is 3:1 LOQ: Limit of quantification is the lowest concentration of the drug that can be quantified with an accuracy and precision. S/n ratio is 10:1 Robustness: Small deliberate changes were made in the method like Mobile phase plus and mobile phase minus (10% of Organic solvent) Flow rate plus and flow rate minus (10%) temperature plus and minus (5%). And sample working solutions were injected and reported as %Relative standard deviation. System suitability: System suitability for that method was tested by five replicate injections of standard preparation. Plate count, tailing factor, resolution and %RSD were reported. Assay: Percentage drug present in a tablet was found by performing assay of the tablet Edarbyclor. Sample working solution containing 400ppm of azilsartan and 125ppm of chlorthalidone was prepared by taking the average of 5 tablets 50ml diluents and further 10 times dilution with diluents.

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RESULTS AND DISCUSSION In the process of method development for azilsartan and chlorthalidone different buffers, different mobile phase composition, and different columns were used but the results are satisfactory with mobile phase composition 30:70 of 0.1%OPA buffer and acetonitrile across the ODS 250mm column with a flow rate of 1ml/min. volume of injection was 10µl and optimized wavelength was 230nm. The above method satisfied all the system suitability parameters like resolution, tailing factor, and plate count are within range. Chlorthalidone and Azilsartan were eluted at 2.266min and 4.568min respectively with good resolution of 11. Plate count, tailing factor of chlorthalidone and azilsartan were 2320, 0.95 and 9730, 1.09 respectively. %RSD of intraday, inter day precision of Chlorthalidone and Azilsartan was found to be 0.72, 0.19 and 0.64, 0.1 respectively. Calibration plot was done and the linearity equation obtained was y = 20261x + 2072 for chlorthalidone and y = 13573x + 1593 for azilsartan with co-relation coefficient 0.999. % Recovery of the chlorthalidone was 99.93% and azilsartan was 99.85%. LOD, LOQ of chlorthalidone and azilsartan was 0.34, 1.02 and 0.39, 1.17 respectively. %RSD of robustness

was found to be within range. Assay of tablet was performed and %Amount of drug present in the tablet was found to be 100.15 for chlorthalidone and 100.08 for azilsartan.

Figure 1. Azilsartan Medoxomil

Figure 2. Chlorthalidone

Figure 3. Chromatogram of sample solution

Figure 4. Calibration curve of chlorthalidone

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Figure 5. Calibration curve of azilsartan medoxomil

Table 1. Validation parameters Parameters Recovery Intraday precision Inter day precision LOD LOQ Specificity Robustness Solvent stability

Chlorthalidone 99.93% 0.72 0.19 0.34ppm 1.02ppm Specific 1.2 Stable for 24 hrs

Azilsartan 99.85% 0.64 0.1 0.39ppm 1.17 Specific 1.5 Stable for 24 hrs

Table 2. Calibration Data Parameters Optimized Wavelength Linearity range Intercept Slope Correlation Coefficient Linearity Equation

Chlorthalidone

Azilsartan

230nm

230nm

31.25ppm-187.5ppm 2072 20261

100ppm-600ppm 1593 13573

0.999

0.999

y = 20261x + 2072

y = 13573x + 1593

Table 3. Robustness Data Parameters Flow minus Flow Plus Mobile phase minus Mobile phase plus Temperature minus Temperature Plus

Chlorthalidone 0.1 0.22 0.08 0.17 0.55 0.26

Azilsartan 0.18 1.05 0.18 0.33 0.2 0.24

Table 4. Robustness Data Parameters Level of Recovery %Recovery STDEV %RSD

Chlorthalidone 50% 100% 150% 99.94 99.69 100.16 0.439 0.624 0.428 0.43 0.62 0.42

50% 99.80 0.753 0.75

Azilsartan 100% 150% 99.68 100.05 0.676 0.731 0.67 0.73

Table 5. Assay table Formulation Edarbyclor

Label Claim CHL AZIL 12.5 40

Amount recovered CHL AZIL 12.52 40.03

% Assay CHL AZIL 100.15 100.08

Table 6. System suitability Parameters Retention time Plate count Tailing Factor Resolution %RSD

Chlorthalidone 2.2 ±0.3 min 2106 0.90 10 0.7

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Azilsartan 4.5 ±0.3 min 9570 1.09 11 0.6

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