Int J Curr Pharm Res, Vol 10, Issue 6, 21-24Original Article


DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF AZILSARTAN MEDOXOMIL AND CHLORTHALIDONE IN BULK AND TABLET DOSAGE FORM

S. S. AHER1*, R. B. SAUDAGAR, HEMANT KOTHARI

1R. G. Sapkal College of Pharmacy, Anjaneri, Nashik, 2Pacific Academy of Higher Education and Research University, Udaipur
Email: smitarohokale@yahoo.com

Received: 11 Jul 2018, Revised and Accepted: 07 Sep 2018


ABSTRACT

Objective: A simple, precise, accurate method was developed for the simultaneous estimation of azilsartan and chlorthalidone in bulk and tablet dosage form by RP-HPLC technique.

Methods: Acetonitrile and water in the ratio of (70:30) pH 2.8 used as mobile phase run through (Cosmosil C18 (4.6ID x 250 mm, Particle size: 5 micron) column with a flow rate of 0.9 ml/min. The temperature of the column oven was maintained at 30 °C. Wavelength was selected 244 nm. Stock and working solutions were prepared by using the diluents water and acetonitrile in the ratio of 50:50. Runtime was fixed to 9 min.

Results: Chlorthalidone and azilsartan were eluted at 2.02 and 3.92 with good resolution the plate count, tailing factor and all system suitability parameters are within ICH range. Azilsartan Medoxomil and Chlorthalidone were found to be linear low in concentration range of 80-400μg/ ml and 25-125μg/ ml respectively in the linearity study, regression equation and coefficient of correlation for Azilsartan Medoxomil and Chlorthalidone were found to be (y = 28695x+15397 r²=0.995) and (y=13444+27405 r² = 0.996) Percentage recovery for both Azilsartan Medoxomil and Chlorthalidone was found in range of 99.89%-99.96% indicating accuracy of the proposed work. Assay of the tablet was performed and found as 100.15%.

Conclusion: 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

This Azilsartan Medoxomil and Chlorthalidone fixed-dose combination is found to show superior antihypertensive efficacy in blood pressure reduction in patients with stage 2 hypertension Azilsartan Medoxomil is an Angiotensin II receptor antagonist [1, 2] which has the chemical name (5–Methyl–2–Oxo-1,3–dioxol-4–yl) methyl 2–ethoxy-1–{[ 2’-(5-Oxo-4,5–dihydro-1, 2, 4–oxadiazol-3-yl) biphenyl–4–yl ] methyl }-1H–benzimidazole-7-carboxylate mono-potassium salt [3, 4]. It is a white crystalline powder which is practically insoluble in water, freely soluble in methanol, dimethyl sulfoxide and dimethylformamide, soluble in acetic acid, slightly soluble in acetone and Acetonitrile and very slightly soluble in Tetra Hydro furan and 1-octanol [5, 6].

Fig. 1: Structure of azilsartan medoxomil

Fig. 2: Structure of chlorthalidone

MATERIALS AND METHODS

Instrumentation

A high-performance liquid chromatography system consisting of HPLC Binary Gradient System Model no HPLC 3000 Series Company: Analytical Technologies Ltd. UV detector-300 Reciprocating (40MPa) pump with Cosmosil C18 (4.6ID x 250 mm, Particle size: 5 micron) column Software HPLC Workstation High Precision Balance Model: max 100 gm Min: 0.001 gm.

Reagents and chemicals

HPLC grade solvents methanol, Acetonitrile and water were obtained from Merck Specialities Pvt Ltd, India. Water was deionized and further purified by means of Milli-Q plus water purification system; AR grade Potassium dihydrogen Orthophosphate was obtained from Ranchem Pharmaceuticals India Ltd.

Azilsartan Medoxomil and Chlorthalidone were obtained as pure standards and tablets of Azilsartan Medoxomil (40 mg) and Chlorthalidone (25 mg)] from Hetero Labs Pvt Ltd, Hyderabad, India.

Preparation of stock, working standard

Standard stock solutions containing Azilsartan Medoxomil and Chlorthalidone prepared individually by dissolving 10 mg of Azilsartan Medoximil and 10 mg of Chlorthalidone separately in 100 ml of mobile phase. It was then sonicated for 15 min, and the final volume of both the solutions was made up to 100 ml with methanol to get stock solutions containing 1000 μg/ml.

Fig. 3: Standard chromatogram of azilsartan medoxomil and chlorthalidone

Method validation

The objective of the method validation is to demonstrate that the method is suitable for its intended purpose as it is stated in ICH guidelines. The method was validated for linearity, precision, accuracy, robustness and system suitability.

Linearity

The developed method has been validated as per ICH guidelines. Working standard solutions of Chlorthalidone and Azilsartan Medoxomil in the mass concentration range of 80-400 ppm and 25-125 ppm was injected into the chromatographic system. The chromatograms were developed and the peak area was determined for each concentration of the drug solution. Calibration curve of Azilsartan Medoxomil and Chlorthalidone was obtained by plotting the peak area ratio versus the applied concentrations. The linear correlation coefficient was found to be 0.996and0.995 resp.

Fig. 4: Linearity graph of azilsartan medoxomil

Table 1: Linearity results showing correlation coefficient for azilsartan medoxomil

Conc(μg/ml) Conc(μg/ml)
80 2224150
160 4961110
240 7269323
320 9132435
400 11616294
Corre. Coeff 0.996
Intercept 15397

Table 2: Linearity results showing correlation coefficient for chlorthalidone

Conc(μg/ml) Conc(μg/ml)
25 270195
50 663033
75 1019758
100 1338698
125 1612897
Corre. Coeff. 0.995
Intercept 27405

Fig. 5: Linearity graph of chlorthalidone

Precision and robustness

Precision

The reproducibility of the proposed method was determined by performing tablet assay at different time intervals (morning, afternoon and evening) on the same day (Intra-day assay precision) and on different days (Inter-day precision). Result of intra-day and inter-day precision is expressed in % RSD. Percent RSD for Intraday assay precision was found to be 1.64 Chlorthalidone, and 0.98Azilsartan medoxomil Inter-day assay precision was found to be 1.08 Chlorthalidone and 0.50Azilsartan medoxomil.

Table 3: Result of precision

Conc. of azilsartan medoxomil and chlorthalidone Interday precision Day1 Day 2 Mean %RSD
75: 240 PPM 1019758 1006574 1011017 1004354 986824 1006748 1005878 1.08%
75:240 PPM 7269353 7309656 7279342 7314070 7293703 7371677 7306295 0.50%
Intraday Morning Evening
75:240 PPM 1019758 1006874 1011017 1020586 995025 1011221 1016197 1.64%
75:240 PPM 7269323 7309656 7279342 7387730 7247470 7427650 7320195 0.98%

Average of three readings

Table 4: Results of robustness

Parameter % mean±SD % RSD*
Robustness Azilsartan medoxomil and Chlorthalidone
75:240 PPM 98.62±0.10 99.03±0.21

Accuracy

The validity and reliability of proposed methods were assessed by recovery studies. The recovery of added standards (80%, 100%, and 120%) was found at three replicate and three concentrations level. The values of % mean just close to 100, SD and % RSD are less than 2 indicate the accuracy of the method. The result of recovery study showed in table 5.

Table 5: Results of recovery

Drug Level Conc. of std. (µg/ml) Conc. of solution (µg/ml) % recovery %RSD
Azilisartan Medoximil 80% 25 20 99.36 0.324
25 25
100% 25 30 98.89 0.524
Chlorthalidone 80 64
120% 80 80 99.26 0.216
80 96

Average of three readings

RESULTS AND DISCUSSION

The present work describes RP-HPLC method for simultaneous estimation of Azilsartan Medoxomil and Chlorthalidone in tablet dosage form on RP C-18 Column, Cosmosil C18 (4.6ID x 250 mm, Particle size: 5 micron) using Acetonitrile and Water (70:30v/v, pH 2.8) as mobile phase at a flow rate of 0.9 ml/min and the detection wavelength was 244 nm [8]. The retention time for Azilsartan Medoxomil and Chlorthalidone was found to be 2.0 and 3.92 min respectively detection response for both Azilsartan Medoxomil and Chlorthalidone were found to be linear low in concentration range of 80-400μg/ml and 25-125μg/ml respectively in the linearity study, regression equation and coefficient of correlation for Azilsartan Medoxomil and Chlorthalidone were found to be and (y=13444+27405 r=0.995 and (y=13444+27405 r² = 0.996) Percentage recovery for both Azilsartan Medoxomil and Chlorthalidone was found in range of 99.89%-99.96% indicating accuracy of the proposed work.

The percentage RSD for both the tablet analysis and recovery studies is less than 2% indicating high degree of precision. The results of recovery studies were found to be linear in 80-120% of final assay concentration range indicating linearity and range of the proposed method [9].

The robustness of the proposed method determined by analyzing the same batch of Azilisartan Medoximil and Chlorthalidone tablets by changing the wavelength the overall mean, standard deviation, and % RSD of the assay values were found to be less than 2% which shows the ruggedness of our method [10].

A sample solution of Azilisartan Medoximil and Chlorthalidone tablets was prepared as per the proposed method and analyzed initially and also analyzed at different time intervals by keeping the solution at room temperature. The cumulative %RSD for the area counts of Azilisartan Medoximil and Chlorthalidone was found to be less than 2% The results of the robustness study also indicated that the method is robust and is unaffected by deliberate variation in the chromatographic conditions. Hence, it can be concluded that the developed RP-HPLC method is accurate, precise, and selective and can be employed successfully for the estimation of Azilisartan Medoximil and Chlorthalidone in tablet dosage formulation.

CONCLUSION

A simple rapid, precise and reliable method was developed for the estimation of the Azilisartan Medoximil and Chlorthalidone tablet dosage formulation. The results obtained are within the specified limit by the ICH guidelines. Analytical column used and the mobile phase provides good separation and gives the sharp results. The retention time observed for both the drugs was good hence the method can be used for routine analysis in quality control laboratories.

ACKNOWLEDGMENT

Thanks to Hetero Laboratories Ltd, Hyderabad, India for providing drug sample of Azilsartan Medoximil and Chlorthalidone.

AUTHORS CONTRIBUTIONS

All the author have contributed equally

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. “FDA approves Edarbi to treat high blood pressure “(press release). U. S. Food and Drug Administration (FDA); 2011.

  2. Bakris Gl, Sica D, Weber M, White WB, Roberts A, Prez A, et al. The comparative effects of azilsartan medoxomil and chlorthalidone on ambulatory and clinical blood pressure. J Clin Hypertension (Green Wich) 2011;13:81-8.

  3. USP 25-NF 20, “Validation of conmpendial Methods Section (1225) (United States Pharmacopoeial Convention”, Rockville, Maryland, USA; 2002. p. 2256.

  4. Madhu Babu, Kasimala Bikshal Babu Kasimala. RP-HPLC method development and validation for the simultaneous estimation of Azilsartan medoxomil and Chlortalidone in pharmaceutical dosage forms. Am J PharmTech Res 2012;2:117-26.

  5. GA Shabir. Validation of high-performance liquid chromatography methods for pharmaceutical analysis Understanding the differences and similarities between the validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. J Chromatography A 2003;987:57–66w.

  6. Requirements of FDA, the US pharmacopeia and the ICH. J Chromatography A 2003;987:57-66.

  7. International Conference on Harmonization, "Q2A: Text on Validation of Analytical Procedures. Federal Register 1995;60:11260–2.

  8. International Conference on Harmonization, "Q2B: Validation of analytical procedures: Methodology. Federal Register 1996; 62:27463–7.

  9. FDA approves Edarbi to treat high blood pressure. (Press release). U. S. Food and Drug Administration (FDA); 2011.

  10. http://www.drugs.com/azilsartan-medoxomil.html. [Last accessed on 02 Jun 2018].



About this article

Title

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF AZILSARTAN MEDOXOMIL AND CHLORTHALIDONE IN BULK AND TABLET DOSAGE FORM

Keywords

Azilsartan, Chlorthalidone, ICH guidelines, RP-HPLC

DOI

10.22159/ijcpr.2018v10i6.30967

Date

30-11-2018

Additional Links

Manuscript Submission

Journal

International Journal of Current Pharmaceutical Research
Vol 10, Issue 6 (Nov-Dec), 2018 Page: 21-24

Online ISSN

0975-7066

Authors & Affiliations

S. S. Aher
R. G. Sapkal College of Pharmacy, Anjaneri, Nashik,

R. B. Saudagar
Pacific Academy of Higher Education and Research University, Udaipur

Hemant Kothari
Pacific Academy of Higher Education and Research University, Udaipur


Refbacks

  • There are currently no refbacks.