Azilsartan: A Review of Analytical Methods for estimation in Pharmaceutical Formulation

 

Zamir G. Khan, Sanjaykumar B. Bari, Sarang N. Gujarathi, Shivam B. Gujarathi,

Prashant B. Patil*

Department of Pharmaceutical Chemistry, H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist: Dhule (MS) 425405 India

*Corresponding Author E-mail: pbp6388@gmail.com

 

ABSTRACT:

Azilsartan medoxomil was approved by the United States Food and Drug Administration in 2011. AZL is used for the treatment of essential hypertension and has publicized promising results in blood pressure (BP) reduction and tolerability, but has not yet been considered into practice as compared to other angiotensin II receptor blockers (ARBs). AZL lowers blood pressure by blocking the action of angiotensin II at the AT1 receptor, a hormone that contracts blood vessels and reduces water excretion through the kidneys. There have been numerous methods developed so far for quantitative estimation of AZL in bulk and pharmaceutical dosage form. Pharmaceutical analytical methods include UV Visible Spectrophotometry, High Pressure Thin Layer Chromatography, Reversed Phase High Performance Liquid Chromatography and hyphenated techniques like Liquid chromatography- Mass Spectrometry. This review paper includes publications form 2010 to 2018. The chromatographic methods primarily RP-HPLC and LC-MS found more accurate, precise and sensitive compared to other pharmaceutical methods. AZL shows greater extent of degradation up to 40% with hydrogen peroxide. AZL is degraded up to 38% and 31% with acidic and neutral pH respectively while it shows greater photo stability and dry heat.

 

KEYWORDS: UV – Visible Spectrophotometry, HPTLC, RP-HPLC, LC-MS, Azilsartan, Validation, stability indicating methods.

 

 


INTRODUCTION:

Azilsartan medoxomil (AZL) is a chemically 2-ethoxy-3-[[4-[2-(5-oxo-2H - 1, 2, 4-oxadiazol-3-yl) phenyl] phenyl] methyl] benzimidazole-4-carboxylic acid. It was approved by the U.S.  Food and Drug Administration (FDA) as Edarby tablets on 25th February 2011to treat hypertension in adults.1

 

 

Azilsartan is an Angiotensin 2 Receptor Blocker. The mechanism of action of azilsartan is as an Angiotensin 2 Type 1 Receptor Antagonist. The physiologic effect of azilsartan is by means of Decreased Blood Pressure. The azilsartan is mainly used in the therapy of hypertension. It is associated with a low rate of transient serum aminotransferase elevations, but has yet to be linked to instances of acute liver injury and in lowering blood pressure reduces the risk of fatal and nonfatal cardiovascular events, primarily strokes and myocardial infarctions. These benefits have been seen in controlled trials of antihypertensive drugs from a wide variety of pharmacologic classes. The chemical structure of Azilsartan is shown in Figure 1.2

 

Figure 1: Chemical structure of Azilsartan

 

Methods for pharmaceutical analysis are significantly much simpler comparatively with metabolites in biological samples as in urine, blood and plasma. The estimation of a drug is to a great extent important in complex matrices because the pharmaceutical product quality is directly associated to patient wellbeing. In the drug development and pharmaceutical control, chemical analysis plays a key role to ensure a high efficacy and safety for patients.3

 

AZL is rapidly hydrolysed to the active moiety azilsartan by esterases in the gastrointestinal tract and/or during drug absorption. The enzyme carboxymethylene butenolidase is a recently discovered hydrolysis mechanism for AZL in the intestine and liver. Azilsartan is an inverse agonist of the AT1 receptor, is a highly potent, selective and competitive antagonist of the angiotensin II type 1 receptor. Molecular basis of AZL reveals that it may be responsible for its clinical  efficacy. 4,17,24

 

Analytical methods for estimation of Lurasidone:

1.      UV – Visible Spectrophotometry:

UV/V Spectrophotometric method is used to as an analytical method of estimation of Azilsartan. UV/ V spectrophotometry is employed as it is ease to handle and accurate. It has accuracy, speed, gives sensitive and precise results. However, UV–Visible Spectrophotometry method is not able to give broad spectra for estimation of certain drugs. It consists of calculating and plotting first order and second order derivative of the mathematical expression of a spectral curve.5,23,25,28

 

Table 1: UV/V Spectrophotometric by AUC method

Method

Solvent

λmax (nm)

Linearity (μg/ml)

r2

% Recovery

Reference

Area Under Curve

Methanol

249

4-20

0.9989

99.91

5

*r2- Correlation coefficient

 

According to above mentioned study UV/V Spectrophotometric method is used for estimation of impurity in AZL.

 

Poonam P. Patil, Dr. Veena S. Kasture and Dr. K. Vanitha Prakash has developed a UV Spectrophotometric method for estimation of 2- Chloromethyl-1h Benzimidazole impurity in AZL in bulk and formulation. This process is referred to identify the impurity of AZL 2-Chloromethyl-1 H benzimidazole in bulk and formulation which was synthesized, further characterized and later UV method was developed. The evaluation was done on laboratory scale i.e. melting point, TLC and elemental analysis. The TLC of Azilsartan was performed by solvents as Chloroform: Methanol in the ratio of 9:1 v/v and the Rf value was found to be 0.68 for confirmation of structure of AZI more sophisticated instruments were used as FT-IR, NMR.6

 

Walid M. Ebeid, Ehab F. Elkady1, Asmaa A. El-Zaher, Ramzia I. El-Bagaryand Gabor Patonay was said about spectrophotometric along with Spectrofluorimetric Study of AZL and Chlorthalidone was utilized in their Determined in Pharmaceuticals. The UV-spectrophotometric technique is dependent on measurement of the first derivative spectra for Azilsartan in combination with Chlorthalidone in methanol. The spectrofluorimetric technique is dependent on measurement of the fourth derivative of the synchronous spectra intensities of Azilsartan in combination with chlorthalidone in same solvent as mentioned above.7

 

Pradeepthi. J, Masthanamma. SK*, Alekhya.G was said four methods as Method A - zero order spectroscopy; Method B - area under curve, Method C - first order derivative, Method D- second order derivative. The proposed methods were concluded as simple, accurate and precise and can employed in routine quality control analysis and have been validated Spectrophotometric Method for Determination of AZL in Pharmaceutical Dosage Form.8

 

Table 2: A summary of above mentioned methods with validation parameters

Parameter

Method A

Method B

Method C

Method D

λmax

243 nm

238 – 248 nm

254 nm

247 nm

Beer’s limit (μg/ml)

5-30

5-30

5-30

5-30

r2

0.992

0.993

0.994

0.996

Precision indicated by % RSD

1.1

0.79

0.66

0.72

Accuracy indicated by %RSD

99.2

101.3

100.5

100.4

*r2- Correlation coefficient

% RSD- Percent Relative Standard Deviation

 

2.      Chromatographic Methods:

2.1 High- Performance Thin- Layer Chromatography (HPTLC):

High performance thin layer chromatography (HPTLC) is an instrumental sophisticated technique dependent on the inclusive capabilities of thin layer chromatography. The advantages of automation, full optimization, scanning, minimum sample preparation, selective detection principle, hyphenation, etc. facilitate it to be a powerful analytical tool for chromatographic information of complex mixtures of organic, inorganic, and biomolecules. Analytical chemists focus on new applications, new methods and discoveries of analysis to boost up the specificity and sensitivity of a method. Many methods, just the once developed, are kept deliberately static so that data can be compared over long periods of time. HPTLC has strong potentials as a substitute chromatographic model for estimating partitioning properties in support of environmental, combinatorial chemistry, and health effect studies.9,18, 23

 

Raja Gorla, Ch Venkata Raju, B Sreenivasulu, N Sreenivas, Naga srinivas K Sharma, Hemantkumar, Raghu Babu have developed HPTLC Method for estimation of AZL in Bulk and Pharmaceutical Dosage Forms. In this estimation the separation of the drug was carried out using ethyl acetate and n-hexane in ratio of 7:3 v/v as mobile phase on precoated silica gel 60 F254 plates. The retention factor (Rf) for AZL was 0.65 ± 0.05. Limit of Detection and limit of quantification were found to be as 3.3 σ/S and 10σ /S respectively; where σ is the standard deviation of the response (y-intercept) and S is the slope of the calibration plot.10

 

Table 3: Summary of the stress degradation studies is mentioned below

Sr. No.

Stress degradation conditions

% Recovery of Azilsartan

% degraded

1

Base (0.01 N NaOH Methanolic),

Kept for 0.5 hr.

83.58

16.42

2

Acid (0.5 N HCl Methanolic),

Kept for 2 hrs

6.21

38.79

3

Neutral (kept for 1 hr.)

68.76

31.23

4

H2O2, 30%

(kept for 2 hrs)

59.70

40.29

5

Dry heat

(60C for 72 hrs)

95.80

4.20

6

Photo stability (UV, 200 watt hrs/square meter and Florescence,

1.2 million Lux. Hrs)

95.18

4.28

 

S. V. Gandhi, P. S. Mittal, A. R. Pahade, S. W. Rege have been developed accurate and validated stability indicating HPTLC method for estimation of AZL in bulk and pharmaceutical dosage form. The drug was subjected to acid, base, neutral hydrolysis, oxidation, thermal degradation and photolysis. The data of linear regression analysis indicated a good linear relationship over the range of 100–800 ng/band concentrations with correlation coefficient 0.994. Stability studies were carried out to find out the stress of environmental influence on quality of drug such as hydrolysis, temperature, oxidation, shelf life, retest period, storage conditions, etc.11

 

Raja Gorla, Ch. VenkataRaju, B Sreenivasulu, N Sreenivas, Nagasrinivas K Sharma, Hemantkumar, Raghu Babu have been reported their work on stability indicating HPTLC assay Method for determination of AZL. This method is validated with the analytical parameters such as linearity, accuracy, precision, robustness, and forced degradation studies. While performing the forced degradation study they have found that the azilsartan was liable acid, base hydrolysis, oxidative stress and stable in photolytic and thermal stress. The calibration curve obtained was linear in concentration range of 100-700 ng per band with correlation coefficient as 0.999. After performing recovery studies, results were reported in range from 99.26 – 100.65 % along with RSD values ranging from 0.183 – 2.23%. This method can be extended to study the degradation kinetics of AZL and its estimation in plasma and other biological fluids. The degradation study results and HPTLC method results are sequentially shown in table 4 and 5.12

 

Table 4: Results of performed degradation studies

Stress Condition

% recovery

Rf of de-gradant

In-ference

Acidic, 0.1 N HCl/15min, 85°C reflux

77.15

0.41

Labile

pH-6.8 phosphate buffer/1 h, 85°C reflux

80.76

0.29

Labile

Oxidation, 10 % v/v H2O2/1h 85°C reflux

81.22

0.28

Labile

Photolytic (UV, 200 watt hours/m2 fluorescent light 1.2 million Lux hours)

99.65

-

Stable

Dry-Induced/2 h, oven at 105°C

99.75

-

Stable

Wet-Induced/2hr/85°C reflux

99.55

-

Stable

 

2.2 Reversed-Phase High-Performance Liquid Chromatography19,20,21,22:

K. Lavanya, V. Srinivasa Rao, P. Sunitha, K. Pavani Sai Rama have been reported their work on reversed phase HPLC method is developed for the determination of Azilsartan medoximil and chlorthalidone in pharmaceutical dosage forms was carried out on a C18 column 4.6 x 100mm, 5μm.


 

 

Table 5: High- Performance Thin- Layer Chromatography method (HPTLC)

Stationary phase

Diluent

Mobile phase

Scanning(nm)

Rf value

r2

% Recovery

Reference.

Silica Gel 60F254

methanol

ethylacetate-n-hexane 7:3 (v/v)

249

0.65 ± 0.05

0.999

99.26 - 100.65

10

Silica Gel 60F254

Methanol

Toluene: Methanol: Ethyl acetate (8:2:1 v/v/v)

250

0.50 ± 0.03

0.994

99.25 – 100.34

11

Silica Gel 60F254

Methanol

ethylacetate-n-hexane 7:3 (v/v)

249

0.65 ±0.05

0.999

100.2-100.5

12

 


The mobile phase used as at a flow rate of 1.0 ml/min mixture of potassium Di hydrogen ortho phosphate buffer and acetonitrile (35:65 v/v). Detection was passed out at 273 nm. The retention time was 2.59±0.1mins and 3.85±0.5 min respectively for both drugs. The linearity was reported in range of 2.5-15 μg/ml and 10-60 μg/ml with a correlation coefficient of AZL and Chlorthalidone were 0.996 and 0.999.13

 

Sravani P, Rubesh Kumar S, Duganath N, Devanna N. have been reported their work on a simple, precise, accurate method was developed for the simultaneous estimation of azilsartan and Chlorthalidone by RP-HPLC technique. Mobile phase was 0.1% Ortho phosphoric acid buffer and acetonitrile in the ratio of (30:70 v/v) run through ODS (250mm: 4.6mm, 5μ) column and flow rate was flow rate of 1ml/min. Wavelength was adjusted to 230nm. The required solutions were prepared by using the diluents water and acetonitrile in the ratio of 50:50 v/v and run time was fixed to 9min. Chlorthalidone and azilsartan were eluted at 2.266 and 4.551 with good resolution of 11. % RSD of chlorthalidone and azilsartan was obtained to be 0.72 and 0.68 respectively and % Recovery was 99.92% and 99.845%, the correlation coefficient was found as 0.999. Assay of the tablet was found as 100.15%.14

 

Kayesh R, Jahan MS, Sultan M Z has been reported their work on stability-indicating reversed-phase high-performance liquid chromatography method by using a photodiode array detector. Separation was achieved on a double end-capped C18 column (150 mm × 4 mm, 5 μm). The effects of % acetonitrile (v/v) and buffer salt concentrations on the retention time of the two drugs and on their resolution were examined and improved. The optimum chromatographic condition within the design space was found to be isocratic mobile phase consisting of 10 mM. Tris (hydroxymethyl) amino methane buffer (pH 7.7) and acetonitrile at ratio of 60:40 (v/v) with flow rate of 1 mL min−1 for 7 min. The retention times of CLR and AZL were found to be 2.6 and 4.9 min, respectively.15

 

Sandeep Kumar Sohni, Robin Kumar, MymoonaAkhtar, ChandaRanjan, Gita Chawla has been reported their work on development and validation of RP-HPLC method for simultaneous estimation of AZL and chlorthalidone in bulk form and formulation using quality by design. The simultaneous estimation of the drugs-azilsartan and Chlorthalidone was performed using C8 column having dimensions 150×4.6 mm×5 μm, injection volume 10 μl, flow rate 0.8 ml/min., runtime 10 min., column temperature 20oC and sampler temperature 5 oC and ultraviolet detection using a photodiode array detector at 220 nm as constant. The retention times for Chlorthalidone and AZL were 2.4 min. and 5.1 min. respectively with resolution 17. The linearity of chlortalidone and AZL was in the range of 6.3 to 15 μg/ml and 20 to 48 μg/ml respectively and potency of the formulation was found to be 108.12 % and 98.20 % respectively for both drugs.16


 

Table 6: Reversed Phase High- Performance Liquid Chromatography (RP-HPLC) 26, 27

Column

Dimension (mm×mm×µ)

Mob phase

λmax (nm)

Linearity  μg/ml

% Recovery

LOD, LOQ (µg/ml)

Detector

r2

% Assay

Diluent

Reference

BDS C18

4.6 x 100 × 5.0

potassium Di hydrogen ortho phosphate buffer

and acetonitrile (35:65 v/v)

273

2.5-15

98.7 – 100.2

0.02,

0.04

PDA

0.996

100.8

Mob phase

13

ODS

4.6  x 250 × 5.0

0.1% Ortho phosphoric acid buffer and acetonitrile

30:70

230

100 - 600

99.92 - 99.84

0.39,

1.17

PDA

0.999

100.15

water and ACN

14

Double

end-capped C18

4 × 150 × 5.0

Tris(hydroxymethyl)aminomethane

buffer (pH 7.7) and acetonitrile at ratio of 60:40 (v/v)

249

6.25 - 25

99.94 -  100.00

0.023, 0.077

PDA

0.999

100.23

Acetate buffer solution

(pH 4.0) and  ACN

15

C8

150×4.6 × 5.0

ACN: water (90:10) and potassium dihydrogen phosphate (KH2PO4) buffer solution (10 mmol)

 

220

 

6.3 - 15

98.101 - 99.347

1.537

4.657

PDA

0.999

108.116

ACN with water (90:10) and10 mmol KH2PO4 buffer in the ratio of (69.5:30.5)

16

ODS- Octadecyl Silica, BDS- base deactivated Silanol, ACN- Acetonitrile, MeOH- Methanol, PDA- Photo Diode Array, Mob phase- Mobile phase, LOD- Limit Of Detection, LOQ- Limit Of Quantitation.

 

 

Figure 2: Analytical methods for estimation of Azilsartan medoxomil in bulk and pharmaceutical dosage forms

 

 

 


CONCLUSION:

In this paper, recent analytical methods employed for quantitative analysis of azilsartan medoxomil in pharmaceutical formulations chiefly from 2010 to 2017 were reviewed. Several techniques like UV - Visible spectrophotometry including Area under curve, Chromatographic methods primarily (high - performance liquid - chromatography, high - performance thin - layer chromatography), Liquid chromatography is the major techniques that have been used, of which it is observed a development to use quicker techniques with cost savings and lessening in solvent consumption. From this work, it has been observed that High Performance Liquid Chromatography is extensively utilised for estimation of azilsartan medoxomil in bulk material and pharmaceutical formulations. Further there has been always greater need to develop more sophisticated method to determine content of Azilsartan in bulk and pharmaceutical dosage form.

 

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Received on 20.11.2018       Accepted on 30.11.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(4): 227-232.

DOI: 10.5958/2231-5675.2018.00041.8