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Method Development and Validation for the Simultaneous Estimation of

Azilsartan and Chlorthalidone by RP-HPLC in Pharmaceutical Dosage Form

Article · September 2014

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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 methanol, alcohol, methanol and DMSO. Pka was 9.57

Azilsartan is an anti hypertensive drug used in the [2]. According to literature two methods were available
treatment of hypertention. It is a angiotensin II in which madhu et al., the retention time for
receptor antagonist. It blocks the action of angiotensin Chlortalidone and Azilsartan Medoxomil were
receptor by vasopressor harmone that prevents 3.923min and 7.208 min respectively [3]. Naazneen et
vasoconstriction and decreases the blood pressure. Its al., the retention time for Chlortalidone and Azilsartan
IUPAC name was (5-methyl-2-oxo-1,3-dioxol-4- Medoxomil were 2.36±0.1 mins and 5.54±0.5 mins
yl)methyl 2-ethoxy-1-([2'-(5-oxo-4,5-dihydro-1,2,4- respectively [4].
oxadiazol-3-yl)biphenyl-4-yl]methyl)-1H-benzimidazol
-7-carboxylate and molecular formula C30H23KN4O8. MATERIALS AND METHODS
Azilsartan was practicaly insoluble in aqueous Materials and reagents: Azilsartan and chlorthalidone
solutions soluble in DMSO and methanol. Pka of the API were gifted samples by spectrum pharma research
drug was 9.21 [1]. Chlorthalidone is a diuretic drug of solutions, HPLC grade water and Acetonitrile from
thiazide diuretics category used in the treatment of Merk, and the combination tablet Edarbyclor with lable
hypertension. Chlorthalidone inhibits Na+ and Cl- ions claim 40mg and 12.5mg of Azilsartan and
re-absorption in the distal convoluted tubule by Chlorthalidone from Takeda.
blocking the Na+ /Cl- Symporter. IUPAC name was (RS)-
2-Chloro-5-(1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol- Instruments
1-yl)benzene-1-sulfonamide with molecular formula High performance liquid chromatography of waters
C14H11ClN2O4S. Chlorthalidone was soluble in water, 2695 with quaternary pumps, Auto sampler and PDA

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detector. Software integrated with the HPLC was and made up with diluents results in solutions with
Waters software Empower 2. Double beam Labindia UV 100ppm, 200ppm, 300ppm, 400ppm, 500ppm, 600ppm
spectrophotometer integrated with UV Win 5 software, azilsartan and 31.25ppm, 62.5ppm, 93.75ppm,
Labman ultra sonic cleaner and Denver Digital balance. 125ppm, 156.25ppm, 187.5ppm of chlorthalidone
respectively in six volumetric flasks.
Preparation of Buffer: (0.1%OPA)
Accurately measured and transferred 1ml of Conc. Precision:
Orthophosphoric acid in a 1000ml of volumetric flask, Intraday precision:
about 900ml of milli-Q water added, sonicate to degass It is also called repeatability, sample working solution
and finally made up the volume with water. was prepared by multiple sampling from a
homogeneous mixture six samples were prepared,
Preparation of standard working solution: injected and reported as %Relative standard deviation.
Accurately Weighed and transferred 40mg and 12.5mg
of Azilsartan and Chlorthalidone API into two 10 ml Inter day precision:
clean dry volumetric flasks, add 7ml of diluent(Water It is also called intermediate precision, day-day
and acetonitrile 50:50), sonicated for 30 minutes and precison, analyst-analyst precision. Sample working
made up to the final volume with diluent. From the solution was prepared by multiple sampling from a
above stock solution, 1 ml was pipette out in to a 10ml homogeneous mixture six samples were prepared and
volumetric flask and then made up to the final volume injected on the next day. It was expressed as % Relative
with diluents results in standard working solution standard deviation.
containing 400ppm of azilsartan and 125ppm of
chlorthalidone. Accuracy:
Three levels of sample solution were prepared
Preparation of sample working solution: 50%(200ppm of azilsartan and 62.5ppm of
5 tablets were weighed and powdered, tablet powder chlorthalidone), 100%(400ppm of azilsartan and
weight equivalent to 62.5mg of chlorthalidone and 125ppm of chlorthalidone), 150%(600ppm of
200mg of azilsartan was weighed and transferred into azilsartan and 187.5ppm of chlorthalidone) and
a 50ml volumetric flask, 30ml of diluent added and injected. %recoverey was calculated and reported.
sonicated for 25 min, further the volume made up with
diluent and filtered. From the filtered solution 1ml was LOD:
pipette out into a 10 ml volumetric flask and made up Limit of detection is the lowest concentration of the
to 10ml with diluents results in sample working drug that can be detected at the detector level without
solution containing 400ppm of azilsartan and 125ppm necessary quantification. S/n ratio is 3:1
of chlorthalidone.
LOQ:
Chromatographic conditions: Limit of quantification is the lowest concentration of
10µl of Sample was injected in to mobile phase line the drug that can be quantified with an accuracy and
composed of buffer and acetonitrile in the ratio of precision. S/n ratio is 10:1
30:70 pumped with a flow rate of 1ml/min through
ODS 250 Column at 30°C oven temperature. Robustness: Small deliberate changes were made in
Wavelength optimized for these two drugs was 230nm. the method like Mobile phase plus and mobile phase
Drugs were eluted at their best retention times of minus (10% of Organic solvent) Flow rate plus and
possible with good resolution. flow rate minus (10%) temperature plus and minus
(5%). And sample working solutions were injected and
Method Validation: reported as %Relative standard deviation.
The developed method was validated as per ICH
guidelines including the parameters specificity, System suitability:
linearity, precision, accuracy, Limit of detection, Limit System suitability for that method was tested by five
of quantification and Robustness. replicate injections of standard preparation. Plate
count, tailing factor, resolution and %RSD were
Specificity: reported.
Specificity determines the placebo interference of the
related substances or the excipients like diluents, Assay:
glidants, lubricants and binders in the process of Percentage drug present in a tablet was found by
determination of the drug. The excipients were spiked performing assay of the tablet Edarbyclor. Sample
to the drug concentrations and interference was working solution containing 400ppm of azilsartan and
estimated. 125ppm of chlorthalidone was prepared by taking the
average of 5 tablets 50ml diluents and further 10 times
Linearity: dilution with diluents.
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

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RESULTS AND DISCUSSION was found to be within range. Assay of tablet was
In the process of method development for azilsartan performed and %Amount of drug present in the tablet
and chlorthalidone different buffers, different mobile was found to be 100.15 for chlorthalidone and 100.08
phase composition, and different columns were used for azilsartan.
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. Figure 1. Azilsartan Medoxomil
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, Figure 2. Chlorthalidone
1.02 and 0.39, 1.17 respectively. %RSD of robustness

Figure 3. Chromatogram of sample solution

Figure 4. Calibration curve of chlorthalidone

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 P. Sravani et. al. / Int J Pharma Sci. 2014, 4(5): 725-729

Figure 5. Calibration curve of azilsartan medoxomil

Table 1. Validation parameters

Parameters Chlorthalidone Azilsartan
Recovery 99.93% 99.85%
Intraday precision 0.72 0.64
Inter day precision 0.19 0.1
LOD 0.34ppm 0.39ppm
LOQ 1.02ppm 1.17
Specificity Specific Specific
Robustness 1.2 1.5
Solvent stability Stable for 24 hrs Stable for 24 hrs

Table 2. Calibration Data

Parameters Chlorthalidone Azilsartan
Optimized
230nm 230nm
Wavelength
Linearity range 31.25ppm-187.5ppm 100ppm-600ppm
Intercept 2072 1593
Slope 20261 13573
Correlation
0.999 0.999
Coefficient
Linearity Equation y = 20261x + 2072 y = 13573x + 1593

Table 3. Robustness Data

Parameters Chlorthalidone Azilsartan
Flow minus 0.1 0.18
Flow Plus 0.22 1.05
Mobile phase minus 0.08 0.18
Mobile phase plus 0.17 0.33
Temperature minus 0.55 0.2
Temperature Plus 0.26 0.24

Table 4. Robustness Data

Parameters Chlorthalidone Azilsartan
Level of Recovery 50% 100% 150% 50% 100% 150%
%Recovery 99.94 99.69 100.16 99.80 99.68 100.05
STDEV 0.439 0.624 0.428 0.753 0.676 0.731
%RSD 0.43 0.62 0.42 0.75 0.67 0.73

Table 5. Assay table

Label Claim Amount recovered % Assay
Formulation CHL AZIL CHL AZIL CHL AZIL
Edarbyclor 12.5 40 12.52 40.03 100.15 100.08

Table 6. System suitability

Parameters Chlorthalidone Azilsartan
Retention time 2.2 ±0.3 min 4.5 ±0.3 min
Plate count 2106 9570
Tailing Factor 0.90 1.09
Resolution 10 11
%RSD 0.7 0.6

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