INTRODUCTION:

Candesartan is an angiotensin-receptor blocker (ARB) that may be used alone or with other agents to treat hypertension. It is administered orally as the prodrug, candesartan cilexetil, which is rapidly converted to its active metabolite, candesartan, during absorption in the gastrointestinal tract. Candesartan lowers blood pressure by antagonizing the renin-angiotensin-aldosterone system (RAAS); it competes with angiotensin II for binding to the type-1 angiotensin II receptor (AT1) subtype and prevents the blood pressure increasing effects of angiotensin II.

Unlike angiotensin-converting enzyme (ACE) inhibitors, ARBs do not have the adverse effect of dry cough.

Fig.1: Candesartan

Several methods are available in the literature for the determination of candesartan like UV and HPLC.^1-7

MATERIAL AND METHODS:^8-17

Material:

Candesartan was a gift sample from Dr. Reddys Lab, Hyderabad. All chemicals (Distilled water, Methanol) and reagents used were of analytical grade and purchased from Qualigens Fine Chemicals, Mumbai, India.

Apparatus:

A Labindia UV–visible spectrophotometer (UV-T60-India) was used for all absorbance measurements with matched quartz cells.

METHODOLOGY:

Preparation of standard stock solution:

10mg Amount of standard was mixed with 10ml acetonitrile to +2ml of rat plasma (untreated) then vertically shaked for 30 min then centrifuged at 5000rpm for 1 hr. Then it was filterated using membrane filters to get clear organic solution. Then it was filled in to the sample vials of HPLC and loaded on to HPLC for Run.

Accurately weighed 10mg of Candesartan was transferred to a 100ml volumetric flask, dissolved in 20 ml distilled water by shaking manually for 10 min. The volume was adjusted with the same up to the mark to give the final strength, i.e. 100μg/ml.

Selection of wavelength for analysis of Candesartan:

Appropriate volume 1ml of standard stock solution of Candesartan was transferred into a 10ml volumetric flask, diluted to a mark with distilled water to give concentration of 10μg/ml (and also 20, 30μg/ml). The resulting solution was scanned in the UV range (200–400nm). In spectrum Candesartan showed absorbance maximum at 245nm

Validation of the method^18-19

The method was validated in terms of linearity, accuracy, precision, and ruggedness.

Linearity study:

Different aliquots of Candesartan in the range 0.5–3ml were transferred into series of 10ml volumetric flasks, and the volume was made up to the mark with distilled water to get concentrations 5, 10, 15, 20, 25, and 30 μg/ml, respectively. The solutions were scanned on a spectrophotometer in the UV range 200–400nm. The spectrum was recorded at 245nm. The calibration plot was constructed as concentration vs. absorbance

Accuracy:

To the preanalysed sample solutions, a known amount of standard stock solution was added at different levels, i.e. 50%, 100%, and 150%. The solutions were reanalyzed by the proposed method.

Precision:

Precision of the method was studied as intraday and interday variations. Intraday precision was determined by analyzing the 10, 15 and 20μg/ml of Candesartan solutions for three times in the same day. Interday precision was determined by analyzing the 10, 15, and 20μg/ml of Candesartan solutions daily for 3 days over the period of week.

Sensitivity:

The sensitivity of measurements of Candesartan by the use of the proposed method was estimated in terms of the limit of quantification (LOQ) and limit of detection (LOD). The LOQ and LOD were calculated using equation LOD = 3 × N/B and LOQ = 10 × N/B, where ‘N’ is standard deviation of the peak areas of the drugs (n = 3), taken as a measure of noise, and ‘B’ is the slope of the corresponding calibration curve.

Repeatability:

Repeatability was determined by analyzing 20μg/ml concentration of Candesartan solution for six times.

Ruggedness:

Ruggedness of the proposed method is determined for 20 μg/ml concentration of Candesartan by analysis of aliquots from a homogenous slot by two analysts using same operational and environmental conditions.

RESULTS AND DISCUSSION:

Solubility studies:

Table 1: Solubility studies

Solvent	Solubility(mg/ml)
Ehanol	2.0
Methanol	1.2
Alcohol	1.3
Chloroform	1.7
Acetone	1.4
Distilled water	0.7

The pure API of Candesartan is freely soluble in in organic solvents and sparingly soluble in aqueous solutions

Selection of wavelength for analysis of Candesartan:

During the development phase, the use of ethanol as the diluent resulted in preferable outcome in UV analysis. The pre-determined wavelength of maximum absorption (λmax) was 245 nm.

Fig.2: UV –visible spectrum

stocks	Wavelentgth of stocks	Absorbance
5 μg/ml	245	0.301
10 μg/ml	245	0.514
15 μg/ml	245	0.715

Method Validation:

Linearity:

Table 2: Results of Linearity

Concentration (ug/ml)	Absorbance (nm)
0	0
5	0.265
10	0.492
15	0.706
20	0.933
25	1.141
30	1.365

Fig.3: Results of Calibration graph

Accuracy:

N=3

Fig 4: Accuracy 50%

N=3

Fig.5: Accuracy 100%

N=3

Fig.6: Accuracy 150%

Sensitivity:

The linearity equation was found to be y=0.0449x + 0.0266. The LOQ and LOD for Candesartan were found to be 1.54μg and 0.541μg, respectively.

Repeatability:

Fig.8: Results of Repeatability

Table 5: Results of Repeatability

Concentration (μg/mL)

Absorbance measured (Mean ± SD)

Amount Found

(%)

RSD (%)

0.7154±0.024

99.74

0.05

Repeatability was determined by analyzing 20μg/ml concentration of Candesartan solution for six times and the % amount found was 99.74 and % RSD was found to be < 2.

Ruggedness:

Fig.9: Results of Ruggedness

The peak area was measured for same concentration solutions, six times. The results are in the acceptable range for both the drugs. The result showed that the % RSD was less than 2%.

Table 6: Results of Ruggedness

Analyst

Concentration (μg/mL)

Absorbance measured (Mean ± SD)

Amount Found

(%)

RSD (%)

0.7114±0.0241

98.94

0.04

0.7215±0.0154

99.16

0.02

ACKNOWLEDGEMENT:

The authors are highly thankful to Dr. Reddys Lab, Hyderabad for gift sample and Sanjivani College of Pharmaceutical Education and Research, Kopargaon for providing necessary support and facilities for this research work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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18. ICH topic Q2B, validation of analytical procedure & methodology, The European agency for evaluation of medicinal products, human medicines evaluation unit 1996.

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Received on 24.12.2020 Revised on 08.02.2021

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):79-83.

DOI: 10.52711/2231-5675.2021.00015

%Concentration (at specification Level) N=3	absorbance	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	0.354	5.0	4.995	99.9	99.92
100%	0.601	10	9.992	99.92
150%	0.695	15	14.991	99.94

Concentration (μg/mL)	Intra-day precision			Inter-day precision
Concentration (μg/mL)	Absorbance measured	RSD (%)	Average (%)	Absorbance measured	RSD (%)	Average (%)
5	0.357±0.12	1.33	99.15	0.364±0.17	1.21	99.10
10	0.605±0.21	0.91	98.75	0.615±0.25	1.24	99.14
15	0.699±0.25	1.01	99.14	0.684±0.20	1.19	98.95