INTRODUCTION:

Fimasartan is an orally active, non-peptide angiotensin II type 1 (AT1) receptor antagonist developed for the treatment of essential hypertension. It is a newer member of the angiotensin receptor blocker (ARB) class, which functions by selectively blocking the binding of angiotensin II to the AT1 receptor, thereby promoting vasodilation and reducing blood pressure without affecting heart rate^1,2. Chemically, Fimasartan is described as (2-butyl-5-dimethylaminothiocarbonyl methyl-6-methyl-3-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazo[4,5-b]pyridine). Its structural design incorporates key pharmacophores from earlier ARBs with modifications to enhance efficacy, metabolic stability, and bioavailability³.

Fig no. 1 Structure of Fimasartan⁴.

Fimasartan demonstrates rapid absorption after oral administration, with peak plasma concentrations typically achieved within 0.5 to 3hours. It has a relatively long elimination half-life (5 to 16hours), enabling once-daily dosing. The drug is primarily eliminated via biliary excretion, and its pharmacokinetics is not significantly altered in patients with mild-to-moderate renal impairment^5,6. Due to its therapeutic significance and increasing clinical use, the development of robust and reliable analytical methods for the quantification of Fimasartan in pharmaceutical formulations and biological matrices has become essential. Analytical method development and validation are critical components in the quality control of pharmaceutical products, ensuring the accuracy, precision, specificity, and reproducibility of results in accordance with ICH guidelines^5,6.

Various analytical techniques have been employed for the estimation of Fimasartan, including UV spectrophotometry⁷, RP-HPLC⁸, UPLC⁹, and LC–MS/MS methods¹⁰. Among these, reversed-phase high-performance liquid chromatography (RP-HPLC) has gained prominence due to its simplicity, high resolution, and wide applicability in routine quality control testing. Method validation parameters such as linearity, precision, accuracy, limit of detection (LOD), limit of quantitation (LOQ), robustness, and specificity are crucial to confirm that the developed method is suitable for its intended purpose⁶.

Materials and Instruments:

Table No 1: List of Instruments used

Double beam UV – Visible Spectrophotometer
Model	UV 550
Make	Jasco
Software	Spectra manager

HPLC System HPLC Quaternary Gradient System
Model No.	1260Infinity II
Make	Agilent
Column	Phenomenex
Software	Openlab EZ Chrome

Table No 2: List of Reagents Used

Sr. No.	Chemicals/ Reagents/ Solvents	Supplier	Grade
1	Acetonitrile	Merck	HPLC grade
2	Water	Siddhi Lab	HPLC grade
3	Orthophosphoric acid	Thermofisher Scientific	Analytical grade

HPLC Method Development:

Preparation of standard stock solution for Chromatographic development:

A standard stock solution of Fimasartan was prepared by accurately weighing 11.83mg of Fimasartan Potassium Trihydrate, corresponding to 10mg of Fimasartan base, and transferring it into a clean, dry 20mL volumetric flask. Approximately 15mL of distilled water was added to dissolve the substance completely, and the volume was adjusted to the mark with the same solvent, resulting in a final concentration of 500µg/mL (PPM). From this stock solution, 2mL was further diluted to 10 mL using the mobile phase to obtain a working standard solution with a concentration of 100µg/mL. This solution was freshly prepared using the mobile phase employed in each trial and injected during method development studies.

Preparation of System suitability test (Fimasartan standard solution):

Approximately 23.67mg of Fimasartan Potassium Trihydrate, equivalent to about 20mg of Fimasartan, was accurately weighed and transferred to a clean 50mL volumetric flask. Around 35mL of distilled water was added, and the mixture was sonicated to ensure complete dissolution. The solution was then diluted to volume with distilled water to obtain the standard stock solution. From this, 1.0mL of the stock solution was pipetted into a 20mL volumetric flask and diluted to the mark with the mobile phase, yielding a working concentration of 20 µg/mL. Chromatographic analysis was carried out by injecting this solution.

To ensure the performance of the chromatographic system, system suitability tests were conducted as per pharmacopoeial guidelines. These tests verify the reliability and efficiency of the system prior to sample analysis. System suitability was assessed by injecting the standard solution five times, and the data obtained were evaluated and recorded accordingly.

Analysis of marketed Test sample:

Marketed test sample Having Name Fimagen 60mg tablets are selected for analysis and for doing validation.

Sample preparation of Marketed test sample:

Twenty Fimasartan tablets were accurately weighed and finely powdered using a mortar and pestle. The powdered material was mixed thoroughly using butter paper to ensure uniform distribution. An amount of the powder equivalent to 20mg of Fimasartan was weighed and transferred to a clean, dry 50mL volumetric flask. Approximately 35mL of distilled water was added, and the mixture was sonicated for 10minutes with intermittent shaking to facilitate dissolution. After sonication, the solution was allowed to cool to room temperature and then diluted to volume with distilled water.

The resulting solution was filtered through a 0.45 µm syringe filter, discarding the first 3–5 mL of the filtrate to ensure clarity and accuracy. A 1.0 mL portion of the filtered solution was further diluted to 20 mL using the mobile phase, resulting in a final concentration of 20 µg/mL of Fimasartan. The prepared solution was then injected into the chromatographic system, and the chromatograms were recorded for analysis.

Table No. 3 Sample Prepared in duplicate and Summary of sample preparation as follows:

Sample	Sample (mg)	Diluted to (mL)	Volume taken	Diluted to (mL)
Sample 1	59.3	50	1	20
Sample 2	58.6	50	1	20

Formula for % Assay calculation:

Validation of RP-HPLC Method:

1) Filtration study:

An analytical procedure's filtration study verifies the filter's compatibility with the sample, deposition on the filter bed, and interference from extraneous components.
The Fimasartan Test sample (tablet solution) was used in this investigation. An unfiltered and a filtered test solution were used in a filtering investigation. Five milliliters of the aliquot sample were discarded in order to use 0.45µm PVDF and 0.45µm nylon syringe filters for the filtration process.

2) Stability of analytical solution:

Both the standard and test sample solutions underwent stability testing. Under standard laboratory settings, a stability investigation was conducted. After 12 and 24 hours, the solution was examined under standard, well-lit laboratory settings. By computing the difference between the test solution's results at each stability time point and the beginning results, a stability analysis of the standard and test solutions was carried out.

3) Specificity:

The capacity to clearly identify the analyte in the presence of potentially anticipated components is known as specificity. To demonstrate the method's specificity, the following solution will be made and injected.

1. Blank (diluent in the mobile phase)

2. Placebo

Placebo Sample solution preparation:

35.23mg of the placebo material, or 20mg of Fimasartan, was weighed and then placed to a 50mL volumetric flask that had been cleaned and dried. 35mL of water was added, and it was sonicated for ten minutes while being shaken occasionally. After ten minutes, let the solution cool to room temperature and add water to bring the volume up to par. 3-5mL of the first filtrate were discarded after the solution was filtered using an appropriate 0.45µ PVDF syringe filter. Chromatograms were recorded after injecting the resulting solution after further diluting 1.0ml of the filtered stock solution to 20 ml with mobile phase.

4) Linearity and range:

Preparation of linearity solution:

The capacity of an analytical process to produce test findings that are exactly proportionate to the concentration (quantity) of analyte in the sample (within a specified range) is known as linearity. Five linearity levels, ranging from 50% to 150% of working concentration, were carried out.

5) Limit of Detection (LOD) and Limit of Quantitation (LOQ):

Detection limit:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

Quantitation limit:

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

As per ICH Q2R1 guidelines LOD and LOQ was determined by using the approach Based on the Calibration Curve in which residual standard deviation of a regression line was calculated and determined the LOD and LOQ by using following formula:

LOD = 3.3 σ / S

LOQ = 10 σ / S

Where,

σ = residual standard deviation of a regression line

S = Slope of regression line

6) Accuracy (% Recovery):

The degree of agreement between the value found and the value that is recognized as either a conventional true value or an acceptable reference value is a measure of the analytical procedure's accuracy. Between 50% and 150% of working concentration will be used for accuracy. Three copies of each accuracy level's solution were made. determined the percentage recovery for every sample, the mean percentage recovery for every level, and the overall recovery. Additionally, the percentage RSD for each level and the percentage RSD for the overall recovery were computed.

7) Precision:

Precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous test under the prescribed conditions. Precision is of two types, Repeatability and Intermediate precision. It is performed on tablet test sample.

1. Repeatibility

2. Intermediate precision.

8) Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

RESULT AND DISCUSSION:

Selection of analytical wavelength:

Fig No. 2 UV spectrum of Fimasartan

Optimized Chromatographic Conditions:

Table No. 3 Chromatographic Conditions for RP-HPLC Method

Parameter	Description
Mode	Isocratic
Column Name	Phenomenex C18, 250 mm*4.6mm, 5µ
Detector	UV Detector
Injection Volume	20 µl
Wavelength	262 nm
Column Oven temp	35ºC
Mobile Phase	Acetonitrile: 0.05% OPA in Water (75:25 % V/V)
Flow Rate	1.0 ml/min
Run time	7 Minutes

Fig No. 3: Typical chromatogram of Standard solution of Fimasartan

Validation of RP-HPLC method:

1) Filtration study:

An analytical procedure's filtration study verifies the filter's compatibility with the sample, deposition on the filter bed, and interference from extraneous components. Conducted on a test sample of tablets.

Table No. 4 Results of Filter study:

Sample description	Area	% Absolute difference
Unfiltered	7752306	NA
0.45 µ PVDF filter	7787261	0.45
0.45 µ Nylon filter	7706925	0.59

2) Solution stability:

Stability study was conducted for Standard as well as Test Sample. Stability study was performed at normal laboratory conditions. The solution was stored at normal illuminated laboratory conditions and analyzed at initial, after 12 hours and 24 hours.

3) Specificity:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present. Blank and Placebo solution prepared and injected to check interference at R.T. of Fimasartan.

Table No. 6 Results of Specificity:

Description	Observation
Blank	No interference at R.T. of Fimasartan due to blank
Placebo	No interference at R.T. of Fimasartan due to placebo

Fig No. 4: Typical chromatogram of Blank solution

Fig No. 5: Typical chromatogram of Placebo solution

4) Linearity and Range:

Linearity of an analytical method is its ability to elicit test results that are proportional to the concentration of analyte in samples within a given range.

Fig No. 6: Calibration curve of Fimasartan

Table No. 7 Data of linearity of Fimasartan:

Sr no.	Parameter	Result value	Acceptance criteria
1	Beer's linearity range	10.0 -30.0 µg/mL	NA
2	Correlation coefficient (R²)	0.99998	NLT 0.98
3	Intercept	-61244.800	To be report
4	Slope	394975.9089	To be report
5	% RSD for area at each level	NA	NMT 2.0

The respective linear equation for Fimasartan was:

Y = M X + C

Y = 394975.9089 X + -61244.800

Where,

X = concentration of Analyte in µg/mL

Y = is area of peak.

M = Slope

C= Intercept

5) Limit of Detection (LOD) and Limit of Quantitation (LOQ):

σ = 18658.61424 (Residual standard deviation of a regression line)

s = 394975.9089

Detection limit (LOD):

LOD = 3.3 σ / S

LOD = 3.3 x 18658.61424 / 394975.9089

LOD = 0.156 µg/mL

Quantitation limit (LOQ):

LOQ = 10 σ / S

LOQ = 10 x 18658.61424 / 394975.9089

LOQ = 0.472µg/Ml

6) Accuracy (recovery):

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method is determined by applying the method to analyzed samples to which known amounts of analyte have been added¹¹.

Table No. 8 Result and statistical data of Accuracy of Fimasartan

Level (%)	Area	Recovered conc (µg/ mL)	Added conc (µg/ mL)	% Recovery	Mean Recovery	% RSD
50	3925300	9.99	10.06	99.30	99.44	0.625
	3971642	10.11	10.22	98.90
	3957405	10.07	10.06	100.12
100	7874512	20.04	20.11	99.66	99.60	0.559
	7880254	20.06	20.03	100.13
	7859210	20.00	20.20	99.02
150	11700410	29.78	30.00	99.26	99.86	0.895
	11923972	30.35	30.08	100.89
	11753049	29.91	30.08	99.44

Overall Recovery: 99.64 %

% RSD for Overall Recovery: 0.641

Chromatograms:

Fig No. 7: Typical chromatogram of Accuracy 50%.

Fig No. 8: Typical chromatogram of Accuracy 100%.

Fig No. 9: Typical chromatogram of Accuracy 150%.

Acceptance criteria:

% Recovery for each level and overall recovery: 98.0 to 102.0%

% RSD for each level and overall recovery: NMT 2.0

7) Precision:

Precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogenous sample. Precision of an analytical method is usually expressed as standard deviation or relative standard deviation. Precision was performed on Test sample¹². (table-9)

8) Robustness:

The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Following changes made under Robustness:

· Change in Wavelength

· Change in flow rate

· Change in column oven temperature

CONCLUSION:

Fimasartan can be estimated in pharmaceutical dosage forms and bulk using the established RP-HPLC method, which is easy to use, precise, accurate, and economical. With Acetonitrile: 0.05% OPA in water (75:25) as the mobile phase and a Phenomenex C18 column, the procedure showed outstanding linearity, specificity, and repeatability. The method's dependability for routine quality control analysis was validated in compliance with ICH Q2(R1) criteria. Fimasartan is efficiently quantified without the involvement of excipients, which makes it appropriate for use in both industrial and research settings. For formulations based on fimasartan, this study guarantees strong pharmaceutical evaluation and regulatory compliance.

REFERENCES:

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5. International Conference on Harmonisation (ICH). (2005). Validation of Analytical Procedures: Text and Methodology Q2 (R1).

6. Blessy, M., Patel, R. D., Prajapati, P. N., and Agrawal, Y. K. Development of forced degradation and stability indicating studies of drugs—A review. Journal of Pharmaceutical Analysis. 2014; 4(3): 159–165.

7. Shaikh, K. A., Patil, S. D., and Devkhile, A. B. Development and validation of UV spectrophotometric method for estimation of Fimasartan in tablet dosage form. International Journal of Pharmaceutical Sciences and Research. 2013; 4(5): 1767–1771

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Received on 24.06.2025 Revised on 24.07.2025

Accepted on 18.08.2025 Published on 08.10.2025

Available online from October 15, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(4):249-255.

DOI: 10.52711/2231-5675.2025.00039

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sample solution			Standard solution
Time point	Area	% Absolute difference	Time point	Area	% Absolute difference
Initial	7787035	NA	Initial	7849625	NA
12 Hours	7725236	0.79	12 Hours	7801360	0.61
24 Hours	7716230	0.91	24 Hours	7789236	0.77

Sr. No	Parameters	Intraday Precision	Interday Precision
1.	Mean	98.62	99.06
2.	STD	1.285862	1.191835
3.	% RSD	1.304	1.203

Change in Parameter	R.T.	Standard area	Asymmetry	Theoretical plates
Wavelength by +3 NM (265 NM)	3.18	7625813	1.26	7802
Wavelength by -3 NM (259 NM)	3.17	7491053	1.22	8116
Flow rate by +10% (1.1 mL/min)	2.87	7168405	1.20	6913
Flow rate by -10% (0.9 mL/min)	3.50	8439125	1.27	7342
Column oven temp by +2ºC (37 ºC)	3.26	7780684	1.25	6981
Column oven temp by -2ºC (33 ºC)	3.27	7789236	1.27	7053