A Green and Stability-Indicating RP-HPLC Approach for the Quantification of Fimasartan Potassium Trihydrate and Indapamide in Synthetic Mixtures

 

Deep Savsani1*, Mitali Dalwadi1, Priyanka Patil2, Umesh Upadhyay3

1PG Scholar, M.Pharm (Q.A.), Sigma Institute of Pharmacy, (Faculty of Pharmacy),

Sigma University, Bakrol, Vadodara – 390019.

1Assistant Professor, Sigma Institute of Pharmacy, Sigma University, Bakrol, Vadodara – 390019.

2Dean, Sigma Institute of Pharmacy, Sigma University.

3ProVC, Sigma University Bakrol, Vadodara – 390019.

*Corresponding Author E-mail: deepsavsani123@gmail.com, mitaliswami7@gmail.com, priyanka.ph@sigmauniversity.ac.in, umesh.ph@sigmauniversity.ac.in

 

ABSTRACT:

Robust RP-HPLC method was established and validated for the simultaneous quantification of Fimasartan potassium trihydrate and Indapamide in a synthetic mixture. Analytes were separated on a C18 column with a mobile phase of phosphate buffer (pH 3.5), methanol, and acetonitrile (45:25:30 v/v/v) at 1.0mL/min, and detected at 248nm. The method showed excellent linearity, precision, and accuracy across the tested concentration ranges, with low LOD and LOQ values. System suitability and specificity were confirmed, including successful separation of both drugs from their degradation products under various stress conditions. The validated method is rapid, sensitive, and suitable for stability and quality control studies of these antihypertensive agents in combination. The developed method also adheres to green analytical chemistry principles. It minimizes solvent consumption and environmental impact while ensuring reliable performance.

 

KEYWORDS: Fimasartan, Indapamide, RP-HPLC, Stability-Indicating, Method Validation, Forced Degradation, Green analytical Chemistry.

 

 

INTRODUCTION: 

Hypertension is commonly managed by combination therapy using agents with complementary mechanisms. Fimasartan potassium trihydrate (FMST) is a selective angiotensin II type-1 receptor blocker (ARB) that lowers blood pressure by preventing vasoconstriction. Indapamide (IND) is a thiazide like diuretic that reduces blood volume by inhibiting renal sodium reabsorption.1-5

 

Combining an ARB with a diuretic, such as FMST and IND, provides synergistic antihypertensive effects by acting on distinct pathways. Despite individual methods for these drugs, no stability-indicating RP-HPLC method has been reported for their simultaneous quantification in a synthetic mixture. Existing literature describes HPLC or spectrophotometric assays for Fimasartan or Indapamide separately or in other combinations, but a validated method addressing both analytes and their degradation is lacking.6-13 This study aims to develop a simple, precise, accurate, and stability-indicating RP-HPLC method accordance with ICH Q2(R1) guidelines. No single stability-indicating RP-HPLC method existed for the simultaneous quantification of Fimasartan potassium trihydrate and Indapamide, despite the widespread use of these agents in combination antihypertensive therapy. This method not only addresses quality control and regulatory requirements but also aligns with green chemistry principles, reducing environmental impact while ensuring reliable stability assessment.13-20

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Reference standards of Fimasartan potassium trihydrate and Indapamide were obtained as pure solids. The APIs employed in the study possessed a purity of ≥99. % (w/w), as certified by the supplier’s Certificate of Analysis (CoA). HPLC-grade methanol, acetonitrile, potassium dihydrogen phosphate and ortho phosphoric acid were of analytical grade.21

 

Instrumentation:

A gradient HPLC system with UV detection was used. Preliminary UV scans of each drug in methanol showed λ_max at 263nm for FMST and 236nm for IND. For simultaneous detection, 248nm was selected as a compromise wavelength.22

 

Chromatographic Conditions:

eparation was performed on a Shimpack ODS C1823 column with a 0.02M phosphate buffer (pH 3.5): methanol: acetonitrile (45:25:30, v/v/v) mobile phase at 1.0mL/min, 248nm detection, and 10μL injection volume. Methanol was used as diluent for analyte solubility.

 

Preparation of Standards and Samples:

Stock solutions (1000µg/mL) of FMST and IND were independently prepared in methanol. Calibration standards were generated by serial dilution, yielding 30–180µg/mL for FMST and 2.5–15µg/mL for IND. System suitability was assessed using a mixed standard (600µg/mL FMST, 50µg/mL IND). Forced degradation samples were obtained by subjecting this mixture to various stress conditions.

 

Method Validation:8

The method was validated per ICH Q2(R1), assessing linearity over six levels for each drug. Accuracy was confirmed by recovery at 50%, 100%, and 150%. Precision (repeatability and intermediate precision) was determined via multiple replicates on different days. LOD and LOQ were calculated statistically, and system suitability criteria—tailing factor (<2) and resolution (>2)—were confirmed across six injections.

 

Forced Degradation Studies:

Stability-indicating ability was assessed by subjecting the drugs to stress conditions: acid hydrolysis (0.1N HCl, reflux 2h), base hydrolysis (0.1N NaOH, 2h), oxidation (3% H₂O₂, room temp 2h), thermal (dry heat 80°C, 24h), and photolytic (UV light, 254nm, 24h). After appropriate neutralization, samples were diluted and analyzed by HPLC. Degradation was quantified by comparing remaining assay to control.

RESULTS:

System Suitability:

The chromatographic system showed excellent suitability. FMST and IND had retention times of ~2.54 and 9.19min (mean, n=6) with RSD ≤1.4%. Tailing factors were 1.27(FMST) and 1.137(IND), and theoretical plates were >5800 for both. Resolution between FMST and IND peaks was 9.409 (RSD = 1.13%). These values meet acceptance criteria, confirming system performance.

 

Linearity:

The method was linear over 30–180 µg/mL for FMST and 2.5–15µg/mL for IND. Linear regression equations were y = 52482x+706.3 (IND) and y = 15293x – 7196.5 (FIMA), with correlation coefficients R˛ = 0.9998 and 0.9999, respectively. Calibration plots (Fig. 1-2) showed even distribution of residuals, indicating good fit.

 

 

Figure 1: Calibration curve of 30-180 µg/mL for Fimasartan potassium trihydrate (FMST)

 

 

Figure 2: Calibration Curve of 2.5- 15 µg/mL of Indapamide

 

Precision and Accuracy: Repeatability was excellent, with %RSD of peak areas <1% at all levels. Recovery studies at 50%, 100%, and 150% levels gave mean recoveries of 98.69–101.39% for FMST and 98.85–101.19% for IND, demonstrating accuracy. Intermediate precision (day-to-day) also showed RSD less than 2%, Showing in table no 1-3.

 

Table 1: Accuracy data of Indapamide (IND) and Fimasartan potassium trihydrate (FMST)

Concentration Level (%)

Preset Concentration (µg/mL)

Fortified Concentration (µg/mL)

Aggregate Concentration (µg/mL)

Peak Area

± SD

Detected Concentration (µg/mL)

Percentage Recovered

Indapamide (IND)

0

5

0

5

264863.14 ± 1188.90

5.03 ± 0.02

100.67±0.02

50

5

2.5

7.5

395257.60 ± 4965.27

7.52 ± 0.09

100.24±0.09

100

5

5

10

519515.56 ± 3305.23

9.89 ± 0.06

98.85±0.06

150

5

7.5

12.5

664515.29 ± 3786.10

12.65 ± 0.07

101.19±0.07

Fimasartan potassium trihydrate (FMST)

0

60

0

60

898390.19 ± 9788.73

59.22 ± 0.64

98.69 ± 1.06

50

60

30

90

1388363.34 ± 6315.96

91.25 ± 0.41

101.39±0.41

100

60

60

120

1811633.19 ± 2586.82

118.93 ± 0.17

99.11±0.17

150

60

90

150

2287441.46 ± 9398.07

150.04 ± 0.64

100.03±0.61

N=3

 

Table 2: Repeatability Data of Indapamide (IND) and Fimasartan potassium trihydrate (FMST)

Fimasartan potassium trihydrate (FMST) (90 μg/ml)

Indapamide (IND) (7.5 μg/ml)

Sr. No

Area

Sr. No

Area

1

1367534

1

392588

2

1367805

2

394600

3

1374441

3

391302

4

1376697

4

390653

5

1360966

5

395370

6

1366993

6

390653

Average

1369073

Average

392516

SD

5673.96

SD

2044.60

% RSD

0.41

% RSD

0.52

 

Table :3 Intraday and Interday precision of method

Fimasartan potassium trihydrate (FMST)

Conc.

(µg/mL)

Intraday precision

Interday precision

Peak Area

(Mean ± SD) n

%RSD

Peak Area

(Mean ± SD) n

%RSD

30

448454.81±4977.05

1.11

453427.13±4662.25

1.03

90

1367036.86±21856.93

1.60

1376743.77±20836.73

1.51

180

2753292.27±42776.17

1.55

2746936.97±48926.38

1.78

Indapamide (IND)

2.5

129721.5±1553.52

1.20

130290.97±1948.92

1.50

7.5

394695.78±4126.48

1.05

389264.11±6419.6

1.65

15

788458.74±6804.36

0.86

778786.74±8750.55

1.12

 

Sensitivity:

LOD and LOQ were calculated 4.39 and 13.31 µg/mLfor FMST, and 0.31 and 0.94 µg/mLfor IND. These low values indicate the method’s suitability for trace-level detection.

 

 

Robustness:

Method robustness was demonstrated by introducing minor variations in organic solvent ratio (±5%), flow rate (±0.1 mL/min), and detection wavelength (±2 nm). These adjustments produced negligible changes in retention time and peak morphology, with all %RSD values remaining below 2% (see Table 4).

 

 

Table 4: Robustness study of Indapamide (IND) and Fimasartan potassium trihydrate (FMST)

Experimental Factors

Experimental Modification

IND

FMST

Peak Area

%RSD

Peak Area

%RSD

Mobile Phase composition

buffer (pH 3.5, adjusted using 0.01N OPA), methanol, and acetonitrile in a volumetric ratio of 45:25:30 (v/v/v)

40:30:30 (% v/v/v) 

1369926.18±16958.61

1.24

399331.42±5208.31

1.30

45:25:30 (% v/v/v) 

1360019.52±22730.83

1.67

396444.52±6900.34

1.74

40:25:35 (% v/v/v) 

1366218.65±18157.61

1.33

398998.84±6482.42

1.62

Detection wavelength

245 nm

1349547.67±18880.05

1.40

403892.98±2217.78

0.55

248 nm

1360019.52±22730.83

1.67

396444.52±6900.34

1.74

251 nm

1360384.72±16913.18

1.24

390488.63±6834.3

1.75

Flow rate Changed

0.9 ml/min.

1365945.46±18062.31

1.32

398171.4±5552.16

1.39

1 ml/min.

1360019.52±22730.83

1.67

396444.52±6900.34

1.74

1.1 ml/min.

1350056.99±23608.19

1.75

393410.29±7323.28

1.86

 

ASSAY OF SYNTHETIC MIXTURE

Synthetic mixture of Indapamide (IND) and Fimasartan potassium trihydrate (FMST) containing 2.5 mg and 30 mg when analysed using the developed method, showed 100.95 % assay for Indapamide (IND) and 98.34 % assay for Fimasartan potassium trihydrate (FMST) % assay was given in Figure no 3:

 

 

Figure 3: Assay of synthetic mixture

Forced Degradation (Specificity):

The method successfully separated degradation products. Table 5 summarizes degradation results. Under acidic stress (0.1 N HCl), FMST degraded 15.31% (major degradant at t_R 3.988 min) and IND degraded 11.86% (t_R 11.028 min). Alkaline hydrolysis caused 18.51% FMST and 9.61% IND degradation. Oxidation (H₂O₂) gave 9.60% FMST and 16.67% IND degradation. Thermal stress induced 6.45% FMST and 6.89% IND degradation. Photolytic stress produced 6.53% FMST and 5.56% IND degradation. In all cases, degradant peaks were well resolved from the main peaks (resolution >2) and the assay of the active drugs remained >80%. Blank and placebo (matrix) injections showed no interfering peaks at the analyte retention times.

 

Table 5: Forced degradation results of Fimasartan potassium trihydrate (FMST) and Indapamide (IND) under various stress conditions

Stress

condition

Drug Conc. Taken

Drug Conc. found

Retention time of degradant

Amount of

degraded (%)

Amount of

recovered (%)

FMST

Acidic

90

76.22

3.988

84.69

15.31

Alkali

90

73.34

2.153, 5.590

81.49

18.51

Oxidative

90

81.35

4.904, 6.083

90.40

9.60

Thermal

90

84.19

-

93.55

6.45

Photo stability

90

84.12

-

93.47

6.53

IND

Acidic

7.5

6.61

11.028

88.14

11.86

Alkali

7.5

6.77

12.314

90.39

9.61

Oxidative

7.5

6.24

12.485

83.33

16.67

Thermal

7.5

6.98

-

93.11

6.89

Photo stability

7.5

7.08

-

94.44

5.56

 

The results confirm that the method is stability-indicating, as stress conditions led to partial degradation without co-elution of degradant peaks. Chromatograms of stressed samples showed a peak Figure no 4–14 for FMST and IND.

 

 

Figure 4: Acid Hydrolysis chromatogram of (a) FMST and IND in mixture 

 

Figure 5: Acid Hydrolysis chromatogram of FMST

 

 

Figure 6: Acid Hydrolysis chromatogram of IND              

 

Figure 7: Base Hydrolysis chromatogram of FMST and IND in mixture

 

 

Figure 8: Base Hydrolysis chromatogram of FMST               

 

 

Figure 9: Base Hydrolysis chromatogram of IND

 

Figure 10: Oxidative degradant chromatogram of FMST and IND in mixture    

 

Figure 11: Oxidative degradant chromatogram FMST

 

 

Figure 12: Oxidative degradant chromatogram of IND

 

 

Figure 13: Thermal stress chromatogram

 

 

Figure 14: Photolytic stress chromatogram

 

Eco-Friendliness and Greenness Assessment24-26

In line with Green Analytical Chemistry principles, the developed RP-HPLC method minimizes hazardous solvent use and reduces run time to lower solvent consumption and waste, without compromising separation quality. The method’s environmental impact was assessed using the Green Analytical Procedure Index (GAPI) and AGREE tools.

 

GAPI results showed 53.3% of parameters as green, indicating good eco-compatibility, while a few parameters related to sample volume and waste were yellow or red, suggesting areas for improvement. AGREE scored the method 0.6/1, reflecting substantial alignment with green chemistry despite manual sample handling and offline analysis.

 

Overall, the method achieves a balance between analytical performance and environmental sustainability.

 

 

Figure 15: The evaluation of the greenness of the proposed HPLC for the determination of FIMA and IND by GAPI.

 

Analytical Greenness:

The AGREE pictogram (shown below) evaluates various environmental aspects of the method. Manual sample handling (parameter 1) is marked yellow, while offline instrument operation (parameter 3) is red. Sample volume (parameter 2) and waste generation of 10 mL mobile phase (parameter 7) are highlighted in orange and yellow, respectively. The 15-minute run time (parameter 8) is dark yellow, and the use of non-bio-based reagents (parameter 10) is red. All other parameters are green, reflecting adherence to green chemistry principles. The method achieved an overall AGREE score of 0.6, demonstrating considerable eco-friendliness.

 

 

Figure 16: The evaluation of the greenness of the proposed HPLC for the determination of FIMA and IND by AGREE

 

DISCUSSION:

The newly developed RP-HPLC method provides an effective solution for simultaneous quantification and stability assessment of Fimasartan potassium trihydrate (FMST) and Indapamide (IND). Its rapid chromatographic separation, characterized by retention times of approximately 2.54min for FMST and 9.19 min for IND, substantially enhances analytical efficiency. The selected mobile phase comprising phosphate buffer (pH 3.5), methanol, and acetonitrile (45:25:30 v/v/v) delivered optimal peak resolution and symmetrical peak shapes, ensuring method robustness and reproducibility.

 

The method validation was rigorously performed as per ICH Q2(R1) guidelines, revealing excellent linearity (R˛ > 0.9998) across the investigated concentration ranges. Precision studies confirmed the method's repeatability, indicated by %RSD values consistently below 1%. Accuracy evaluation through recovery studies demonstrated satisfactory recoveries ranging from 98.69% to 101.39% for FMST and 98.85% to 101.19% for IND, thus establishing the reliability of this analytical approach for routine quality assurance.

 

Significantly, the method's stability-indicating capability was thoroughly examined through forced degradation studies under acidic, alkaline, oxidative, thermal, and photolytic conditions. The clearly separated degradation products confirmed the method's suitability for distinguishing active pharmaceutical ingredients from their degradation products. The observed degradation profiles were consistent with previous reports on similar antihypertensive agents, underscoring the method's practicality in stability studies and regulatory compliance.

 

Additionally, the environmental impact of the developed method was evaluated using Green Analytical Procedure Index (GAPI) and AGREE metrics. GAPI analysis indicated satisfactory environmental compatibility, with approximately 53% of evaluated parameters being classified as green. The AGREE analysis yielded an overall score of 0.6 out of 1, reflecting substantial eco-friendliness. These assessments highlight the method's alignment with green analytical chemistry principles, ensuring minimized environmental impact and sustainability.

 

The rationale for combining FMST and IND lies in their complementary mechanisms of action, providing synergistic antihypertensive effects and superior control compared with monotherapy. A clinical trial (ClinicalTrials.gov Identifier: NCT05878561) further supports the therapeutic significance of this combination. To date, no validated, stability-indicating and eco-friendly RP-HPLC method for their simultaneous estimation has been reported, highlighting the novelty of the present work

 

Compared to existing methodologies, this novel method uniquely facilitates simultaneous analysis of FMST and IND with enhanced sensitivity, shorter analysis time, and improved eco-friendliness, thus improving laboratory efficiency, throughput, and sustainability.

 

CONCLUSION:

A validated, stability-indicating RP-HPLC method was established for simultaneous quantification of Fimasartan potassium trihydrate and Indapamide in synthetic mixtures. The method is rapid, precise, and specific, enabling effective separation and degradation product detection under isocratic conditions. All validation parameters met ICH Q2(R1) standards, confirming suitability for routine quality control and stability assessment of combined antihypertensive formulations. In addition to its analytical robustness, the developed method supports the principles of green analytical chemistry. By reducing solvent consumption and minimizing environmental hazards, this eco-friendly approach ensures both sustainability and regulatory compliance.

 

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Received on 04.07.2025      Revised on 08.11.2025

Accepted on 12.01.2026      Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3162-3168.

DOI: 10.52711/0974-360X.2026.00449

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