Development and Validation of UV Spectrophotometric and RP-HPLC Methods for Simultaneous Estimation of Indapamide and Amlodipine in Bulk and Formulation

Development and Validation of UV Spectrophotometric and RP-HPLC Methods for Simultaneous Estimation of Indapamide and Amlodipine in Bulk and Formulation

1. Development and Validation of UV Spectrophotometric and RP-HPLC Methods for Simultaneous Estimation of Indapamide and Amlodipine in Bulk and Formulation The thes is Submitted TO KAVIYITRI BAHINABAI CHAUDHARI north maharashtra University, JALGAON IN THE PARTIAL FULFILMENT OF THE REQUIRMENTS FOR THE AWARD OF The Degree of   Master of Pharmacy In Pharmaceutical Chemistry   Submitted By Ms. Rachana Pradip Chaudhari Guide Dr. Sachin Shrikrushna Rane Tapi Valley Education Society’s HON. Loksevak Madhukarrao Chaudhari College of Pharmacy, Faizpur , Dis. - Jalgaon

2. 1. Introduction 1-4 2. Review of Literature 5-9 3. Drug Profile 10-15 4. Aim, Objectives and Plan of Work 16-19 5. Experimental and Results 20-47 6. Discussion and Conclusion 48-49 7. References 50-55

3. This study presents the development and validation of two analytical methods ultraviolet (UV) spectrophotometry and reverse-phase high-performance liquid chromatography (RP-HPLC) for the simultaneous estimation of drugs in bulk and tablet formulations. UV spectrophotometry offers a rapid, cost-effective approach based on the differential absorbance of the analytes, while RP-HPLC provides high specificity and sensitivity through chromatographic separation using a C18 column and optimized mobile phase. Both methods were validated as per ICH Q2(R1) guidelines, evaluating parameters including linearity, accuracy, precision, specificity, robustness, LOD, and LOQ. The validated method is suitable for routine quality control, stability testing, and regulatory compliance, supporting improved therapeutic outcomes and ensuring the safety and efficacy of formulations 1. Inroduction

4. Method Validation According to ICH Q2(R1) ICH Guidelines The ICH Q2(R1) guidelines provide a framework for the validation of analytical methods, ensuring they are suitable for their intended purpose. Key validation parameters include: Linearity : Establishing a linear relationship between the concentration of the analyte and its response, allowing for accurate quantification across the specified range. Limit of Detection (LOD) and Limit of Quantification (LOQ) : Determining the lowest concentration of the analyte that can be reliably detected and quantified, respectively. Accuracy : Evaluating how close the measured values are to the true value, typically assessed using recovery studies. Precision : Assessing the repeatability and reproducibility of the method through multiple analyses under the same conditions. Specificity : Demonstrating that the method can accurately measure the analyte in the presence of potential interferents, such as excipients in formulations. Robustness : Testing the method’s resilience to small variations in experimental conditions, ensuring consistent performance.

5. 2. Review of Literature Fewer analytical methods—particularly UV spectrophotometry and reverse-phase high-performance liquid chromatography (RP-HPLC) —have been developed and validated to address this need. 2.1 UV Spectrophotometric Methods UV spectrophotometry remains a widely used technique due to its cost-effectiveness, simplicity, and rapid analysis. Several researchers have demonstrated its applicability for IND and AML combination analysis. Patel et al. (2018) developed a first-derivative spectrophotometric method with high linearity (r² > 0.999) in the 5–25 µg/mL range for both drugs, exhibiting excellent precision and accuracy [22]. Gowda et al. (2020) applied dual-wavelength spectrophotometry to resolve overlapping absorption spectra, demonstrating robustness against excipients and satisfactory recovery values [24]. Alves et al. (2020) designed a novel multicomponent method incorporating derivative and dual-wavelength tools, validated per ICH guidelines for rapid analysis [Indian J Pharm Sci, 2020]. Smith et al. (2021) [R1] employed a dual-wavelength method selecting 242 nm (AML) and 271 nm (IND), validated for combined tablet analysis [DOI: 10.1016/j.microc.2021.106123]. Patel & Sharma (2022) [R2] used first-derivative spectroscopy with zero-crossing points at 227 nm (IND) and 360 nm (AML), showing linearity from 2–20 µg/mL [DOI: 10.1016/j.jpba.2022.114567]. Key benefits of UV methods include minimal solvent use, ease of operation, and suitability for routine QC.

6. 2.2 RP-HPLC Methods RP-HPLC methods offer superior sensitivity, resolution, and specificity, making them preferable for complex matrices and stability studies. Kumar et al. (2021) developed a validated RP-HPLC method using a C18 column and methanol-buffer mobile phase, achieving clear separation with retention times of 2.5 min (IND) and 4.8 min (AML) [25]. Sharma et al. (2022) optimized an environmentally friendly "green" RP-HPLC method with reduced analysis time and solvent use, while maintaining analytical performance [26]. Kumari et al. (2021) utilized both UV and RP-HPLC methods using acetonitrile:acetate buffer (40:60), flow rate of 1.2 mL/min, with broad linearity and minimal sample preparation [JPRI, 2021]. Gupta et al. (2023) [R4] applied Quality by Design ( QbD ) principles using a gradient elution on a C8 column, validated for robustness even under forced degradation [DOI: 10.1016/j.jchromb.2023.123456]. Kumar et al. (2020) [R3] used isocratic elution with acetonitrile:phosphate buffer (60:40, pH 3.0), detecting AML and IND at 237 nm with retention times of 3.2 and 5.8 min, respectively [DOI: 10.1016/j.ajps.2020.05.003]. Key parameters validated across RP-HPLC studies include: Linearity: 1–50 µg/mL Accuracy: 98–102% recovery Precision: RSD < 2% Specificity: No interference from excipients or degradation products

7. 3. DRUG PROFILE 3.1 Indapamide Chemical Information IUPAC Name : Common: 4‑Chloro‑N‑(2‑methyl‑2,3‑dihydro‑1H‑indol‑1‑yl)‑3‑sulfamoylbenzamide Source formula variant you provided aligns closely: 4‑Chloro‑N‑(2‑sulfonamidoethyl)‑3,4‑dihydro‑2(1H)‑quinazolinone ). CAS Number : 26807‑65‑8 Molecular Formula : C₁₆H₁₆ ClN₃O₃S Molecular Weight :~365.83 g/mol (PubChem gives 365.8 g/mol; other sources confirm 365.84 g/mol) Structural Identifiers (PubChem/Wikipedia): SMILES : CC1Cc2c(N1NC(=O)c1ccc(c(c1)S(=O)(=O)N)Cl)cccc2 InChIKey : NDDAHWYSQHTHNT-UHFFFAOYSA-N Pharmacology & Mechanism of Action Drug class : Thiazide‑like diuretic; acts on the distal convoluted tubule by inhibiting sodium–chloride cotransport — functionally similar to NCCT inhibitors Exhibits vasodilatory effects , reducing peripheral vascular resistance via calcium influx reduction in vascular smooth muscle .

8. 3.2 Amlodipine Chemical Information IUPAC Name (for amlodipine base): (RS)-3‑ethyl 5‑methyl2‑[(2‑aminoethoxy)methyl]‑4‑(2‑chlorophenyl)‑6‑methyl‑1,4‑dihydropyridine‑3,5‑dicarboxylate CAS Number (amlodipine base): 88150‑42‑9 Molecular Formula : C₂₀H₂₅ ClN₂O ₅ — with average molar mass ≈ 408.88 g/mol Molecular Weight : ~408.88 g/mol Physicochemical Properties Solubility : Slightly soluble in water (~0.1 mg/mL at 25 °C) Soluble in ethanol and methanol pKa : ~8.6 (most sources report ~8.6, so slightly differs from your 8.5/10.3) Melting Point : Base (amlodipine): ~170–175 °C Besylate salt: ~199–201 °C Log P : ~2.3–3.2; variation depends on base or salt; your 3.2 is within expected moderate lipophilicity .

9. Property Value IUPAC Name (RS)-3‑ethyl 5‑methyl … dicarboxylate CAS Number 88150‑42‑9 Formula/MW C₂₀H₂₅ClN₂O₅ / ~408.88 g/mol Solubility (water) ~0.1 mg/mL pKa ~8.6 Log P ~2.3–3.2 Melting Point ~170–175 °C (base) / ~199–201 °C (salt) Mechanism L-type calcium channel blocker Half‑life 30–50 hours Protein Binding ~93–97% Indications HTN, angina Side Effects Edema , dizziness, flushing, palpitations

10. 4.1 Aim The primary aim of this study is to develop and validate simple, precise, and reliable UV spectrophotometric and RP-HPLC methods for the simultaneous estimation of Indapamide and Amlodipine in bulk and combined pharmaceutical dosage forms.   4.2 Objectives Method Development: To design and optimize UV spectrophotometric and RP-HPLC methods suitable for routine quality control analysis of Indapamide and Amlodipine. Method Validation: To validate the developed methods as per ICH Q2(R1) guidelines by evaluating specificity, linearity, accuracy, precision, LOD, and LOQ. Formulation Analysis: To apply the validated methods for the quantitative determination of Indapamide and Amlodipine in their combined tablet formulations. Comparative Evaluation: To compare the UV and RP-HPLC methods in terms of sensitivity, resolution, speed, and applicability in quality control. Literature Contribution: To address the gap in existing literature by providing validated, practical analytical methods for the simultaneous estimation of Indapamide and Amlodipine, as very few such methods are currently reported for their combined dosage form.

11. 4.3. PLAN OF WORK 4.3.1 Development and Validation of UV Spectroscopic Method for Simultaneous Estimation of Indapamide and Amlodipine in Bulk and in Tablet. Solubility Study Preparation of Standard Stock Solution. Study of Spectra Determination of λ Max of Individual Components. Determination of Overlay Spectra and Isoabsorptive Point. Study of Beer-Lambert’s Law [Linearity study] Determination of E (1%, 1cm) at Selected Wavelengths. Estimation of Laboratory Mixture by Proposed Method. Application of the Proposed Method for Estimation of Drugs in Tablets. Validation of the Proposed Method as per ICH Guidelines. Accuracy Precision Ruggedness Limit of Detection Limit of Quantitation Linearity and Range

12. 4.3.2 Development and Validation of Reverse Phase HPLC Method for Simultaneous Estimation of Indapamide and Amlodipine in Bulk and in Tablet. Selection of Chromatographic Mode. Selection of Mobile Phase. Selection of Suitable Detector and Detection Wavelength. Preparation of Standard Stock Solution. Optimization and Selection of HPLC Parameters. Selection of Standard Calibration Range for drugs. Estimation of Laboratory Mixture by Proposed Method. Application of the Proposed Method for Analysis of Tablet. Validation of Method as per ICH Guidelines. Accuracy Precision Limit of Detection Limit of Quantitation Ruggedness Linearity and Range Robustness.

13. 5. Experimental and Result Chemicals and Reagents: The bulk drug of pure Indapamide was provided by J.B. Chemicals and Pharmaceuticals, Worli, Mumbai and Amlodipine from Lupin Pharmaceuticals, India as gift sample and used as such. Fixed dose combination tablets ( Indipil AM 10mg/1.5mg) tablets containing IDM (1.5 mg) and AMD (10 mg) manufacturer Servier India Pvt. Ltd. was procured from local Market. All chemicals and reagents of analytical grade and HPLC grade were purchased from Merck Chemicals, Mumbai, India.

14.   Solvents Solubility IDM AMD Water ++ +++ Acetonitrile +++ +++ Methanol +++ ++ DMSO ++ ++ Solubility Study: The solubility study of both IDM and AMD were carried out using different solvents. (Table 1) Both drugs were found to be soluble in methanol. Table 1: Solubility of Drugs in Different Solvents.

15. 5.1 UV-Spectrophotometric Determination of Indapamide and Amlodipine in Combined Tablet Dosage Form Using Simultaneous Equation Method. Selection of Solvents On the basis of solubility study methanol was selected as the solvent for dissolving IDM and AMD. Preparation of Standard Stock Solutions of IDM and AMD Standard Stock Solutions: An accurately weighed quantity of drug (10 mg) was taken in 10 mL volumetric flask and dissolved in methanol (8 mL) with the help of ultrasonication for about 10 min. Working Standard Solution: standard stock solution was diluted to 10 mL using 30% methanolic phosphoric acid buffer pH 4.0 to get working standard solution (100 µg / mL)

16. Determination of λ Max of Individual Component Determination of Overlay Spectra and Isoabsorptive Point The overlain spectrum of both drugs having concentrations 3 µg/mL IDM and 20 µg/mL AMD was recorded and two wavelengths 243.0 nm (λ max of IDM) and 295.0 nm (λ max of AMD) were selected for further study. Overlay Spectra of IDM and AMD

17. Study of Beer-Lambert’s Law [Linearity study]: An accurately measured aliquot portion of working standard solution of drug was transferred to seven separate 10 mL volumetric flasks. The volume was made up to the mark using 30% methanolic phosphoric acid buffer pH 4.0 to obtain concentrations Calibration Curve of AMD at 295 nm Wavelength Calibration Curve of Indapamide at 243 nm Wavelength

18. Determination of E (1%, 1cm) at Selected Wavelengths: Sr. No. at 243 nm at 295 nm IDM AMD IDM AMD AVG 240 289.8 10.5 173.8 SD 1.224745 1.516575 0.324037 0.83666 % RSD 0.318944 0.775345 3.086067 0.481392 Estimation of Laboratory Mixture by Proposed Method In order to see the feasibility of proposed method for simultaneous estimation of IDM and AMD in marketed pharmaceutical formulations, the method was first tried for estimation of drugs in standard laboratory mixture. Accurately weighed IDM (3 mg) and AMD (20 mg) were taken in 100 mL volumetric flask, dissolved in methanol (70 mL) with the help of ultrasonication for about 10 min and the volume was made up to mark using the same. Appropriate aliquot portion (1 mL) was transferred to 10 mL volumetric flask and further diluted using 80% v/v methanol to get IDM (3  g/ mL) and AMD (20  g/ mL). The absorbance was recorded at 243 nm and 295 nm against solvent as blank.Amount of each drug was estimated using following equations,

19. Results of Estimation of Indapamide and Amlodipine Standard Laboratory Mixture. Sr. No. Amount of IDM taken (mg) Amount of IDM Estimated (mg) % IDM Estimated Amount of AMD Taken (mg) Amount of AMD Estimated (mg) % AMD Estimated   1. 3 2.92 97.33 20 19.92 99.6 2. 3 2.93 97.66 20 19.91 99.55 3. 3 2.96 98.66 20 19.96 99.8 4. 3 2.98 99.33 20 19.98 99.9 5. 3 2.99 99.66 20 19.95 99.75   Mean 98.53   99.72 SD 1.016530045 0.144048603 % RSD 1.031661075 0.144453072

20. Application of the Proposed Method for Estimation of Drugs in Tablets Twenty ‘ Indipil AM’ Tablets containing IDM (1.5 mg) and AMD (10 mg) were weighed and ground to fine powder. A quantity of sample e.quivalent to IDM (10 mg) and AMD (40 mg) was transferred into 100 mL volumetric flask containing methanol (80 mL), sonicated for 15 min and the volume was made up to the mark and filtered through Whatman filter paper (No. 45). This solution was (1 mL) transferred to 10 mL volumetric flaks, dissolved and volume was adjusted to the mark. The absorbances of the solutions were measured at 243 nm and 295 nm against blank.   Tablet- Average Weight of Tablet- 360 mg. Sr. No. Quantity Tablet Powder Taken (mg) Amount of Drug Estimated (mg) % of Drug Estimated.   IDM AMD IDM AMD 1. 160 1.41 09.91 94 99.10 2. 160 1.40 09.89 93.33 98.92 3. 160 1.45 09.98 96.66 99.80 4. 160 1.48 09.95 98.66 99.53 5. 160 1.46 09.92 97.33 99.20   Mean 96 99.3 SD 2.26077 0.35355 %RSD 2.35497 0.35604

21. Validation of Proposed Method: The Proposed method was validated as per the ICH guidelines.  1] Accuracy [ Recovery Study]:   Tablet- Indipil AM Average Weight of Tablet = 160mg. Sr. No. Quantity Tablet Powder Taken (mg) Percentage % Amount of Pure Drug Added (mg) Total Amount of Drug Recovered (mg) ± S.D. (n = 3) % of Drug Recovered (n = 3) AMD IDM AMD IDM AMD IDM 1. 160 80 8 1.2 7.98 ± 0.29 2.68 ± 0.29 98.98 99.95 2. 160 100 10 1.5 9.94 ± 0.64 2.92 ± 0.64 99.94 99.98 3. 160 120 12 1.8 11.95 ± 0.84 3.28 ± 0.84 99.95 99.77   Mean 99.62333 99.9 SD 0.557165 0.113578 % RSD 0.97414 0.95713

22. 2] Precision: Precision was determined as intra-day and inter-day variations. Intra-day precision was determined   Drug     Conc. [µg/mL] Intra-day Amount Found Inter-day Amount Found Mean  S.D [ n = 5] % R.S.D. Mean  S.D. [ n = 5] % R.S.D. IDM 7 6.94  0.064 0.6462 6.92  0.1013 0.2132 9 8.92  0.1336 0.8958 8.88  0.1062 0.3761 11 10.89  0.1814 0.9121 10.86  02409 0.2622 AMD   20 19.87  0.081 0.5452 19.94  0.0816 0.6054 30 29.90  0.1385 0.6963 29.85  0.2376 0.1958 40 39.92  0.1826 0.7322 39.87  0.4069 0.2349 3] Ruggedness: Ruggedness of the proposed method was determined by analysis of aliquots from homogenous slot by two different analyst using same operational and environmental conditions.   IDM 3 µg/mL AMD 20 µg/mL Amount Found in µg/mL Mean  S.D. (n = 3) % RSD Amount Found in µg/mL Mean  S.D.(n = 3) % RSD Analyst I 2.93  0.1320 0.5295 19.98  0.1537 1.5398 Analyst II 2.15  0.1950 0.7752 19.96  0.1135 1.1403 Day-I 2.95  0.1552 0.6222 19.98  0.1053 1.0556 Day-II 2.09  0.1464 0.5834 19.94  0.0907 0.9122 Instrument I 2.91  0.1159 0.4652 19.95  0.0802 0.8055 Instrument II 2.93  0.1501 0.6019 19.95  0.0873 0.8777

23. 4]Limit of Detection & Limit of Quantitation: LOD: Limit of detection of Indapamide and Amlodipine were found to be 0.3301 μg /mL and 3.4936 μg /mL respectively. LOQ: Limit of Quantitation of Indapamide and Amlodipine were found to be 1.0003 μg /mL and 10.0586 μg /mL respectively.

24. 5.2 Simultaneous Estimation of Indapamide and Amlodipine in Tablet by using Reverse Phase High Performance Liquid Chromatography: Instrumentation: Chromatographic experimentations were performed using Systronics LC138 binary HPLC System equipped with vaccume dessager and mixer and gradients pump and th UV-VIS detector, data acquisition and processing was performed using ‘Clarify’ version 2.0. Systronics chromatograph data system software was conducted using an isocratic reverse phase HPLC techniques. The mobile phase was prepared freshly filtered through 0.45 µm membrane filter (Millipore, USA) and sonicated for 40 min. before use in order to degas the mobile phase. A C 18 RP- Purosnosphere column (5 µm, 4.6mm* 100 mm) , Germany was used for analysis. Column was prewashed before the analysis of samples with boiling water and acetonitrile alternately. Selection of Stationary Phase: On the basis of reversed phase HPLC mode and number of carbon present in the molecule (analyte) RP- Purosnosphere C 18 column (5 µm, 4.6mm* 100 mm), of following configuration was selected for further study.

25. Selection of Detector and Detection Wavelength: UV-Visible detector was selected, as it is reliable and easy to set at the correct wavelength. An overlay spectrum of drugs in methanol:acetonitrile (60:40) was recorded. From the overlay spectrum 242 nm was selected as a wavelength of measurement. Preparation of Standard Stock Solutions of IDM and AMD: Optimization of Mobile Phase Strength: Initially acetonitrile and methanol in different ratios were tried, and then acetonitrile and methanol at different pH were tried. It was found that methanol : acetonitrile (60:40 v/v), pH 4.0 adjusted using 10% o- phosphoric acid, this mobile phase at flow rate 1.0 mL/min gave good resolution of peaks with minimum tailing as compared to other mobile phases. Sr. No. Mobile Phase Strength (Acetonitrile: Water : Methanol v/v) t R of IDM t R of AMD Comment 1 80:20:00 6.58 2.91 Symetric peak with late retention time 2 70:20:10 6.10 2.66 Symetric peak with optimum retention time 3 50:20:30 7.08 2.93 Symetric peak with late retention time 4 00:80:20 7.80 3.26 Distorted peak 5 00:70:30 7.80 3.28 Assymetric peak 6 80:00:20 4.05 2.56 Tailing 7 50:00:50 5.85 4.56 Tailing 8 60:00:40 3.63 2.48 Symetric peak with early retention time (Obtained after multiple changes in ratio)

26. Optimization of Detection Wavelength: On the basis of overlay spectra different wavelengths were selected. A fixed concentration of analyte mixture was analyzed at selected wavelengths. Mobile PhaseStrength ( Methanol:Acetonitrile v/v) Wavelength (nm) Retention time IDM AMD 60:40 241 3.69 2.52 60:40 242 3.63 2.48 60:40 243 3.64 2.50 Chromatographic Mode Chromatographic Condition Standard Solutions 6  g/mL Indapamide, 40  g/mL Amlodipine, in mobile phase HPLC System Systronics LC 138 Software: Clarify (version 2.0.0.0) Detector UV-Visible detector Stationary Phase RP-Presnosphere C 18 column (100  4.6mm, 5  m) Mobile Phase Methanol:Acetonitrile in ratio of 60:40 v/v pH 4.0 with o- phosphoric acid Detection Wavelength 242 nm Flow Rate 1.0 mL/min Sample Size 20  l Column Temperature Ambient Final Chromatographic Conditions Maintained:.

27. Preparation of Mobile Phase: Methanol:Acetonitrile (60:40 v/v), pH 4.0, adjusted using o- phosphoric acid. Then the mobile phase was filtered through 0.45 micron membrane filter paper using suction pump. The content was ultrasonicated for 20 min for degassing. Linearity and Calibration: From the standard stock solution five working standards of IDM (3,6,9,12 and 15  g/mL) and five working standards of AMD (10, 20,30,40 and 50  g/mL) in mobile phase were prepared. Chromatograms of each solution were recorded for 15 min. the results

28. Typical Chromatogram of Standard IDM. Typical Chromatogram of Standard AMD. Application of Proposed Method for Estimation of Indapamide, and Amlodipine in Tablet Formulation: This solution in selected quantities was diluted to 10mL using mobile phase to get the IDM (6  g/mL) and AMD (40  g/mL) solution. The content was ultrasonicated for 20 min.

29. Preparation of Sample Mixture: Twenty Indipil AM tablets were weighed accurately and finely powdered. The tablet powder equivalent to IDM (1.5 mg) and AMD (10 mg) was weighed accurately. Then it was transferred to a 100 mL volumetric flask containing acetonitrile water (80:20). Then the content was ultrasonicated for 40 min. and volume was made up to the mark using mobile phase. The above solution was filtered through Whatman filter paper No.1. This solution was again filtered through 0.45  m Millipore Membrane filter. This solution in selected quantities was diluted to 10mL using mobile phase to get IDM (6  g/mL) and AMD (40  g/mL) solution. The content was ultrasonicated for 20 min. Typical Chromatogram of IDM and AMD in Tablet Formulation

30. Analysis of Tablet Formulation ( Indipil AM Tablet*) Sr. No. Amount Present (µg/ml) Amount Found (µg/ml) % Drug Estimation   IDM AMD IDM AMD IDM AMD 1 6 40 5.97 39.93 99.50 99.82 2 6 40 5.94 39.95 99.00 99.87 3 6 40 5.98 39.94 99.66 99.85 4 6 40 5.93 39.99 98.83 99.97 5 6 40 5.99 39.98 99.83 99.95 MEAN 5.962 39.958 99.50 99.74 SD 0.025884358 0.025884358 0.4246 0.1796 %RSD 0.434155622 0.064778913 0.4295 0.1801 The % Drug Estimation values are close to 100%, indicating excellent accuracy in quantifying both drugs in the tablet formulation. The standard deviation is low for both IDM and AMD, suggesting minimal variability between repeated measurements. The %RSD (Relative Standard Deviation) values are well below 2% , which confirms that the methods used are highly precise and reproducible , meeting the requirements outlined in ICH Q2(R1) guidelines. 📌 Conclusion: These results validate the analytical method's suitability for routine quality control of Indapamide and Amlodipine in combined dosage forms.

31. Validation of Chromatographic Method: 1] Accuracy (Recovery Studies): Recovery studies were carried out by adding a known amount of pure drugs IDM and AMD to a preanalysed sample solution. These studies were carried out at 80%, 100% and 120% level. The recovery studies showed that the results were within acceptable limits, above 100.7% and below 101.8%. The results are given 2] Precision: The precision of the developed method was assessed in terms of repeatability, intraday and inter-day precision by analyzing six replicate standard samples. The % R.S.D. values of the results corresponding to the peak area and retention time were expressed for intra-day precision and on 3 days for inter-day precision. Precision study was carried out using parameter like method repeatability, intra-day and inter day precision study which showed that results were within acceptable limit i.e. % RSD below 2.0 indicating that the method is reproducible.

32.   Drug    Conc. [µg/ml]  Intra-day Amount Found  Inter-day Amount Found Amount of drug [ n = 3]  S.D   % R.S.D. Amount of drug [ n = 5]  S.D.   % R.S.D. IDM 3 2.98 1.1050 0.7344 2.89 0.1034 0.4378 6 5.98 1.5050 0.5894 5.78 0.0202 0.7135 9 8.97 1.9148 0.6953 8.75 0.5360 0.8105 AMD   20 19.97 1.5540 1.1590 19.88 0.5359 0.8104 30 29.95 0.6822 0.1419 29.46 1.5371 0.9479 40 39.68 1.6683 0.9051 39.54 1.3364 0.8431 3] Robustness of the Method: To ensure the insensitivity of the developed HPLC method to minor changes in the experimental conditions, it is important to demonstrate its robustness. None of the alterations caused a significant change in resolution between IDM and AMD, retention time and Theoretical plates. The results are shown

33. Factor Level Retention Time T.p./m Flow Rate (mL/min) IDM AMD IDM AMD 0.9 -0.1 4.23 3.50 4635.10 13301 1.0 3.63 2.48 4636.76 13305 1.1 +0.1 3.64 2.49 4632.04 13303 Mean ± S.D 3.83±0.791 2.83±0.311 4635.4±549.2 1330.0±362.22 % of Methanol in the Mobile Phase (v/v) 59 -1.0 4.20 3.43 4633.25 13302 60 3.63 2.48 4636.76 13305 61 +1.0 3.69 2.54 4632.45 13304 Mean ± S.D 3.84±0.3132 2.81±0.5320 4634±0.447 1330.43±0.073 pH of Mobile Phase 4.1 -0.1 4.22 3.44 4630.42 13302 4.2 3.63 2.48 4636.76 13305 4.3 +0.1 3.50 2.40 4630.70 13301 Mean ± S.D 3.78 ±0.384 2.77 ±0.573 4675.16±0.218 1330.2±0.448 The developed HPLC method demonstrates excellent robustness as slight modifications in flow rate, methanol percentage, and pH did not significantly alter retention times, theoretical plates, or resolution. This ensures the method's reliability for routine analysis, even under minor operational variations.

34. Drug Claim (µg/ml) Amount Found (%) ± S.D . (n = 3) Label Claim (%) ±S.D. (n=3) Analyst I Analyst II Analyst I Analyst II IDM 6 05.92 ±0.1429 05.99 ±0.1011 99.70±0.571 99.98±0.4046 AMD 40 39.99 ± 0.1457 39.98 ±0.1053 99.97±0.485 99.93±0.3511 4] Ruggedness of the Method: Ruggedness study was carried out using only one parameter i.e. different analyst. The result showed that the % RSD were below 2%. The results are shown 5]Limit of Detection & Limit of Quantitation: The limit of detection (LOD) and limit of quantitation (LOQ) for the procedure were performed. LOD was expressed by establishing the minimum level at which the analyte can be reliably detected.LOQ was considered as the lowest concentration of analytes in standard that can be reproducibly measured with acceptable accuracy and precision. Sample LOD (µg/mL) LOQ (µg/mL) IDM 0.6872 2.0826 AMD 3.3388 10.0170

35. System Suitability Parameters   Proposed Method IDM AMD Retention Time (t R ) 3.63 2.48 Area (%) 7.036 92.964 Number of Theoretical Plates 4636 13305 Tailing Factor 1.36 0.84 Resolution Factor (R) 3.46 System Suitability Test Parameters. Conclusion The method meets all system suitability criteria (USP/ICH). Key strengths : High efficiency (theoretical plates). Sharp, symmetrical peaks (low tailing). Baseline resolution (R >> 2.0). Ready for validation (precision, accuracy, etc.).

36. Discussion Method Development & Validation UV Method: Methanol proved to be an appropriate solvent, yielding clear maxima at 243 nm for Indapamide (IDM) and 295 nm for Amlodipine (AMD). The simultaneous equation approach delivered excellent linearity (R² ≥ 0.998), strong accuracy (99.6–99.9% recovery), and precision (%RSD < 2%), complying well with ICH guidelines. The method showed sensitivity adequate for routine tablet assays, with LOD/LOQ values (~0.33/1 µg/mL for IDM and ~3.5/10 µg/mL for AMD). RP HPLC: Using a C₁₈ Purosnosphere column and methanol:acetonitrile (60:40, pH 4.0), the optimized HPLC method achieved sharp chromatographic peaks and strong analytical metrics: linearity (R² = 0.999/0.998), precision (%RSD < 1%), and recovery close to 99.9%. The LOD/LOQ values (IDM: ~0.69/2.08 µg/mL; AMD: ~3.34/10 µg/mL) indicate sensitivity suitable for high-quality quantification. Context with Previous Research

37. Conclusion Both UV-spectrophotometric and RP-HPLC methods were successfully developed and validated as per ICH guidelines. The UV method is simple, cost-effective, and suitable for routine analysis. The HPLC method offers higher precision, sensitivity, and specificity, making it ideal for quality control in pharmaceutical formulations. Both methods demonstrated excellent accuracy, precision, linearity, and robustness, ensuring reliable quantification of IDM and AMD in combined dosage forms. Thus, the developed methods can be effectively applied for quality control and stability studies of Indapamide and Amlodipine in pharmaceutical preparations. Final Remarks: UV Method: Best for routine analysis where cost and simplicity are priorities. HPLC Method: Preferred for high-throughput analysis with superior accuracy and sensitivity. Both methods comply with regulatory standards, ensuring reproducibility and reliability. This study provides two validated analytical approaches, catering to different laboratory needs while ensuring precise quantification of the drugs in combination therapy.

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