Development and Validation of Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Dapagliflozin and Eplerenone in Bulk and Synthetic Mixture

Development and Validation of Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Dapagliflozin and Eplerenone in Bulk and Synthetic Mixture | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and Validation of Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Dapagliflozin and Eplerenone in Bulk and Synthetic Mixture Prexa Tandel, HIRALBEN MEHTA This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8677881/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Apr, 2026 Read the published version in BMC Chemistry → Version 1 posted 11 You are reading this latest preprint version Abstract A stability-indicating analytical method utilizing reverse-phase high-performance liquid chromatography was developed and validated for the simultaneous quantification of dapagliflozin and eplerenone in bulk drug materials and a laboratory-prepared synthetic mixture. Chromatographic separation was achieved on a Shim-pack C18 column (250 × 4.6 mm, 5 µm) using an isocratic elution of methanol and water (80:20, v/v) at a constant flow rate of 1.0 mL/min. Detection was carried out with a photodiode array detector set at 265 nm. The method exhibited a linear response across concentration ranges of 1–5 µg/mL for dapagliflozin and 2.5–12.5 µg/mL for eplerenone, with correlation coefficients exceeding 0.999. Validation studies, conducted in accordance with the International Council for Harmonization (ICH) Q2(R2) guidelines, confirmed that the method was precise, accurate, robust, specific, and sensitive. The stability-indicating capability was verified through forced degradation experiments under acidic, alkaline, oxidative, thermal, and photolytic stress conditions, where degradation products were adequately resolved from the parent compounds using the developed method. The validated method was successfully applied to the analysis of a synthetic mixture, yielding assay results of 99.56% for dapagliflozin and 99.48% for eplerenone. Therefore, the proposed method is suitable for routine quality control analysis and stability studies. stability-indicating RP-HPLC dapagliflozin eplerenone synthetic mixture Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Dapagliflozin, a potent sodium-glucose co-transporter-2 (SGLT2) inhibitor, has emerged as a cornerstone in the management of type 2 diabetes mellitus due to its glucose-lowering effects and clinically significant cardiovascular and renoprotective benefits. Its therapeutic scope has expanded to include chronic kidney disease and heart failure, making it a valuable asset in cardio-renal management [ 1 ]. Eplerenone, a selective mineralocorticoid receptor antagonist, is widely used for treating hypertension and improving outcomes in heart failure patient’s post-myocardial infarction [ 2 ]. The complementary mechanisms of action of dapagliflozin and eplerenone have sparked interest in their combined use for managing cardio-renal disorders [ 1 , 2 ]. From an analytical standpoint, dapagliflozin and eplerenone exhibit distinct physicochemical properties, with dapagliflozin showing moderate lipophilicity (log P ≈ 2.7, pKa ≈ 12.6) and eplerenone being more polar (log P ≈ 1.3, pKa ≈ 16.44) [ 3 , 4 ]. These differences pose challenges for their simultaneous separation and quantification using reverse-phase chromatography, necessitating meticulous method optimization. Current literature extensively covers dapagliflozin in various combination therapies [ 5 – 13 ], yet eplerenone is primarily documented through single-drug analysis [ 14 – 17 ]. Due to the absence of a validated technique for their combined measurement in synthetic blends, this work presents a streamlined, ICH-compliant RP-HPLC protocol designed for the dual quantification of these compounds MATERIALS AND METHODS Selection of Detection Wavelength To determine the optimal monitoring wavelength, individual solutions of dapagliflozin (1 µg/mL) and eplerenone (2.5 µg/mL) in methanol. A wavelength of 265 nm was selected for simultaneous detection, after scanning in UV range as it provided the most robust sensitivity and absorbance for both analytes at their clinical dose ratio (10:25). Chemicals and Reagents Alembic Pharmaceutical Research Centre (Vadodara, India) provided a gift sample of dapagliflozin, while Triveni Chemicals Pvt. Ltd. (Vapi, India) provided eplerenone. Merck Pvt. Ltd. (Mumbai, India) provided the HPLC-grade methanol. Every other chemical utilised was of the grade of an analytical reagent. Instrumentation and Chromatographic Conditions The analysis has been performed using a Shimadzu LC-2050C HPLC-PDA detector. Shim-pack C18 column, (250 × 4.6 mm, 5 µm) has been use as a stationary phase. Methanol and Water (80:20, v/v) has been use as a mobile phase with flow rate of 1.0 mL/min. Analysis has been perform at ambient temperature, wavelength at 265nm and run time 10 minutes. Preparation of Standard Stock Solutions Primary stock solutions of dapagliflozin and eplerenone were prepared by using mobile phase to get concentration of (100 µg/ml) and (250 µg/ml). further dilution was prepared to get concentration 10 µg/ml and 25 µg/ml. Subsequent dilutions were performed to reach the final analytical concentrations of 1 µg/mL and 2.5 µg/mL Preparation of Standard Mixture Standard mixture was prepared by using mobile phase to get final concentration of dapagliflozin and eplerenone 1µg/mL and 2.5µg/mL respectively. Preparation of Synthetic Mixture A laboratory-prepared synthetic mixture was formulated to simulate a combined tablet dosage form. The mixture contained dapagliflozin and eplerenone along with commonly used pharmaceutical excipients, including microcrystalline cellulose, starch, croscarmellose sodium, hydroxypropyl cellulose, talc, and magnesium stearate. Method Validation [ 18 , 19 , 20 ] To confirm its suitability for consistent pharmaceutical testing, the newly created RP-HPLC technique for co-detecting eplerenone and dapagliflozin was subjected to comprehensive validation. Adhering to the ICH Q2(R2) [ 18 ] framework, the study assessed vital performance metrics, specifically robustness, sensitivity, accuracy, precision, Working range, specificity, and system suitability. System suitability Prior to sample quantification, system suitability was verified to confirm consistent chromatographic integrity. This was achieved through six replicate injections of a standard mixture containing 1 µg/mL dapagliflozin and 2.5 µg/mL eplerenone. Key metrics, including theoretical plate count, tailing factor, resolution, and retention time, were assessed [ 19 ]. All results remained within established acceptance limits, validating the system’s performance for the intended study. Specificity Method specificity [ 18 ] was evaluated to confirm the absence of interference from formulation excipients. Placebo solutions containing commonly used excipients were analyzed under the optimized chromatographic conditions. In addition, placebo samples spiked with dapagliflozin and eplerenone were examined. The lack of excipient-related responses at the respective retention times confirms that the method enables selective analysis of both drugs. Working Range The analytical working range was established by evaluating standard solutions across concentration intervals of 1–5 µg/mL for dapagliflozin and 2.5–12.5 µg/mL for eplerenone. Linear regression analysis was performed by plotting the peak response against concentration [ 18 ]. The high correlation coefficients satisfy the ICH requirements for a robust linear relationship within the specified ranges. Precision Method precision was assessed by examining repeatability alongside intra-day and inter-day precision studies [ 18 , 20 ]. Repeatability was verified through six replicate injections of the standard mixture at the target concentration, while intermediate precision was determined by analyzing three concentration levels both on a single day and over three consecutive days. Across all evaluations, the percentage relative standard deviation (%RSD) [ 18 ] for peak areas remained consistently below 2%, confirming the method's high reproducibility and suitability for routine analytical use. Accuracy Accuracy was verified using a standard addition technique at three levels: 80%, 100%, and 120% [ 18 ] of the nominal concentration. Known amounts of both drugs were added to pre-analyzed samples to calculate recovery. The recovery percentages were within the acceptable regulatory limits, demonstrating that the method is accurate and free from matrix-induced interference [ 18 , 20 ]. Sensitivity Method sensitivity was defined by calculating the limits of detection (LOD) and quantification (LOQ) [ 18 ]. LOD = 3.3σ/S and LOQ = 10σ/S. [ 18 , 20 ] Robustness The robustness of the method was tested by introducing minor, intentional modifications to the chromatographic parameters. Variations included in flow rate, mobile phase composition, detection wavelength, and column temperature [ 18 ]. The stability of the chromatographic responses and suitability parameters under these conditions indicated the method's resilience to operational variability. Forced Degradation Studies [ 21 , 22 ] To evaluate the stability of the drugs stress studies were performed on a synthetic mixture in compliance with ICH Q1A (R2) guidelines [ 21 ] to confirm the separation of dapagliflozin and eplerenone from potential degradation products. Stress testing involved exposing the mixture to acidic and alkaline conditions using 0.1 N HCl and 0.1 N NaoH at 40°C for 60 minutes, followed by neutralization and filtration through a 0.22 µm membrane filter.[ 20 ] Oxidative stress was induced by treating the mixture with 3% hydrogen peroxide for 3 hours at room temperature [ 22 ], while thermal degradation was assessed by subjecting the sample to dry heat at 80°C for 6 hours in a hot air oven [ 22 ]. For photolytic degradation, the samples were exposed to sun light for 2 intervals of 12 hours to get 24hr [ 20 ] as per regulatory standards. In all stress conditions, the degraded samples were neutralized or diluted with the mobile phase and filtered. Sample Preparation for HPLC Analysis For quantitative analysis, a 160 mg portion of the laboratory-formulated synthetic mixture equivalent to 10 mg of dapagliflozin and 25 mg of eplerenone was accurately weighed and transferred to a 100 mL volumetric flask. The analytes were extracted into a methanol-water mixture (80:20, v/v) through a 20-minute sonication period to ensure exhaustive dissolution. The resulting extract was passed through a 0.45 µm membrane filter to remove insoluble excipients. Aliquots of this filtrate were systematically diluted with the mobile phase to achieve final target concentrations of 1 µg/mL for dapagliflozin and 2.5 µg/mL for eplerenone before being subjected to HPLC analysis under optimized conditions. RESULTS AND DISCUSSION The development of this stability-indicating RP-HPLC protocol was primarily guided by the divergent physicochemical properties of the two drugs, specifically their variations in polarity and chromatographic retention. While dapagliflozin demonstrates moderate lipophilicity, eplerenone possesses a more polar character, which required precise optimization of the mobile phase to ensure effective resolution and peak symmetry. UV-Vis spectral analysis across the 200–400 nm range revealed that both compounds exhibit sufficient absorbance at 265 nm, leading to its selection as the optimal wavelength for simultaneous monitoring. Initial experiments using various methanol-to-water ratios frequently resulted in either excessive retention times or poor peak resolution. However, an mobile phase of 80:20 (v/v) methanol and water prepared without additional pH buffers provided superior chromatographic performance, yielding highly symmetrical peaks within a brief analytical runtime. Using a Shim-pack C18 column at a flow rate of 1.0 mL/min and an injection volume of 20 µL, eplerenone eluted at 3.269 min, followed by dapagliflozin at 4.844 min. This setup achieved high quality resolution between the analytes (Fig. 1 ), as summarized in the final chromatographic parameters in Table 1 . Table 1 Chromatographic conditions evaluated during RP-HPLC method optimization Parameter Experimental conditions Stationary phase Shim-pack C18 column (250 × 4.6 mm, 5 µm) Eluent composition Methanol–water mixture (80:20, v/v) Elution mode Isocratic Flow rate 1.0 mL min⁻¹ Sample volume 20 µL Detection wavelength 265 nm Temperature Maintained at 25°C Total analysis time 10 min Retention time Eplerenone: 3.269 min; Dapagliflozin: 4.844 min Method validation To ensure the reliability of the chromatographic system, system suitability testing was performed. The results for dapagliflozin and eplerenone demonstrated acceptable chromatographic behavior, characterized by tailing factors 2000, and a resolution > 1.5. Methodological robustness was further confirmed by the minimal %RSD values recorded for retention times and peak areas, as detailed in Table 2 . Table 2 System performance evaluation parameters for dapagliflozin and eplerenone Drug Parameter Mean ± SD (n = 6) %RSD Dapagliflozin Peak area 4762 ± 17.52 0.37 Retention time (min) 4.847 ± 0.016 0.34 Theoretical plates 3767.17 ± 28.24 0.75 Tailing factor 1.300 ± 0.011 0.81 Resolution 5.7906 ± 0.00654 0.11 Tailing factor 1.300 ± 0.011 0.81 Eplerenone Peak area 31861 ± 158.30 0.50 Retention time (min) 3.266 ± 0.008 0.24 Theoretical plates 3089.00 ± 10.07 0.33 Resolution 4.357 ± 0.00447 0.10 Tailing Factor 1.368 ± 0.004 0.27 To establish the analytical working range, standard solutions were evaluated across concentration intervals of 1–5 µg/mL for dapagliflozin and 2.5–12.5 µg/mL for eplerenone. The calibration curves demonstrated excellent linear relationships between peak area and concentration, with regression equations of y = 5177.8x for dapagliflozin and y = 12042x for eplerenone. High correlation coefficients (R² =0.9995 for dapagliflozin and 0.9999 for eplerenone) confirmed the linearity of the method within the studied ranges. Representative calibration curves are illustrated in (Fig. 2 ). Method sensitivity was established by calculating the limit of detection (LOD) and the limit of quantification (LOQ). For dapagliflozin, the LOD and LOQ were 0.018 µg/mL and 0.053 µg/mL, respectively. Eplerenone yielded an LOD of 0.031 µg/mL and an LOQ of 0.0093 µg/mL. These results suggest the assay possesses the necessary sensitivity for trace-level quantification of both compounds. Additionally, method precision was validated through repeatability and intermediate precision (intra-day and inter-day) studies. Across all concentration levels, %RSD values remained under 2%, demonstrating the high degree of reliability and consistency of the developed method (Table 3 ). Table 3 Precision assessment of dapagliflozin and eplerenone at different concentration levels Drug Precision Parameter Concentration (µg/mL) Mean Peak Area (n = 3–6) SD %RSD Dapagliflozin Repeatability 1.0 4777 40.19 0.84 Intraday Precision 1.0 4762.33 19.01 0.40 2.0 9683.33 35.79 0.37 3.0 15863.33 73.01 0.46 Interday Precision 1.0 4788.67 25.66 0.54 2.0 9744.00 34.64 0.36 3.0 15835.33 73.05 0.46 Eplerenone Repeatability 2.5 31724 123.53 0.39 Intraday Precision 2.5 31766.00 71.57 0.23 5.0 59869.00 150.07 0.25 7.5 89368.33 405.72 0.45 Interday Precision 2.5 31904.67 71.57 0.22 5.0 59379.66 575.28 0.97 7.5 89349.00 581.00 0.65 Accuracy was verified by spiking at 80%, 100%, and 120% levels using the standard addition method. Mean recovery values for dapagliflozin and eplerenone were within acceptable limits with low %RSD values, confirming the accuracy of the method and the absence of interference from excipients present in the synthetic mixture (Table 4 ). Table 4 Recovery study results obtained by standard addition method Drug Recovery level (%) Nominal concentration (µg/mL) Added concentration (µg/mL) Measured concentration (µg/mL) Recovery (%) ± SD (n = 3) RSD (%) Dapagliflozin 80 1.0 0.8 1.79 99.44 ± 0.56 0.56 100 1.0 1.0 1.98 99.00 ± 0.50 0.50 120 1.0 1.2 2.19 99.55 ± 0.46 0.46 Eplerenone 80 2.5 2.0 4.49 99.56 ± 0.23 0.23 100 2.5 2.5 4.98 99.60 ± 0.20 0.20 120 2.5 3.0 5.47 99.45 ± 0.19 0.19 Developed method robustness was verified by introducing intentional, minor adjustments to the chromatographic parameters, such as the flow rate, mobile phase composition, column temperature, and detection wavelength. These deliberate variations did not lead to significant deviations in system suitability or chromatographic response. Consequently, the results indicate that the method remains stable and reliable despite small fluctuations in operational conditions (Table 5 ). Table 5 Effect of deliberate variations in chromatographic conditions on the performance of the RP-HPLC method Parameter Deliberate Change Dapagliflozin Peak Area (Mean ± SD) %RSD Rt (min) ± SD %RSD Mobile phase composition Methanol: Water (78:22, v/v) 5651 ± 20 0.35 4.902 ± 0.0090 0.18 Methanol: Water (82:18, v/v) 5367 ± 22 0.41 4.781 ± 0.0105 0.22 Flow rate 0.8 mL/min 5998 ± 28 0.47 4.809 ± 0.0096 0.20 1.2 mL/min 5033 ± 23 0.46 4.855 ± 0.0075 0.15 Detection wavelength 263 nm 5487 ± 22 0.40 4.847 ± 0.0090 0.19 267 nm 6121 ± 21 0.34 4.852 ± 0.0092 0.19 Column temperature 23°C 5899 ± 22 0.37 4.859 ± 0.0093 0.19 27°C 5892 ± 24 0.41 4.769 ± 0.0094 0.20 Parameter Deliberate Change Eplerenone Peak Area (Mean ± SD) %RSD Rt (min) ± SD %RSD Mobile phase composition Methanol: Water (78:22, v/v) 25789 ± 134 0.52 3.374 ± 0.0091 0.27 Methanol: Water (82:18, v/v) 32544 ± 144 0.44 3.191 ± 0.0092 0.29 Flow rate 0.8 mL/min 32782 ± 132 0.40 3.281 ± 0.0093 0.28 1.2 mL/min 31924 ± 134 0.42 3.268 ± 0.0097 0.30 Detection wavelength 263 nm 31042 ± 142 0.45 3.257 ± 0.0087 0.27 267 nm 38945 ± 165 0.42 3.256 ± 0.0095 0.29 Column temperature 23°C 33031 ± 141 0.43 3.290 ± 0.0091 0.28 27°C 32639 ± 139 0.43 3.271 ± 0.0102 0.31 We verified method specificity by comparing chromatograms from blank, placebo, standard, and synthetic mixture samples. The absence of co-eluting or interfering peaks at the retention times of dapagliflozin and eplerenone confirms the selectivity of the developed method (Fig. 3 ). The analytical method’s stability-indicating capacity was verified by subjecting samples to forced degradation across several stressors, such as photolytic, thermal, oxidative, alkaline, and acidic conditions. Acidic Stress : Both analytes showed the highest susceptibility to degradation. Alkaline and Photolytic Stress : Moderate degradation levels were observed. Oxidative Stress : Minimal degradation was recorded. In all instances, parent drug peaks were successfully resolved from the degradation products, ensuring that the RP-HPLC method can accurately distinguish intact analytes from their impurities (Fig. 4 , Table 6 ). Table 6 Degradation profiles of dapagliflozin and eplerenone following exposure to various stress conditions Stress environment Experimental stress applied Dapagliflozin degraded (%) Eplerenone degraded (%) Acidic hydrolysis Exposure to hydrochloric acid (0.1 N) at 40°C for 60 min 16.54 17.94 Alkaline hydrolysis Treatment with sodium hydroxide solution (0.1 N) at 40°C for 60 min 13.45 12.97 Oxidative stress Oxidation using hydrogen peroxide solution (3%, 3 h, ambient temperature) 0.73 0.61 Thermal stress Heating at 80°C for 6 h 1.51 6.34 Photolytic stress Exposure to natural sunlight for 24 h 13.74 14.84 Analysis of Synthetic Mixture The validated RP-HPLC technique was employed for the quantitative estimation of dapagliflozin and eplerenone within a laboratory-formulated synthetic mixture. By comparing peak areas of the sample solutions against established standard solutions, the drug content was determined to be 99.56% for dapagliflozin and 99.48% for eplerenone. These results align closely with the nominal concentrations, confirming the method's practical utility for routine quality control applications. CONCLUSION A stability-indicating RP-HPLC technique was successfully implemented for the simultaneous determination of both drugs in laboratory-prepared synthetic matrices and bulk forms. The optimized chromatographic conditions ensured efficient separation with high resolution, symmetrical peaks, and rapid analysis times. Validation in accordance with ICH Q2(R2) guidelines confirmed that the assay is linear, accurate, precise, and robust. Furthermore, forced degradation studies across acidic, alkaline, oxidative, thermal, and photolytic conditions verified the method's ability to resolve analytes from their degradation products. Given its operational simplicity and reliability, this method is well-suited for the routine stability assessment and quality monitoring of these pharmaceutical agents. FUNDING DECLARATION Open access funding provided by Parul University. None received. Abbreviations ICH International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use LOD Limit of detection LOQ Limit of quantification DP Degradation product SD Standard deviation RSD Relative standard deviation Declarations Author Contribution Prexa Tandel has done practical work and prepared initial draft. Dr. Hiralben Mehta has supervised the work and done final review of this research manuscript. Acknowledgement The authors are thankful to Alembic Pharmaceutical ltd, Vadodara, Gujarat, India for providing gratis samples of drugs, and Parul Institute of Pharmacy and Research for providing facilities to perform research. References Abdel-Mooty M. Albuminuria-Lowering Effect of Dapagliflozin, Eplerenone, and Their Combination in Patients with Chronic Kidney Disease: A Randomized Crossover Clinical Trial. J Am Soc Nephrol. 2022;33(8):1569–80. 10.1681/asn.2022020207 . 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MEHTA","email":"data:image/png;base64,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","orcid":"","institution":"Parul University","correspondingAuthor":true,"prefix":"","firstName":"HIRALBEN","middleName":"","lastName":"MEHTA","suffix":""}],"badges":[],"createdAt":"2026-01-23 10:09:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8677881/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8677881/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13065-026-01792-3","type":"published","date":"2026-04-20T15:59:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101664877,"identity":"7af38930-857a-46cc-b67e-7bf18d804017","added_by":"auto","created_at":"2026-02-02 11:35:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":77939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatographic separation of dapagliflozin and eplerenone\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8677881/v1/b1f838f92e4354dc6a518559.png"},{"id":101664874,"identity":"d8747a87-8442-4729-b34a-0bd98c76eb05","added_by":"auto","created_at":"2026-02-02 11:35:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42669,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative calibration curves of dapagliflozin and eplerenone demonstrating method linearity.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8677881/v1/ec76077537bdd2487c17777b.png"},{"id":101664875,"identity":"94bd6ac1-7d8c-49e8-a914-ac8f21c6fbf5","added_by":"auto","created_at":"2026-02-02 11:35:22","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":232862,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative chromatograms used to evaluate method specificity.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8677881/v1/fb123fa9ec7aace39f456c80.jpeg"},{"id":101753008,"identity":"25a5e5ca-e598-499f-85c3-38e3a13b78b3","added_by":"auto","created_at":"2026-02-03 10:38:49","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":640524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative chromatograms obtained following forced degradation of dapagliflozin and eplerenone under various stress conditions\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8677881/v1/5de9dd851d63b3a04c6d70e2.jpeg"},{"id":107928640,"identity":"8fbf0f9a-1bd6-41aa-9caf-b1343853e766","added_by":"auto","created_at":"2026-04-27 16:11:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1361924,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8677881/v1/0f04e4c5-e64c-4f86-83aa-71a73efcad6e.pdf"},{"id":101664878,"identity":"5e2e602e-5a99-42dd-9456-882a3c15acae","added_by":"auto","created_at":"2026-02-02 11:35:22","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":381349,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8677881/v1/001fe007eac5605114e3be6b.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Validation of Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Dapagliflozin and Eplerenone in Bulk and Synthetic Mixture","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDapagliflozin, a potent sodium-glucose co-transporter-2 (SGLT2) inhibitor, has emerged as a cornerstone in the management of type 2 diabetes mellitus due to its glucose-lowering effects and clinically significant cardiovascular and renoprotective benefits. Its therapeutic scope has expanded to include chronic kidney disease and heart failure, making it a valuable asset in cardio-renal management [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Eplerenone, a selective mineralocorticoid receptor antagonist, is widely used for treating hypertension and improving outcomes in heart failure patient\u0026rsquo;s post-myocardial infarction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The complementary mechanisms of action of dapagliflozin and eplerenone have sparked interest in their combined use for managing cardio-renal disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom an analytical standpoint, dapagliflozin and eplerenone exhibit distinct physicochemical properties, with dapagliflozin showing moderate lipophilicity (log P\u0026thinsp;\u0026asymp;\u0026thinsp;2.7, pKa\u0026thinsp;\u0026asymp;\u0026thinsp;12.6) and eplerenone being more polar (log P\u0026thinsp;\u0026asymp;\u0026thinsp;1.3, pKa\u0026thinsp;\u0026asymp;\u0026thinsp;16.44) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These differences pose challenges for their simultaneous separation and quantification using reverse-phase chromatography, necessitating meticulous method optimization.\u003c/p\u003e \u003cp\u003eCurrent literature extensively covers dapagliflozin in various combination therapies [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], yet eplerenone is primarily documented through single-drug analysis [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Due to the absence of a validated technique for their combined measurement in synthetic blends, this work presents a streamlined, ICH-compliant RP-HPLC protocol designed for the dual quantification of these compounds\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSelection of Detection Wavelength\u003c/h2\u003e \u003cp\u003eTo determine the optimal monitoring wavelength, individual solutions of dapagliflozin (1 \u0026micro;g/mL) and eplerenone (2.5 \u0026micro;g/mL) in methanol. A wavelength of 265 nm was selected for simultaneous detection, after scanning in UV range as it provided the most robust sensitivity and absorbance for both analytes at their clinical dose ratio (10:25).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemicals and Reagents\u003c/h3\u003e\n\u003cp\u003eAlembic Pharmaceutical Research Centre (Vadodara, India) provided a gift sample of dapagliflozin, while Triveni Chemicals Pvt. Ltd. (Vapi, India) provided eplerenone. Merck Pvt. Ltd. (Mumbai, India) provided the HPLC-grade methanol. Every other chemical utilised was of the grade of an analytical reagent.\u003c/p\u003e\n\u003ch3\u003eInstrumentation and Chromatographic Conditions\u003c/h3\u003e\n\u003cp\u003eThe analysis has been performed using a Shimadzu LC-2050C HPLC-PDA detector. Shim-pack C18 column, (250 \u0026times; 4.6 mm, 5 \u0026micro;m) has been use as a stationary phase. Methanol and Water (80:20, v/v) has been use as a mobile phase with flow rate of 1.0 mL/min. Analysis has been perform at ambient temperature, wavelength at 265nm and run time 10 minutes.\u003c/p\u003e\n\u003ch3\u003ePreparation of Standard Stock Solutions\u003c/h3\u003e\n\u003cp\u003ePrimary stock solutions of dapagliflozin and eplerenone were prepared by using mobile phase to get concentration of (100 \u0026micro;g/ml) and (250 \u0026micro;g/ml). further dilution was prepared to get concentration 10 \u0026micro;g/ml and 25 \u0026micro;g/ml. Subsequent dilutions were performed to reach the final analytical concentrations of 1 \u0026micro;g/mL and 2.5 \u0026micro;g/mL\u003c/p\u003e\n\u003ch3\u003ePreparation of Standard Mixture\u003c/h3\u003e\n\u003cp\u003eStandard mixture was prepared by using mobile phase to get final concentration of dapagliflozin and eplerenone 1\u0026micro;g/mL and 2.5\u0026micro;g/mL respectively.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Synthetic Mixture\u003c/h2\u003e \u003cp\u003eA laboratory-prepared synthetic mixture was formulated to simulate a combined tablet dosage form. The mixture contained dapagliflozin and eplerenone along with commonly used pharmaceutical excipients, including microcrystalline cellulose, starch, croscarmellose sodium, hydroxypropyl cellulose, talc, and magnesium stearate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethod Validation\u003c/b\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eTo confirm its suitability for consistent pharmaceutical testing, the newly created RP-HPLC technique for co-detecting eplerenone and dapagliflozin was subjected to comprehensive validation. Adhering to the ICH Q2(R2) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] framework, the study assessed vital performance metrics, specifically robustness, sensitivity, accuracy, precision, Working range, specificity, and system suitability.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSystem suitability\u003c/h3\u003e\n\u003cp\u003ePrior to sample quantification, system suitability was verified to confirm consistent chromatographic integrity. This was achieved through six replicate injections of a standard mixture containing 1 \u0026micro;g/mL dapagliflozin and 2.5 \u0026micro;g/mL eplerenone. Key metrics, including theoretical plate count, tailing factor, resolution, and retention time, were assessed [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. All results remained within established acceptance limits, validating the system\u0026rsquo;s performance for the intended study.\u003c/p\u003e\n\u003ch3\u003eSpecificity\u003c/h3\u003e\n\u003cp\u003eMethod specificity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] was evaluated to confirm the absence of interference from formulation excipients. Placebo solutions containing commonly used excipients were analyzed under the optimized chromatographic conditions. In addition, placebo samples spiked with dapagliflozin and eplerenone were examined. The lack of excipient-related responses at the respective retention times confirms that the method enables selective analysis of both drugs.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWorking Range\u003c/h2\u003e \u003cp\u003eThe analytical working range was established by evaluating standard solutions across concentration intervals of 1\u0026ndash;5 \u0026micro;g/mL for dapagliflozin and 2.5\u0026ndash;12.5 \u0026micro;g/mL for eplerenone. Linear regression analysis was performed by plotting the peak response against concentration [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The high correlation coefficients satisfy the ICH requirements for a robust linear relationship within the specified ranges.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePrecision\u003c/h2\u003e \u003cp\u003eMethod precision was assessed by examining repeatability alongside intra-day and inter-day precision studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Repeatability was verified through six replicate injections of the standard mixture at the target concentration, while intermediate precision was determined by analyzing three concentration levels both on a single day and over three consecutive days. Across all evaluations, the percentage relative standard deviation (%RSD) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] for peak areas remained consistently below 2%, confirming the method's high reproducibility and suitability for routine analytical use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAccuracy\u003c/h2\u003e \u003cp\u003eAccuracy was verified using a standard addition technique at three levels: 80%, 100%, and 120% [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] of the nominal concentration. Known amounts of both drugs were added to pre-analyzed samples to calculate recovery. The recovery percentages were within the acceptable regulatory limits, demonstrating that the method is accurate and free from matrix-induced interference [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSensitivity\u003c/h2\u003e \u003cp\u003eMethod sensitivity was defined by calculating the limits of detection (LOD) and quantification (LOQ) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLOD\u0026thinsp;=\u0026thinsp;3.3σ/S and\u003c/p\u003e \u003cp\u003eLOQ\u0026thinsp;=\u0026thinsp;10σ/S. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRobustness\u003c/h2\u003e \u003cp\u003eThe robustness of the method was tested by introducing minor, intentional modifications to the chromatographic parameters. Variations included in flow rate, mobile phase composition, detection wavelength, and column temperature [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The stability of the chromatographic responses and suitability parameters under these conditions indicated the method's resilience to operational variability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eForced Degradation Studies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/h2\u003e \u003cp\u003eTo evaluate the stability of the drugs stress studies were performed on a synthetic mixture in compliance with ICH Q1A (R2) guidelines [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] to confirm the separation of dapagliflozin and eplerenone from potential degradation products. Stress testing involved exposing the mixture to acidic and alkaline conditions using 0.1 N HCl and 0.1 N NaoH at 40\u0026deg;C for 60 minutes, followed by neutralization and filtration through a 0.22 \u0026micro;m membrane filter.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Oxidative stress was induced by treating the mixture with 3% hydrogen peroxide for 3 hours at room temperature [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], while thermal degradation was assessed by subjecting the sample to dry heat at 80\u0026deg;C for 6 hours in a hot air oven [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For photolytic degradation, the samples were exposed to sun light for 2 intervals of 12 hours to get 24hr [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] as per regulatory standards. In all stress conditions, the degraded samples were neutralized or diluted with the mobile phase and filtered.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSample Preparation for HPLC Analysis\u003c/h2\u003e \u003cp\u003eFor quantitative analysis, a 160 mg portion of the laboratory-formulated synthetic mixture equivalent to 10 mg of dapagliflozin and 25 mg of eplerenone was accurately weighed and transferred to a 100 mL volumetric flask. The analytes were extracted into a methanol-water mixture (80:20, v/v) through a 20-minute sonication period to ensure exhaustive dissolution. The resulting extract was passed through a 0.45 \u0026micro;m membrane filter to remove insoluble excipients. Aliquots of this filtrate were systematically diluted with the mobile phase to achieve final target concentrations of 1 \u0026micro;g/mL for dapagliflozin and 2.5 \u0026micro;g/mL for eplerenone before being subjected to HPLC analysis under optimized conditions.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eThe development of this stability-indicating RP-HPLC protocol was primarily guided by the divergent physicochemical properties of the two drugs, specifically their variations in polarity and chromatographic retention. While dapagliflozin demonstrates moderate lipophilicity, eplerenone possesses a more polar character, which required precise optimization of the mobile phase to ensure effective resolution and peak symmetry.\u003c/p\u003e \u003cp\u003eUV-Vis spectral analysis across the 200\u0026ndash;400 nm range revealed that both compounds exhibit sufficient absorbance at 265 nm, leading to its selection as the optimal wavelength for simultaneous monitoring. Initial experiments using various methanol-to-water ratios frequently resulted in either excessive retention times or poor peak resolution. However, an mobile phase of 80:20 (v/v) methanol and water prepared without additional pH buffers provided superior chromatographic performance, yielding highly symmetrical peaks within a brief analytical runtime.\u003c/p\u003e \u003cp\u003eUsing a Shim-pack C18 column at a flow rate of 1.0 mL/min and an injection volume of 20 \u0026micro;L, eplerenone eluted at 3.269 min, followed by dapagliflozin at 4.844 min. This setup achieved high quality resolution between the analytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), as summarized in the final chromatographic parameters in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChromatographic conditions evaluated during RP-HPLC method optimization\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExperimental conditions\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStationary phase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShim-pack C18 column (250 \u0026times; 4.6 mm, 5 \u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEluent composition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethanol\u0026ndash;water mixture (80:20, v/v)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElution mode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsocratic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlow rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0 mL min⁻\u0026sup1;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample volume\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetection wavelength\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e265 nm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaintained at 25\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal analysis time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRetention time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEplerenone: 3.269 min; Dapagliflozin: 4.844 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMethod validation\u003c/h2\u003e \u003cp\u003eTo ensure the reliability of the chromatographic system, system suitability testing was performed. The results for dapagliflozin and eplerenone demonstrated acceptable chromatographic behavior, characterized by tailing factors\u0026thinsp;\u0026lt;\u0026thinsp;2, theoretical plates\u0026thinsp;\u0026gt;\u0026thinsp;2000, and a resolution\u0026thinsp;\u0026gt;\u0026thinsp;1.5. Methodological robustness was further confirmed by the minimal %RSD values recorded for retention times and peak areas, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSystem performance evaluation parameters for dapagliflozin and eplerenone\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDapagliflozin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeak area\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4762\u0026thinsp;\u0026plusmn;\u0026thinsp;17.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRetention time (min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.847\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTheoretical plates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3767.17\u0026thinsp;\u0026plusmn;\u0026thinsp;28.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTailing factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.300\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResolution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.7906\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00654\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTailing factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.300\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEplerenone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeak area\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e31861\u0026thinsp;\u0026plusmn;\u0026thinsp;158.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRetention time (min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.266\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTheoretical plates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3089.00\u0026thinsp;\u0026plusmn;\u0026thinsp;10.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResolution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.357\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00447\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTailing Factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.368\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo establish the analytical working range, standard solutions were evaluated across concentration intervals of 1\u0026ndash;5 \u0026micro;g/mL for dapagliflozin and 2.5\u0026ndash;12.5 \u0026micro;g/mL for eplerenone. The calibration curves demonstrated excellent linear relationships between peak area and concentration, with regression equations of y\u0026thinsp;=\u0026thinsp;5177.8x for dapagliflozin and y\u0026thinsp;=\u0026thinsp;12042x for eplerenone. High correlation coefficients (R\u0026sup2; =0.9995 for dapagliflozin and 0.9999 for eplerenone) confirmed the linearity of the method within the studied ranges. Representative calibration curves are illustrated in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMethod sensitivity was established by calculating the limit of detection (LOD) and the limit of quantification (LOQ). For dapagliflozin, the LOD and LOQ were 0.018 \u0026micro;g/mL and 0.053 \u0026micro;g/mL, respectively. Eplerenone yielded an LOD of 0.031 \u0026micro;g/mL and an LOQ of 0.0093 \u0026micro;g/mL. These results suggest the assay possesses the necessary sensitivity for trace-level quantification of both compounds. Additionally, method precision was validated through repeatability and intermediate precision (intra-day and inter-day) studies. Across all concentration levels, %RSD values remained under 2%, demonstrating the high degree of reliability and consistency of the developed method (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrecision assessment of dapagliflozin and eplerenone at different concentration levels\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrecision Parameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentration (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean Peak Area (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;6)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDapagliflozin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRepeatability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e40.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntraday Precision\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4762.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9683.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15863.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e73.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInterday Precision\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4788.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9744.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15835.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e73.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEplerenone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRepeatability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e123.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntraday Precision\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31766.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e71.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59869.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e150.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89368.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e405.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInterday Precision\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31904.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e71.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59379.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e575.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89349.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e581.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAccuracy was verified by spiking at 80%, 100%, and 120% levels using the standard addition method. Mean recovery values for dapagliflozin and eplerenone were within acceptable limits with low %RSD values, confirming the accuracy of the method and the absence of interference from excipients present in the synthetic mixture (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRecovery study results obtained by standard addition method\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRecovery level (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNominal concentration (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdded concentration (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMeasured concentration (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRecovery (%)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDapagliflozin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e99.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e99.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e99.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEplerenone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e99.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e99.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e99.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDeveloped method robustness was verified by introducing intentional, minor adjustments to the chromatographic parameters, such as the flow rate, mobile phase composition, column temperature, and detection wavelength. These deliberate variations did not lead to significant deviations in system suitability or chromatographic response. Consequently, the results indicate that the method remains stable and reliable despite small fluctuations in operational conditions (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of deliberate variations in chromatographic conditions on the performance of the RP-HPLC method\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDeliberate Change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDapagliflozin\u003c/p\u003e \u003cp\u003ePeak Area (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRt (min)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMobile phase composition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethanol: Water (78:22, v/v)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5651\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.902\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethanol: Water (82:18, v/v)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5367\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.781\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlow rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8 mL/min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5998\u0026thinsp;\u0026plusmn;\u0026thinsp;28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.809\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2 mL/min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5033\u0026thinsp;\u0026plusmn;\u0026thinsp;23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.855\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetection wavelength\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e263 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5487\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.847\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e267 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6121\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.852\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColumn temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5899\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.859\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5892\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.769\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDeliberate Change\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eEplerenone Peak Area (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e%RSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eRt (min)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e%RSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMobile phase composition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethanol: Water (78:22, v/v)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25789\u0026thinsp;\u0026plusmn;\u0026thinsp;134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.374\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethanol: Water (82:18, v/v)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32544\u0026thinsp;\u0026plusmn;\u0026thinsp;144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.191\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlow rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8 mL/min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32782\u0026thinsp;\u0026plusmn;\u0026thinsp;132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.281\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2 mL/min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31924\u0026thinsp;\u0026plusmn;\u0026thinsp;134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.268\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0097\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetection wavelength\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e263 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31042\u0026thinsp;\u0026plusmn;\u0026thinsp;142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.257\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e267 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38945\u0026thinsp;\u0026plusmn;\u0026thinsp;165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.256\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColumn temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33031\u0026thinsp;\u0026plusmn;\u0026thinsp;141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.290\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32639\u0026thinsp;\u0026plusmn;\u0026thinsp;139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.271\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe verified method specificity by comparing chromatograms from blank, placebo, standard, and synthetic mixture samples. The absence of co-eluting or interfering peaks at the retention times of dapagliflozin and eplerenone confirms the selectivity of the developed method (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe analytical method\u0026rsquo;s stability-indicating capacity was verified by subjecting samples to forced degradation across several stressors, such as photolytic, thermal, oxidative, alkaline, and acidic conditions.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eAcidic Stress\u003c/b\u003e: Both analytes showed the highest susceptibility to degradation.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eAlkaline and Photolytic Stress\u003c/b\u003e: Moderate degradation levels were observed.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eOxidative Stress\u003c/b\u003e: Minimal degradation was recorded.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eIn all instances, parent drug peaks were successfully resolved from the degradation products, ensuring that the RP-HPLC method can accurately distinguish intact analytes from their impurities (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDegradation profiles of dapagliflozin and eplerenone following exposure to various stress conditions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStress environment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eExperimental stress applied\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDapagliflozin degraded (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEplerenone degraded (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcidic hydrolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExposure to hydrochloric acid (0.1 N) at 40\u0026deg;C for 60 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e16.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlkaline hydrolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment with sodium hydroxide solution (0.1 N) at 40\u0026deg;C for 60 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e13.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxidative stress\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxidation using hydrogen peroxide solution (3%, 3 h, ambient temperature)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThermal stress\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeating at 80\u0026deg;C for 6 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhotolytic stress\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExposure to natural sunlight for 24 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e13.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Synthetic Mixture\u003c/h2\u003e \u003cp\u003eThe validated RP-HPLC technique was employed for the quantitative estimation of dapagliflozin and eplerenone within a laboratory-formulated synthetic mixture. By comparing peak areas of the sample solutions against established standard solutions, the drug content was determined to be 99.56% for dapagliflozin and 99.48% for eplerenone. These results align closely with the nominal concentrations, confirming the method's practical utility for routine quality control applications.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eA stability-indicating RP-HPLC technique was successfully implemented for the simultaneous determination of both drugs in laboratory-prepared synthetic matrices and bulk forms. The optimized chromatographic conditions ensured efficient separation with high resolution, symmetrical peaks, and rapid analysis times. Validation in accordance with ICH Q2(R2) guidelines confirmed that the assay is linear, accurate, precise, and robust. Furthermore, forced degradation studies across acidic, alkaline, oxidative, thermal, and photolytic conditions verified the method's ability to resolve analytes from their degradation products. Given its operational simplicity and reliability, this method is well-suited for the routine stability assessment and quality monitoring of these pharmaceutical agents.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFUNDING DECLARATION\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOpen access funding provided by Parul University. None received.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInternational Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLimit of detection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLOQ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLimit of quantification\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDegradation product\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRelative standard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePrexa Tandel has done practical work and prepared initial draft. Dr. Hiralben Mehta has supervised the work and done final review of this research manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are thankful to Alembic Pharmaceutical ltd, Vadodara, Gujarat, India for providing gratis samples of drugs, and Parul Institute of Pharmacy and Research for providing facilities to perform research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel-Mooty M. Albuminuria-Lowering Effect of Dapagliflozin, Eplerenone, and Their Combination in Patients with Chronic Kidney Disease: A Randomized Crossover Clinical Trial. 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A novel RP-HPLC approach for simultaneous determination of dapagliflozin, linagliptin, and metformin in pharmaceutical formulations. BMC Chem. 2025;19:257. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13065-025-01620-0\u003c/span\u003e\u003cspan address=\"10.1186/s13065-025-01620-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"stability-indicating RP-HPLC, dapagliflozin, eplerenone, synthetic mixture","lastPublishedDoi":"10.21203/rs.3.rs-8677881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8677881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA stability-indicating analytical method utilizing reverse-phase high-performance liquid chromatography was developed and validated for the simultaneous quantification of dapagliflozin and eplerenone in bulk drug materials and a laboratory-prepared synthetic mixture. Chromatographic separation was achieved on a Shim-pack C18 column (250 \u0026times; 4.6 mm, 5 \u0026micro;m) using an isocratic elution of methanol and water (80:20, v/v) at a constant flow rate of 1.0 mL/min. Detection was carried out with a photodiode array detector set at 265 nm. The method exhibited a linear response across concentration ranges of 1\u0026ndash;5 \u0026micro;g/mL for dapagliflozin and 2.5\u0026ndash;12.5 \u0026micro;g/mL for eplerenone, with correlation coefficients exceeding 0.999. Validation studies, conducted in accordance with the International Council for Harmonization (ICH) Q2(R2) guidelines, confirmed that the method was precise, accurate, robust, specific, and sensitive. The stability-indicating capability was verified through forced degradation experiments under acidic, alkaline, oxidative, thermal, and photolytic stress conditions, where degradation products were adequately resolved from the parent compounds using the developed method. The validated method was successfully applied to the analysis of a synthetic mixture, yielding assay results of 99.56% for dapagliflozin and 99.48% for eplerenone. Therefore, the proposed method is suitable for routine quality control analysis and stability studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Development and Validation of Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Dapagliflozin and Eplerenone in Bulk and Synthetic Mixture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 11:35:13","doi":"10.21203/rs.3.rs-8677881/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-19T15:33:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-19T09:03:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-05T12:54:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330231504097042917015375667949864317247","date":"2026-01-31T19:02:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163156334267252701390072536130860660863","date":"2026-01-31T18:37:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89497782908481507639793916283354040108","date":"2026-01-30T09:34:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-29T18:35:27+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-28T14:23:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-28T12:54:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-28T12:51:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Chemistry","date":"2026-01-23T09:57:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5cdb7c95-2c96-4780-9be9-5b58fd92b887","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:09:08+00:00","versionOfRecord":{"articleIdentity":"rs-8677881","link":"https://doi.org/10.1186/s13065-026-01792-3","journal":{"identity":"bmc-chemistry","isVorOnly":false,"title":"BMC Chemistry"},"publishedOn":"2026-04-20 15:59:25","publishedOnDateReadable":"April 20th, 2026"},"versionCreatedAt":"2026-02-02 11:35:13","video":"","vorDoi":"10.1186/s13065-026-01792-3","vorDoiUrl":"https://doi.org/10.1186/s13065-026-01792-3","workflowStages":[]},"version":"v1","identity":"rs-8677881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8677881","identity":"rs-8677881","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}