1. INTRODUCTION:

Dapagliflozin Propanediol Monohydrate (DAPA) is a C-glucoside derivative that acts as a selective SGLT2 inhibitors. By preventing glucose from being reabsorbed in the renal system, it assists in Maintaining stable blood sugar concentrations in individuals diagnosed with type 2 diabetes and provides additional benefits for cardiovascular and renal outcomes. Chemically, it is designated as (2S) – Propane -1,2 - Diol (2S,3R,4R,5S,6R) – 2 - {4 - Chloro - 3- [(4 – ethoxy phenyl) Methyl] Phenyl} – 6 - (Hydroxy methyl) Oxane - 3,4,5 - Triol Hydrate, with a structural formula of C₂₄H₃₅ClO₉ and an empirical mass of 502.99 Da.

DAPA appears as an Off-white to light yellow crystalline powdered form, is non-hygroscopic, and exhibits very limited water solubility¹. It shows slight solubility in ethanol and acetonitrile, sparing solubility in methanol, and is practically insoluble in toluene. The USFDA granted clearance for DAPA in 2014 as a therapeutic use in type 2 diabetes². Bisoprolol Fumarate (BSF), on the other hand, is a β1-selective adrenergic receptor blocker used primarily to manage hypertension and chronic cardiovascular conditions. It is chemically described as (2E) – But – 2 - Enedioic Acid; Bis (1 - [(Propan – 2 - yl) Amino] – 3 - [4 - {[2 - (Propan - 2-yloxy) Ethoxy] Methyl} Phenoxy] Propan - 2-ol), with the structural formula C₄₀H₆₆N₂O₁₂ and an empirical mass of 766.97 Da³. The compound is a hygroscopic Fine crystalline powder, white to off-white in color and is officially listed in various pharmacopeias, including the USP-NF⁴, British Pharmacopoeia (BP)⁵, and European Pharmacopoeia (Ph. Eur.)⁶. The combined extended-release dosage form ensures sustained effects, enhancing compliance in chronic cardiovascular conditions. Extensive literature reviews indicated that UV-Visible Spectrophotometry^7-11, HPLC-UV^12-13, HPLC-PDA^14-20, HPTLC^21-24, RP-UPLC-PDA²⁵ have been extensively employed for the analysis of both DAPA and BSF, Individually or combined with other drugs, in both bulk substance and finished pharmaceutical products. Figure 1 illustrates the molecular structures of both drugs. This research aims to establish and perform validation of an RP-HPLC method of This research aims to establish and perform validation of an RP-HPLC method proficient of concurrently quantifying DAPA and BSF concurrently quantifying DAPA and BSF under stability-indicating conditions, as guided by the ICH Q2(R1) framework. The proposed novel combination tablet of DAPA (10 mg) and BSF (5 mg) is currently being evaluated in clinical trials III initiated by Eris Lifesciences Ltd, North Guwahati, Assam (File No. FDC/MA/23/000162). This combination is hypothesized to enhance anti-diabetic and anti-hypertensive efficacy through complementary mechanisms of action. Bilayer tablets were prepared in the laboratory to resemble the actual formulation, as the commercial formulation is not yet available. Stress degradation testing, and method validation, complied with ICH regulatory guidelines ^26-27.

Fig 1: Chemical Structure of (a) Dapagliflozin Propanediol monohydrateand (b) Bisoprolol Fumarate

2. METHODS AND MATERIALS:

2.1 Reagents and chemicals:

The DAPA pure drugs was procured from Zydus Pharma Ltd in Ahmedabad, Gujarat, India, and BSF was procured from Sun Pharmaceutical Industries Ltd in Vadodara, Gujarat, India, respectively. Chromatographic solvents including water, methanol, and acetonitrile and analytical-grade reagents, including sodium hydroxide, hydrogen peroxide, and hydrochloric acid, potassium dihydrogen phosphate was procured from Ranchem Ltd in Mumbai, Maharashtra, India.

2.2 Instrumentation:

Chromatographic evaluation was carried out on a shimadzu P-series integrated hplc system (Model LC-20 AD), equipped with a quaternary pump, solvent delivery module (LC-20 AD), online degassing unit (DGU-20AR), temperature-controlled column oven (CTO-10ASVP), PDA detector (SPD-M40), and a programmable auto-sampler (SIL-20AC). The system was operated using LAB SOLUTION software. For chromatographic separation, a Shim pack Octadecylsilane C18 stationary phase (250 × 4.6mm, 5µm) was employed, ensuring high precision and reproducibility. Buffer pH was adjusted using a pH meter (Model PHS-3C, BR Biochem Life Sciences Pvt. Ltd., India), and pharmaceutical ingredients were precisely weighed using an analytical balance (Model AUW 220D, Shimadzu Ltd., Japan).

2.3 Mobile phase preparation:

Phosphate buffer (OPA was used to bring the pH to 4.0), methanol, and acetonitrile were mixed in a 50:35:15 v/v/v ratio to serve as the mobile solvent. Filtration and degassing were performed on the mixture before use.

2.4 Method for Preparing Standard and Working Solutions:

Ten milligrams of DAPA and five milligrams of BSF were accurately quantified and placed into a 100mL calibrated glass flask. The compounds were completely solubilized in 50mL of methanol with the aid of ultra-sonication until complete dissolution was achieved. Diluent (methanol) was added to bring the total volume to the mark, resulting in stock solutions of 100µg/mL of DAPA and 50µg/mL of BSF. To obtain working concentrations of 20µg/mL for DAPA and 10µg/mL for BSF, 2mL from each stock was diluted up to 10mL.

2.5 Analysis of synthetic mixture:

A blend simulating 10mg of DAPA and 5mg of BSF was precisely weighed and placed into a Calibrated glass flask of 100mL volume. To promote solubility, the compounds were added to 50mL of distilled water and sonication for 5 minutes. After being passed through a 0.45µm filter, the solution was topped up to the final volume with distilled water. To achieve final concentrations of 40µg/mL of DAPA and 20µg/mL of BSF, 4mL of the solution was diluted to 10mL with the mobile phase. A volume of 10µL was administered into the HPLC system in accordance with the predetermined chromatographic settings.

2.6 Preparation of Degradation Samples:

A prepared solution comprising 1000 µg/mL DAPA and 500µg/mL BSF was formulated in the selected diluent.

Acid hydrolysis:

For acid stress testing, a 1.5 mL prepared solution was combined with 1.5 mL of 0.1 N HCl in a 10 mL calibrated glass flask and heated at 60 °C for one hour. Once cooled, 1.5 mL of the degraded sample was placed into a separate ten milliliters calibrated flask, after pH adjustment to neutrality with 0.1 N NaOH, the solution was diluted to the final volume with the methanol.

Base Hydrolysis:

A similar approach to the acidic condition was followed, except that 0.1 N NaOH was used instead of HCl, and the neutralization was performed using 0.1 N HCl.

Oxidative Degradation:

To induce oxidative degradation, 1.5mL sample of the prepared solution was combined with an equal volume of 3% H₂O₂ in a 10mL calibrated flask and incubation at ambient temperature for 1 hour.

Thermal Degradation:

A powder blend consisting of Ten milligrams of DAPA and five milligrams of BSF were subjected to dry heat at 60°C for 2hours in a glass Petri dish. The degraded residue was then dissolved in methanol, sonicated thoroughly, and suitably diluted for analysis.

Photolytic Degradation:

A drug powder blend was subjected to ultraviolet light for 6hours, followed by dissolution in methanol and dilution with the diluent. All stressed samples were appropriately diluted to yield final concentrations for DAPA (60µg/mL) and BSF (30µg/mL). Peak purity was evaluated using a photodiode array (PDA) detector to ensure that no co-eluting decomposition impurities interfered with the quantification of the target substances.

2.7 Validation of analytical method:

The designed analytical method for concurrent quantification of DAPA and BSF in a combined mixture was thoroughly validation conducted according to ICH Q2(R1) criteria.

2.7.1 Specificity:

Chromatograms of blank, standard, and formulation samples were analyzed to assess the RP-HPLC method’s specificity. Peak purity analysis of DAPA and BSF was performed utilizing a PDA detector. The established method's specificity was confirmed when the peaks for DAPA and BSF were well-resolved, with no interference from inactive ingredients or impurities, demonstrating the method's ability to selectively measure the active ingredients.

2.7.2 Linearity:

A working sample containing of DAPA (200µg/mL) and BSF (100µg/mL) was made to assess the method's linearity. Samples from the stock solution were diluted to prepare six different concentration series ranges, from 20 to 120µg/mL for DAPA and 10 to 60µg/mL for BSF. Each sample was introduced into the analytical system and analysed under the optimal condition parameters. A calibration plot of concentration versus peak area was created to determine the regression coefficient.

2.7.3 Precision:

Repeatability was assessed by testing a sample containing a combination of DAPA of 60 (µg/mL) and BSF of 30 (µg/mL). The detected peak responses of the solution at the same concentration were measured six times under optimal chromatographic conditions, and the %RSD was determined. For intraday precision, samples containing DAPA (20 60, 120µg/mL) and BSF (10, 30, 60µg/mL) were tested at 3 different times interval throughout the same day. For interday precision, the same concentrations of DAPA and BSF were tested across three consecutive days, and the %RSD was determined.

2.7.4 Accuracy:

Percentage recovery investigation by introducing known quantities of DAPA and BSF standard drugs to pre-tested sample mixtures to evaluate the recovery. Standard solutions of DAPA and BSF were added to pre evaluated sample consisting of DAPA (40 µg/mL) and BSF (20 µg/mL), achieving target concentrations of 50%, 100%, and 150%. The prepared recovery solutions were analyzed by injection into the quaternary HPLC system. Recovery studies were assessed by evaluating the observed concentrations against the theoretical concentrations of the spiked drugs.

2.7.5 LOD and LOQ:

Triplicate measurements were used to generate the standard plot equation, which served as the basis for assessing the detection and quantitation limits.

2.7.6 Robustness:

A standard sample solution of for DAPA (60µg/mL) and BSF (30µg/mL) was analyzed under deliberate variations to assess the reliability of the validated method. Robustness was determining by making minor and deliberated deviation in chromatographic parameters, including changes in the detection wavelength (226±2 nm), flow rate (1.0mL/Minute ± 0.1 mL), and mobile phase ratio (±5mL). The effects of these variations were assessed based on system suitability and repeatability parameters to determine the method’s reliability under slight changes in analytical conditions. The results were reported as the % RSD. A %RSD value below 2% confirmed the method was considered robust and reliably reproducible.

2.7.7 System suitability parameter:

The chromatographic system's performance was measured by assessing system suitability. The first standard drug solution was injected five times, while the second standard solution was introduced into the system once. The % RSD of the Peak response area for the first standard solution and the %RD between the two standard solutions were determined. Additionally, retention period, theoretical plate numbers, minimal tailing and resolution from the first injection of the first standard solution were measured and documented.

3. RESULTS AND DISCUSSION:

A RP-HPLC method with stability indicating capability was developed using a PDA detector for concurrent analysis of DAPA and BSF. Optimal chromatographic conditions were identified by considering physicochemical properties such as solubility, pH, and pKa values of both the drugs. Key parameters including various mobile phase composition, stationary phase and pH levels, flow rate, column temperature, diluents, and detection wavelength were optimized via trial and error. The stationary phase Shim pack Octadecylsilane C18 column was used to ensured high specificity and reproducibility, achieving efficient separation with retention times of 4.632 minutes for DAPA and 3.031 minutes for BSF. The mobile phase pH was optimized by evaluating various buffer systems across a range of pH values. Phosphate buffer, with its pH brought to 4 with 0.1% OPA (orthophosphoric acid) was identified as the superior choice for Providing enhanced resolution and symmetrical peak profiles. Methanol was selected as the preferred solvent due to superior solubility, among organic modifiers, acetonitrile showed superior performance in the mobile phase. The finalized mobile phase component—comprising buffer (pH modified to 4.0 using OPA) and Methanol, Acetonitrile(ACN) in a 50:35:15 (%v/v/v) ratio—exhibited excellent repeatability under isocratic elution conditions. Maintaining the pump flow at 1.0mL/min achieved a suitable balance between chromatographic resolution and analysis time. The detection wavelength of 226nm was selected as it demonstrated optimal absorbance for both drugs, indicating a good sensitivity for their detection. Method validation, conducted per ICH guidelines, confirmed its robustness and suitability for routine pharmaceutical quality control.

3.1 Selection of analytical Wavelength:

DAPA and BSF were separately dissolved in methanol at a 10µg/mL. Both drug solutions were examined using the double beam UV-Vis spectrophotometer within the 200nm–400nm wavelength range. Based on the overlay spectra of DAPA and BSF, the analytical wavelength of 226nm was selected, as both compounds exhibited significant absorbance at this wavelength.

3.2 Linearity:

Linearity was observed for the analytical method applicable within the concentration range of 20 to 120 µg/mL for DAPA (R² = 0.9981) and 10 to 60µg/mL for BSF (R² = 0.9964), confirming its suitability for analysis.

3.3 Precision:

The validated method consistently delivered precise and reliable results., with %RSD for the assay consistently under 2.0 for both DAPA and BSF, demonstrating the robustness of the proposed method.

3.4 Accuracy:

Accuracy were performed at spiked concentrations of 50%, 100%, and 150%, the average recovery results were 101.18%, 98.99%, and 99.00 % for DAPA, and 101.50%, 98.88%, and 101.90 % for BSF, respectively. These observations confirmed that the formulated method was consistent and unaffected by interference from inactive ingredients.

3.5 LOQ and LOD:

The detection limits were established as 2.81 for DAPA and 1.33 (µg/mL) for BSF, demonstrating the proposed method's capability to measure even trace amounts of both compounds. The quantitation limits were established as 8.53 for DAPA and 4.05 for BSF, demonstrating the method's capability to quantify small amounts of both substances.

3.6 Robustness:

The chromatographic method parameters varied within the acceptable limits for retention time changes, system suitability, and repeatability under all tested conditions. The findings confirmed that the proposed method is robust.

3.7 Specificity:

The validated method demonstrated specificity, as no interfering peaks were observed from blanks, standards, or formulation samples. Matrix components did not affect retention times, and peak purity was acceptable under all conditions (peak purity threshold 0.995647 of DAPA and 0.867547 of BSF).

3.8 Assay:

The assay percentage was calculated by performing triplicate injections (sample solution) into the HPLC system. The mean assay percentages were calculated as 102.79% for BSF and 102.54% for DAPA.

3.9 Stressed degradation studies:

To evaluate the stressed degradation studies of the developed method and understand the Stability degradation pathways and intrinsic stability of DAPA and BSF, stress studies were conducted as per ICH stability guidelines. DAPA and BSF were exposed to different stressed environments, including acid hydrolysis, base hydrolysis, hydrogen peroxide, dry heat, and photolytic environments. The stressed degradation conditions were optimized to induce degradation ranging from 5% to 20%. Degradation conditions, results, and chromatograms were presented in Table 2. The stressed degradation studies verified that the optimized method was suitable for batch release testing and long-term stability evaluation.

4. CONCLUSION:

The developed RP-HPLC method not only effectively separated and quantified these components with high accuracy but also provided valuable insights into their stability under a variety of stress conditions. The compounds were subjected to stressed degradation under multiple stress environments, among these conditions, the highest degradation of Dapagliflozin was observed under acidic and oxidative condition and Bisoprolol fumarate was observed under oxidative condition. The validated method underwent validation in compliance with ICH Q2(R1) standards. The designed method was demonstrated to be effective for the regular pharmaceutical testing of dapagliflozin propanediol monohydrate and Bisoprolol fumarate in synthetic formulations, offering a reliable tool for quality control in pharmaceutical applications and stability testing in pharmaceutical industries.

Table 2: Summary of Forced Degradation

Sress Condition	DAPA (60 μg/ml)				BSF (30 μg/ml)
Sress Condition	Area	% Drug Recovered	% Drug degraded	Degradant Retention time (min)	Area	% Drug Recovered	% Drug degrade	Degradant Retention time (min)
Acid hydrolysis (0.1N HCl) Duration = 2 hr at 60⁰C	1281953	86.36	13.64	2.779, 5.969	905807	87.91	12.09	2.431, 2.575
Base hydrolysis (0.1N NaOH) Duration = 2 hr at 60⁰C	1322052	89.76	10.24	1.689, 5.918	932612	90.78	9.22	2.679, 7.778
Hydrogen peroxide (3% H₂O₂) Duration = 2 hr at 60⁰C	1306421	88.43	11.57	1.378, 2.475, 5.975, 7.692	892063	86.44	13.56	2.239, 10.075
Light Degradation (under UV light254nm wavelength for 6 hrs)	1369024	93.74	6.26	-	963312	94.06	5.94	-
Dry Heat Degradation (3 hrs of heating at 60⁰ C)	1367982	93.65	6.35	-	953172	92.98	7.02	-

Fig 2: (a) Chromatogram of acid stressed of DAPA and BSF mixtures (b) Chromatogram of Base stressed of DAPA and BSF mixtures (c) Chromatogram of Hydrogen peroxide stressed of DAPA and BSF mixtures (d) Chromatogram of thermal stressed of DAPA and BSF mixtures (e) Chromatogram of photolytic stressed of DAPA and BSF mixtures.

ACKNOWLEDGEMENT:

We sincerely acknowledge the guidance and support received during this research of this article.

CONFLICTS OF INTERESTS:

No conflicts of interests.

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Received on 05.12.2024 Revised on 11.04.2025

Accepted on 08.09.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):420-425.

DOI: 10.52711/0974-360X.2026.00061

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