Stability-Indicating RP-HPLC Method for Determination of Empagliflozin using AQbD Approach

Shital Chaudhari, Dr. Vishwas Bhagat, Dr. R. V. Shete*

Department of Pharmaceutical Quality Assurance, Rajgad Dnyanpeeth's College of Pharmacy, Bhor.

*Correspondence: shitalc2904p@gmail.com;

DOI: https://doi.org/10.71431/IJRPAS.2026.5410   

INTRODUCTION

Empagliflozin is an orally active, highly selective inhibitor of the sodium–glucose co-transporter-2 (SGLT2), widely prescribed for the management of Type 2 Diabetes Mellitus. By inhibiting SGLT2 in the proximal renal tubules, empagliflozin reduces glucose reabsorption and promotes urinary glucose excretion, thereby lowering blood glucose levels independently of insulin secretion. In addition to glycemic control, it has demonstrated cardiovascular and renal protective effects, making it an important therapeutic option in modern diabetes management.

The increasing clinical use of empagliflozin necessitates the development of reliable and robust analytical methods for its quantitative determination in pharmaceutical dosage forms. Among various analytical techniques, Reverse Phase High Performance Liquid Chromatography has emerged as a preferred method due to its high sensitivity, specificity, accuracy, and reproducibility. However, conventional method development approaches often rely on trial-and-error strategies, which may not ensure optimal method performance or robustness.

In recent years, the concept of Analytical Quality by Design has gained significant attention in pharmaceutical analysis. AQbD is a systematic, science- and risk-based approach that emphasizes understanding the method and controlling variability to ensure consistent performance. It involves defining an Analytical Target Profile (ATP), identifying Critical Quality Attributes (CQAs), and evaluating Critical Method Parameters (CMPs) using structured tools such as Design of Experiments. This approach enhances method robustness, reduces variability, and ensures regulatory compliance.

A stability-indicating method is essential for assessing the degradation behavior of drug substances under various stress conditions such as hydrolytic, oxidative, photolytic, and thermal degradation. Such methods are crucial for ensuring drug safety, efficacy, and shelf-life. Regulatory guidelines, including ICH Q2(R1) and ICH Q14, emphasize the importance of validated, stability-indicating analytical procedures in pharmaceutical development.

Therefore, the present study aims to develop and optimize a stability-indicating RP-HPLC method for the determination of empagliflozin using an AQbD approach. The method is designed to achieve high accuracy, precision, and robustness while effectively separating degradation products, thereby making it suitable for routine quality control and stability studies of pharmaceutical formulations.

MATERIALS AND METHODS

Column: C18 column (250 mm × 4.6 mm, 5 µm)

Mobile Phase: Acetonitrile : Phosphate buffer (pH 3.0 adjusted with orthophosphoric acid) (60:40 v/v)

Flow Rate: 1.0 mL/min

Detection Wavelength: 225 nm

Injection Volume: 20 µL

Column Temperature: 30°C

Run Time: 10 min

Analytical Quality by Design (AQbD) Approach

Analytical Target Profile (ATP)

The method was developed to:

Accurately quantify empagliflozin in pharmaceutical dosage form

Separate degradation products effectively

Provide reproducible and robust results

Critical Quality Attributes (CQAs)

Retention time

Peak area

Resolution (>2)

Tailing factor (<2)

Critical Method Parameters (CMPs)

Mobile phase composition

Flow rate

Detection wavelength

pH of buffer.

Design of Experiments (DoE)

A Central Composite Design (CCD) was employed.

Factors:

A: Mobile phase ratio

B: Flow rate

C: pH

Levels: Low (-1), Medium (0), High (+1)

Responses:

Retention time

Peak area

Resolution

Statistical analysis was performed using ANOVA to evaluate model significance

Risk Assessment

Risk assessment was conducted using:

Fishbone (Ishikawa) Diagram

Failure Mode and Effects Analysis (FMEA)

High-risk parameters were selected for DoE optimization.

RESULTS AND DISCUSSION

The model showed statistical significance (p < 0.05)

ANOVA confirmed adequacy of model

Lack-of-fit was found to be insignificant

METHOD VALIDATION

Parameter                   Result

Linearity .                    2–10 µg/mL (R² = 0.999)

Precision.                    %RSD < 2

Accuracy                     98–102% recovery

LOD.                             0.15 µg/mL

LOQ.                             0.45 µg/mL

Calibration Curve

The calibration curve demonstrated linearity over 2–12 µg/mL (R² = 0.999). Precision studies showed %RSD<2%. Recovery ranged between 98–102%. LOD and LOQ were 0.45 and 1.40 µg/mL. Forced degradation confirmed stability-indicating nature. Results are consistent with recent studies (Kumar et al., 2024; Madure et al., 2025; ICH Q14, 2023).

Chromatogram

CONCLUSION

A stability-indicating RP-HPLC method for the determination of Empagliflozin was successfully developed and optimized using the Analytical Quality by Design approach. The application of systematic experimental design, particularly Central Composite Design, enabled effective evaluation and optimization of critical method parameters, ensuring a thorough understanding of method performance.

The developed method demonstrated excellent linearity, accuracy, precision, and sensitivity in accordance with ICH Q2(R1) requirements. Forced degradation studies confirmed the specificity and stability-indicating capability of the method, as it efficiently separated the drug from its degradation products under various stress conditions. Statistical analysis, including ANOVA, validated the robustness and reliability of the optimized method within the defined design space.

Overall, the AQbD-based RP-HPLC method proved to be simple, precise, reproducible, and robust. It is highly suitable for routine quality control, stability testing, and regulatory compliance in pharmaceutical analysis of empagliflozin formulations.

REFERENCES

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