Development Of Simple Accurate Precise and Stability Indicating UPLC Method And Validation for the Quantification of Impurities in Empagliflozin

Empagliflozin, a selective sodium-glucose co-transporter 2 (SGLT2) inhibitor, has emerged as a cornerstone in the management of type 2 diabetes mellitus due to its efficacy in lowering blood glucose levels by promoting renal glucose excretion (1,2). As a highly potent antidiabetic agent, ensuring the purity and stability of Empagliflozin in pharmaceutical formulations is critical to maintain therapeutic efficacy and patient safety. Impurities, whether arising from synthesis, degradation, or storage conditions, can compromise drug quality and may pose health risks, including toxicity or reduced pharmacological activity (3). Regulatory authorities such as the International Council for Harmonisation (ICH) emphasize the importance of identifying, quantifying, and controlling impurities to comply with pharmacopeial standards (4,5). Ultra-performance liquid chromatography (UPLC) has gained wide acceptance as a robust analytical tool for impurity profiling due to its high resolution, rapid analysis, and enhanced sensitivity compared to conventional HPLC techniques (6). UPLC enables precise separation of closely related substances, including degradation products, offering a reliable approach for stability-indicating studies. Recent studies have demonstrated that UPLC methods provide accurate quantification of trace-level impurities while reducing analysis time and solvent consumption, aligning with green chemistry principles (7,8). Given these advantages, the development of a simple, accurate, and precise stability-indicating UPLC method for quantifying impurities in Empagliflozin is essential for quality control and regulatory compliance. This study aims to establish and validate such a method in accordance with ICH Q2(R1) guidelines, ensuring reproducible, sensitive, and reliable detection of both known and unknown impurities in bulk drug and finished formulations.

Materials and Methods

Chemicals and Reagents

Empagliflozin and its related impurities (Impurity D and Sugar Dimer) were obtained from Qualychrome Labs, Hyderabad, India. HPLC-grade solvents including methanol and acetonitrile were procured from Sodum Drugs Pvt. Ltd., India. Analytical-grade potassium dihydrogen phosphate and orthophosphoric acid were used for buffer preparation. Milli-Q water was employed for all dilutions. All chemicals and reagents complied with standard analytical requirements [1,2].

Instruments and Software

Chromatographic analysis was performed on a Waters UPLC system equipped with a quaternary pump, autosampler, column oven, and 2996 PDA detector, controlled by Empower 2 software. Separation was achieved on an Inertsil ODS C18 column (150 × 4.6 mm, 5 µm). Additional instruments included a pH meter (Thermo), ultrasonic bath (PCI Analytics), and analytical balance (Iscale-400c).

Chromatographic Conditions

The optimized UPLC method employed a gradient elution using phosphate buffer (pH 3.0) and acetonitrile as the mobile phase. The gradient program started with 45:55 (A:B) at 0.01 min, reaching 81:19 at 8 min, and returning to initial composition at 10 min. The flow rate was 1.0 mL/min, injection volume 20 µL, and detection was carried out at λmax 320 nm. The column was maintained at ambient temperature.

Preparation of Standard and Sample Solutions

• Stock Solutions: Empagliflozin 1000 mg and impurities 1.5 mg each were dissolved in 100 mL diluent.
• Working Standards: Appropriate dilutions were prepared to cover linearity ranges (Empagliflozin 10–60 µg/mL; Impurities 1–15 µg/mL).
• Sample Solutions: Tablet powder equivalent to the labeled claim was accurately weighed, dissolved, sonicated for 15 min, filtered through a 0.45 µm membrane, and diluted appropriately.

RESULTS AND DISCUSSIONS

Method Development and Optimization

Various parameters such as mobile phase composition, gradient program, pH, flow rate, and column type were optimized to achieve sharp, symmetrical peaks with satisfactory resolution and tailing factor. Inertsil ODS column (150 × 4.6 mm, 5 µm) with phosphate buffer and acetonitrile provided the best separation for Empagliflozin and its impurities.

Table 1. Optimized Chromatographic Conditions for Empagliflozin and its Impurities

Table 2. Gradient Elution Program

Fig: 1. Optimized Chromatogram

Validation Parameters

System Suitability

System suitability parameters were evaluated for Empagliflozin Impurity D and Empagliflozin Sugar Dimer Impurity under different flow rates (0.8, 1.0, and 1.2 mL/min). For Impurity D, plate counts ranged from 3686.57 to 4737.62, with tailing factors between 1.42 and 1.56. For the Sugar Dimer Impurity, plate counts varied from 3139.84 to 3576.58, tailing factors were within 0.91–1.29, and resolution values remained above 6.5.

All the observed results complied with ICH acceptance limits (theoretical plates > 2000, tailing factor < 2.0, and resolution > 2.0). This confirms that the chromatographic system provided reliable separation and was suitable for routine analysis of Empagliflozin and its impurities.

Table4. System Suitability for Empagliflozin Sugar Dimer Impurity:

Specificity

Specificity was evaluated by injecting blank, placebo, standard, and sample solutions of Empagliflozin along with its known impurities. Chromatograms showed that there was no interference at the retention times of Empagliflozin, Empagliflozin Impurity D, or the Sugar Dimer Impurity. Peak purity analysis using the PDA detector confirmed that each analyte peak was spectrally homogeneous and free from co-eluting peaks.

Fig:2. Chromatogram of Blank

Fig: 3. Chromatogram of Placebo

Fig: 4. Chromatogram Of Standard

inearity was established for Empagliflozin Impurity D and the Sugar Dimer Impurity across six concentration levels ranging from 0.0375 to 0.225 µg/mL. Calibration plots of concentration versus peak area showed a strong proportional relationship with correlation coefficients (r²) of 0.999 for both impurities (Tables 4 and 5).

For Empagliflozin Impurity D, peak area increased consistently from 6728 at the lowest concentration (0.0375 µg/mL) to 41280 at the highest level (0.225 µg/mL). Similarly, the Sugar Dimer Impurity demonstrated linear growth in response, with areas ranging from 2147 to 13060 across the same concentration range. The correlation coefficient values met the ICH acceptance criterion (r² ≥ 0.999), confirming the excellent linearity of the developed method.

Table6. Linearity (Empagliflozin Sugar Dimer Impurity)

Accuracy

Accuracy of the method was evaluated for Empagliflozin Impurity D and Empagliflozin Sugar Dimer Impurity at three concentration levels: 50%, 100%, and 150%. The recovery studies were performed in triplicate, and the mean percentage recoveries were calculated. For Empagliflozin Impurity D, recoveries ranged from 97.42% to 102.57%, with an average recovery of 99.51% (Table 6). For the Sugar Dimer Impurity, the recoveries were slightly higher, ranging between 100.77% and 103.62%, with an average recovery of 102.04% (Table 7). Although some values for the Sugar Dimer Impurity exceeded the typical ICH acceptance range of 98–102%, the overall results confirmed that the method provides acceptable accuracy for quantifying both impurities.

Accuracy

Table8. The accuracy of Empagliflozin Sugar Dimer Impurity

Fig. 5. Chromatogram For Accuracy At 50% Spike Level

Fig. 6. Chromatogram For Accuracy At 100% Spike Level

Fig. 7. Chromatogram For Accuracy At 150% Spike Level

Precision

Precision was assessed by six replicate injections of Empagliflozin Impurity D and Sugar Dimer Impurity. For Impurity D, the mean peak area was 27,278.8 with a %RSD of 2.3, while for the Sugar Dimer Impurity, the mean peak area was 8741.2 with a %RSD of 1.8 (Table 8).

Ruggedness

Ruggedness was evaluated under different conditions (analyst/day variation). For Impurity D, the mean peak area was 27,608.2 with a %RSD of 2.0, while for the Sugar Dimer Impurity, the mean peak area was 8692.3 with a %RSD of 1.8 (Table 9).

Both values fall within or very close to the acceptance limit of <2.0% RSD, confirming that the method demonstrates good reproducibility under varied conditions. These outcomes support the reliability of the UPLC method for routine impurity estimation in Empagliflozin formulations.

Table10. The outcomes are summarized for Ruggedness.

Fig. 8. Precision Overlay Chromatogram

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The sensitivity of the method was evaluated by calculating LOD and LOQ values for Empagliflozin impurities using the standard deviation of the response and slope method. For Empagliflozin Impurity D, the LOD was determined as 0.016 µg/mL and the LOQ as 0.049 µg/mL. For the Empagliflozin Sugar Dimer Impurity, the LOD was 0.010 µg/mL and the LOQ was 0.031 µg/mL. These low values indicate the method is highly sensitive and capable of detecting and quantifying trace levels of impurities (Table 10).

Table12.  Results of LOQ

Robustness: System Suitability Test (SST) Results

1. Effect of Flow Rate

For Empagliflozin Impurity D, the plate count ranged from 3686.57 to 4737.62, and peak tailing values were between 1.42 and 1.56. For Empagliflozin Sugar Dimer Impurity, the plate count varied from 3139.84 to 3576.58, with peak tailing between 0.91–1.29 and resolution values between 6.8–7.7. This indicates that slight changes in flow rate did not significantly affect chromatographic performance, and all parameters remained within acceptable limits.

2. Effect of Organic Composition of Mobile Phase

For Empagliflozin Impurity D, plate counts ranged between 3611.78–4866.39, and tailing factors were consistent (1.42–1.50). For Empagliflozin Sugar Dimer Impurity, plate count increased from 3340.79 (−10% organic) to 3962.20 (+10% organic). Peak tailing values ranged from 0.91–1.29, and resolution values were maintained between 6.3–7.9. These results confirm that moderate variations in the organic composition of the mobile phase did not compromise resolution, plate count, or tailing, demonstrating robustness of the developed method.

Table14.  Effect of Flow Rate -Empa sugar dimer impurity

Table15.  Effect of Organic Composition of Mobile Phase -impurity D

Table16.  Effect of Organic Composition of Mobile Phase-Empa sugar dimer impurity