Analytical Method Development and Validation of Dapagliflozin Enantiomer and Other Related Impurities in API Form with Cost Effective and Green Approach

Received: 01-Jan-2025, Manuscript No. DPC-25-166938; Editor assigned: 14-Jan-2025, Pre QC No. DPC-25-166938; Reviewed: 28-Jan-2025, QC No. DPC-25-166938; Revised: 01-Jul-2025, Manuscript No. DPC-25-166938 (R); Published: 28-Jul-2025, DOI: DOI: 10.4172/0975-413X.17.2.646-656

Introduction

Type 2 diabetes mellitus (T2DM) is among the most prevalent chronic metabolic disorder in Western countries [1]. Millions of individuals rely on pharmacological treatments to control the symptoms of T2DM, while substantial financial resources are invested globally in the development of novel therapeutics agents. Dapagliflozin is one such agent-a highly selective orally bioavailable and reversible inhibitor of the human Sodium Glucose Co-Transporter 2(SGLT2), which plays a key role in renal glucose reabsorption [2]. Dapagliflozin enhances glycemic control in patients with type 2 diabetes by inhibiting the Sodium-Glucose Co-Transporter 2 (SGLT2), thereby reducing renal glucose reabsorption. This inhibition promotes increased urinary glucose excretion, which in turn leads to a reduction in plasma glucose levels [3]. As a result of its unique mechanism, Dapagliflozin is widely used in the treatments of patients with type 2 diabetes. Unlike many conventional antidiabetic agents, Dapagliflozin acts through an insulin-independent pathway by promoting glucose elimination via the kidneys. It belongs to a novel class of drugs known as SGLT2 inhibitors or ‘flozins’, which rely on plasma glucose levels and renal function to exert their therapeutic effects. Beyond glycemic control, Dapagliflozin offers additional clinical benefits, including modest reduction in body weight and blood pressure, along with improvements in insulin sensitivity and β-cell function. The drug is formulated as an oral tablet, making it convenient for long-term use. Dapagliflozin is effective both as a monotherapy and in combination with other antidiabetic agents, including insulin and oral hypoglycemic drugs. Its ability to lower blood glucose, body weight and blood pressure makes it a valuable option in the comprehensive management of type 2 diabetes [4-6]. According to previous literature, various chromatographic techniques have been employed for the estimation of Dapagliflozin [7,8] such as liquid chromatography-mass spectrometry [9, 10], normal and reverse phase high-performance liquid chromatography [11-15], ultra-performance liquid chromatography for simultaneous determination [16, 17] and by different spectroscopic methods such ultraviolet (UV) [18-20] and several methods are there for the determination of pharmacological action.

During the synthetic process of Dapagliflozin, it was observed that an enantiomeric form of the compound was also generated as by-product. To date, no official analytical method has been reported for the specific determination of this Dapagliflozin enantiomer. Therefore, the objective of the present study was to develop and validate a precise, accurate, linear, robust, specific and rapid analytical method for the estimation of the Dapagliflozin enantiomer along with its related impurities (bromo Dapagliflozin, ethyl Dapagliflozin and Dapagliflozin Alfa isomer), in the Active Pharmaceutical Ingredient (API) form (Figures 1-4).

Figure 1: Structure of Dapagliflozin enantiomer.

Figure 2: Structure of ethyl Dapagliflozin.

Figure 3: Dapagliflozin alfa isomer.

Figure 4: Bromo Dapagliflozin (Dapagliflozin related compound A).

Materials and Methods

Materials and reagents

Pharmaceutical grade Dapagliflozin API and Dapagliflozin Propanediol Monohydrate standard (purity 95.7%) used Inhouse, Bromo Dapagliflozin (Dapagliflozin related compound A) from USP, Dapagliflozin enantiomer and Ethyl Dapagliflozin was received Aquigen Bio Science pvt ltd, Dapagliflozin Alfa Isomer received from Simson pharma limited, acetonitrile and methanol used are from Rankem make and MiliQ grade water used for preparation of mobile phase (Mili Q system, Merck/Milipore Molsheim france).

Instruments

The analysis was performed on waters HPLC (with Empower 3 software), fitted with a gradient pump UV detector and Chiralcel 0J-3R (150 mm x 4.6 mm, 3 μm) column which is maintained at 40°C temperature. The mobile phase-A composition for water and acetonitrile was (95:5 v/v) and for mobile phase-B was (5:95 v/v). The mobile phase was run at a flow rate of 1 mL/min. The injection volume was 10 μL. The chromatographic run time was 40 mins. The wavelength of the detector was set at 227 nm for the analysis of the process impurity Dapagliflozin enantiomer along with Bromo Dapagliflozin (Dapagliflozin related compound A), Dapagliflozin enantiomer, Ethyl Dapagliflozin, Dapagliflozin Alfa Isomer.

Preparation of mobile phase

The mobile phase-A was prepared by mixing water and acetonitrile in ratio of 95:5 v/v and mobile phase-B was prepared in ratio of 5:95 v/v. The prepared mobile phase was shaken well and degassed by sonication. The gradient program as follows (Table 1).

Table 1: Gradient program.

Preparation of diluent

The solubility of impurities was found to be in acetonitrile. In order to avoid drift in the chromatogram, a mixture of acetonitrile and water in a ratio of 50:50 v/v was prepared.

Preparation of standard solution, system suitability solution and sample solution

The stock solution of Dapagliflozin enantiomer impurity, Bromo Dapagliflozin ( Dapagliflozin related compound A), Ethyl Dapagliflozin and Dapagliflozin Alfa Isomer was prepared in diluent at 0.0075 mg/mL 100 mL. The standard solutions were prepared from stock solutions after adequate dilution with diluent.

To prepare the system suitability solution, weighed and transferred accurately about 62.0 mg of Dapagliflozin Propanediol monohydrate standard into a 50 mL volumetric flask, added about 30 mL of Diluent, then sonicated to dissolve. Pipetted out 1.0 mL of Standard Stock solution into the above flask and made up the volume with diluent and mixed. Sample solution prepared 1 mg/mL with diluent.

Study of system suitability parameter

After equilibration of the column with mobile phase, System suitability, six replicate injections of standard solution were injected into the system and the chromatogram was recorded and the peak area was measured (Figures 5,6).

Figure 5: Chromatogram of system suitability.

Figure 6: Chromatogram of standard solution.

Construction of calibration curve

Aliquots of different concentrations of standard solution were prepared and their chromatograms were recorded at the optimized chromatographic conditions. The mean peak areas at different concentration levels were calculated from the chromatograms. The calibration curve was plotted between concentrations versus peak area having correlation coefficient (r²) 1.000 (Figure 7).

Figure 7: Calibration Plot of Dapagliflozin at different concentrations plotted between concentration and peak area.

Results and Discussion

Method validation

The developed method was validated according to the International Conference of Harmonization (ICH) Q2 (R2) Guideline for linearity, accuracy, precision, limit of detection, limit of quantitation, robustness, Specificity and solution stability parameters as described in ICH guidelines [21].

Linearity and range

From standard stock solutions prepared 0.45 g/mL, 0.75 g/mL, 1.2 g/mL, 1.5 g/mL and 2.25 g/mL solutions as per specification and their respective chromatograms were recorded. From the recorded chromatograms, their respective mean peak areas were calculated and the linearity plot was constructed using the mean peak areas at their respective concentrations. The correlation coefficient (r2) was found to be 1.000. The linearity data of Dapagliflozin was shown in Table 2 and the calibration plot was shown in Figure 8.

Table 2: Linearity data of Dapagliflozin enantiomer and other impurities.

Figure 8: Calibration plot of Dapagliflozin.

Accuracy

The accuracy of the proposed method was ascertained on the basis of recovery studies performed by the standard edition method. The sample preparation was done as above and the known amount of standard stock solution was spiked at different levels from 30%, 100% and 120%. The resultant solution was re-analyzed by the developed method. At each concentration, each sample was analyzed thrice at each level to check repeatability and from the data it was analyzed that the method was found to be accurate (Table 3 and Figure 9).

Table 3: Recovery Study Result of Dapagliflozin Enantiomer and other impurities

Figure 9: Plots for Accuracy. Note: A. As such sample solution, B. Accuracy at LOQ level (30%) spike sample solution, C. Accuracy at 100% level spike sample solution and D. Accuracy at 120% level spike sample solution.

Precision

The precision of the method was evaluated by carrying repeatability in the same day precision study. Precision of any analytical method was expressed as SD and RSD of a series of measurements. Precision of estimation of Dapagliflozin by proposed method was ascertained by replicate analysis of homogenous sample solutions (6 replicates). The percentage relative standard deviation (%RSD) of each study was calculated and was found to be less than 1% showing the method was precise.

Table 4: Precision study result of Dapagliflozin enantiomer and other impurities.

LOD and LOQ

Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the method were recorded based on signal to noise ratio. The LOQ was validated by triplicate analysis of samples prepared at a concentration near to that experimentally obtained. So from the LOD and LOQ results we can conclude that this method is capable for the detection and quantification of Dapagliflozin Enantiomer and related impurities in the Dapagliflozin sample.

Figure 10: Plots for LOD and LOQ. Note: A. Standard solution at LOD Level and B. Standard solution at LOQ level.

Robustness

The method can remain unaffected by small but deliberate variations in method parameters. Robustness of the method was determined by slightly changing the column temperature from the optimized chromatographic conditions (Figure 11 and Table 5).

Figure 11: Representative chromatogram of Dapagliflozin spiked sample solution under different column temperatures. Note: A. Column temperature at 38°C and B. Column temperature at 42°C.

Table 5: Robustness study of Dapagliflozin enantiomer and other related impurities.

Specificity

The specificity of the method was evaluated by spiking of Dapagliflozin sample solution with Dapagliflozin enantiomer, Bromo Dapagliflozin, Ethyl Dapagliflozin, Dapagliflozin Alfa Isomer along other process related impurities which is Dapagliflozin hydroxy, Oxo Dapagliflozin and Dapagliflozin acetate at theoretical concentration of 1.5 μg/mL and the chromatogram was compared with the standard solution (Figure 12 and Table 6).

Figure 12: Representative chromatogram of Dapagliflozin isomers.

Table 6: Details of spiked Sample solution in specificity parameter.

Solution stability

In order to check the solution stability of Dapagliflozin enantiomer and related impurities we prepared standard solution and sample solution as per analytical procedure and injected the standard solution and sample solution at regular time intervals. Calculated area and % content change with respect to initial results (Tables 7 and 8).

Table 7: Solution stability of standard solution.

Table 8: Solution stability of samples solution.

Cost effective and green approach

After the development and validation of the current method, the evaluation of its cost effectiveness was to a critical aim. As mainly from phase preparation to sample preparation we can see that throughout the analysis, the use of acetonitrile and water has only been used. Mixture of acetonitrile and water which are the only two solvents used in this method is considered to be very cost effective in nature in chemical and pharmaceutical industry. The greenness of the method was evaluated using the AGREE metric tool via relevant software was utilized. This system was proposed in 2020 [22] and is based on the 12 basic principles of Green Analytical Chemistry, embedded in a clock-like diagram. Each of the 12 segments has a colour related to the score obtained between 0 and 1 where 0 is red, 1 is green and value within this range-yellow. The final score represents the degree of greenness and the value closest to one means that the method is green. In the current method, the overall score of 0.64, as shown in Figure 13 (the middle of the pictogram), indicates that the proposed method is green. This result is above 0.5 (which is also considered green) due to the content of acetonitrile, lack of any salts or acids and short run time.

Figure 13: Greenness results for the proposed method by the AGREE metric tool.

Conclusion

The developed RP-HPLC method for Dapagliflozin enantiomer and related impurities was found to be simple, precise, accurate, reproducible and cost effective. Statistical analysis of the developed method confirms that the proposed method is an appropriate and it can be useful for the routine analysis. This method gives the basic idea to the researcher who is working in areas such as product development and finish product testing. The developed Method can also be used regularly for the in process quality control of the sample. By studying all these validation parameters, we have concluded that the method was linear, accurate, precise, robust, specific and rapid for the determination of Dapagliflozin enantiomer in API along with other related impurities. Hence the method can be successfully applied for the estimation of Dapagliflozin enantiomer and related impurities in API.

Data Availability

All the data generated or analysed during this study are included in this published article. All the data included in this manuscript have been generated at USV Private Limited and does not include any third-party data/analysis.

Conflicts of Interest

There are no conflicts to declare. To the best of our knowledge, the contents of this manuscript do not conflict with any third-party rights/interests.

Acknowledgment

We acknowledge USV private limited, management for support during the entire work of method development and method validation. The content and the information provided herein are proprietary and developed for USV private limited. Copying and circulation of the content are strictly prohibited.