Stability Indicating Method Development and Validation of Vildagliptin and Dapagliflozin in Bulk and in Marketed Formulation by UHPLC Method

One of the most prevalent and complicated metabolic disorders is Type 2 Diabetes Mellitus (T2DM), which typically requires the combination of multiple pharmaceutical approaches to control hyperglycemia.¹ Two main reasons contribute to T2DM: The β-cells impaired insulin secretion and inadequate response of insulin-sensitive tissues to insulin. In India, T2DM accounts for over 90% of Diabetes cases.²

Vildagliptin is a potent di-peptidyl peptidase IV (dip-IV) inhibitor, which binds covalently to the catalytic sites of dipeptidyl peptidase-4(dpp-4), eliciting prolonged enzyme inhibition. DPP-4 inhibitors are a novel class of oral antihyperglycemic drugs used to treat type 2 diabetes mellitus.³ Dapagliflozin (DAPA) in Biopharmaceutics Classification System is classified as Class II drug, being highly soluble and poorly permeable. DAPA is a Sodium-Glucose Co-Transporter 2 (SGLT2) inhibitor that is highly potent, selective and reversible. It enhances urine glucose excretion by decreasing the kidney’s reabsorption of glucose, improving glycemic management in individuals with type 2 diabetes mellitus.⁴

A review of the literature indicates that there aren’t many techniques for estimating dapagliflozin and vildagliptin simultaneously. The literature conveyed that one RP-HPLC and HPTLC have been reported for the estimation of the VILDA and DAPA simultaneously.^5,6 There have been publications of several novel UV spectroscopic techniques for simultaneous assessment of dapagliflozin and vildagliptin in combination dose forms.⁷ Vildagliptin and Dapagliflozin, either alone or in combination with pharmaceutical moieties, could be analyzed using a number of analytical techniques outlined in the literature.^8–12

This is the first publication, as far as we are aware, of a stability suggesting UHPLC approach for simultaneous estimation of dapagliflozin and vildagliptin in bulk and therapeutic dosage formulation. Therefore, in accordance with ICH recommendations, the current study work was focused on developing a stability-indicating UHPLC method for the simultaneous assessment of dapagliflozin and vildagliptin in bulk and pharmaceutical formulation.

Agilent UHPLC system connected with quaternary pumps, DAD detector, CHEMSTATION software and with autosampler is used for the study.

Standard samples of Vildagliptin and Dapagliflozin were procured from Swapnroop Drugs and Pharmaceuticals, Sambhajinagar.

Marketed formulation of VILDAR-D (100-10 mg) from Elder Pharma Pvt. Ltd., was employed in the research.

Methanol, water (HPLC grade), hydrogen peroxide (AR grade), hydrochloric acid and sodium hydroxide chemicals were procured from Merck.

The mobile phase is made up of 0.1% OPA and methanol at a 78:22 ratio. To de-gas the mixture, a 0.45 μ membrane filter is used for filtration, followed by sonication.

Stock solutions of both drugs were prepared by dissolving 100 mg of Vildagliptin and 10 mg of Dapagliflozin in methanol and sonicated until fully dissolved. Using methanol in a 10 mL volumetric flask, the volume was further adjusted. According to the requirements, the stock solution was diluted.

The equivalent weights of DAPA and vildagliptin were determined. Weight of powder equal to 100 mg of Vildagliptin and 10 mg of Dapagliflozin was taken in a 100 mL volumetric flask, and sonicated for 15 min. Volume was made up of methanol. The solution was filtered via a 0.45 μm membrane filter.

The UV spectrum of VILDA and DAPA in the range of 200-400 nm was recorded in order to determine the appropriate wavelength of chromatographic separation. The iso-absorptive point was found at 234 nm and 262 nm from the obtained overlaying spectrum given in Figure 1. The 234 nm wavelength was used for chromatographic due to better resolution.

Figure 1:
The isobestic point of both drugs in UV-visible Spectroscopy.

In order to improve chromatographic conditions and produce a symmetrical peak and improve drug resolution, a number of system appropriateness parameters were examined. The mobile phase including methanol and 0.1% OPA (78:22%, v/v) at a flow rate of 1 mL/min produced the more symmetrical and resolved peaks at 234 nm as given in Figure 2. This was achieved by combining several appropriate solvents in different ratios to optimize the mobile phase. Table 1 lists the ideal chromatographic conditions.

Figure 2:
The developed chromatogram of the optimized method for VILDA and DAPA.

The suggested method’s validation was completed in accordance with ICH Q2 (R1) requirements and validation metrics included system suitability parameters, LOD, LOQ, robustness, specificity, precision, accuracy and linearity.

Forced degradation experiments were conducted in accordance with ICH Q1A (R2) and Q1B recommendations, covering alkali hydrolysis, peroxide hydrolysis, acid hydrolysis, hydrolytic degradation and photolysis.

The linearity denotes direct proportionality between the input concentrations and the peak regions of the technique. The existing approach produced linear graphs for both VILDA and DAPA between peak areas and employing concentrations between 50-250 mg/mL of VILDA and between 5-25 μg/mL of DAPA. The regression coefficient (r²) values were evaluated.

The precision of the method was assessed by using intra- and inter-day precision. Three distinct concentrations were analyzed in order to assess the intra-day precision of VILDA (100, 150, 200 μg/mL) and DAPA (10, 15, 20 μg/mL) using UHPLC on the same day. Similarly, the same concentrations were analyzed for inter-day precision. Relative standard deviation was used to express precision.

Accuracy is expressed as % recovery. The percentage recovery was obtained by adding a known amount of vildagliptin and dapagliflozin standard at three different concentrations (80%, 100% and 120%), to pre-analyzed samples.

The standard deviation and the slope of the calibration curve were used to determine the LOD and LOQ. By using the slope approach, the method’s sensitivity to the LOD and LOQ for Vildagliptin and Dapagliflozin was determined. LOD and LOQ were computed with LOD=3.3σ/S and LOQ=10σ/S, where S is the slope of the standard curve and σ is the standard deviation.

The robustness of the approach was confirmed by deliberately altering the process specifications, including the mobile phase, detecting wavelength and flow rate.

The marketed formulation, VILDAR-D, including 100 mg of Vildagliptin and 10 mg of Dapagliflozin, was assayed. To record the chromatogram, 20 μL of the sample solution and standard solution were introduced into the UHPLC independently. The peak area of the chromatograms that were recorded was used to calculate the drug concentrations that were present.

Studies on forced degradation were carried out to evaluate the proposed method’s stability-indicating property. The standard was subjected to a range of deterioration conditions, including acidic stress (0.1 N), alkaline stress (0.1 N NaOH), peroxide (3% H₂O₂) and photolytic stress (1 day). The system appropriateness characteristics were examined along with the percentage degradations after the exposed solutions were injected into duplicates

The developed chromatogram of the optimized method for Vildagliptin and Dapagliflozin, as illustrated in Figure 2, shows that the blank has no peak at the retention time of Vildagliptin and Dapagliflozin, indicating that the peaks obtained in the standard solutions at working concentrations are solely due to the drugs.

Aliquots at 50, 100, 150, 200, 250 μg/mL for VILDA and 5, 10, 15, 20 and 25 μg/mL for DAPA were prepared for the determination of linearity. Vildagliptin and Dapagliflozin obeyed the linearity for the concentration range of 50-250 μg/mL and 5-25 μg/mL correspondingly. The regression equations for VILDA and DAPA were discovered to be y=11.1686x+113.413 and y=19.08x+18.027 respectively. The correlation coefficient was discovered to be 0.9995 and 0.9999 for Vilda and DAPA respectively. Linearity graphs of VILDA and DAPA are shown in Figures 3 and 4 respectively. Results are represented in Table 2.

Figure 3:
The calibration curve of Vildagliptin at 234 nm.

Figure 4:
The calibration curve of Dapagliflozin at 234 nm.

The inter-day and intra-day precisions were estimated by calculating the percentage RSD. The %RSD for intra-day and inter-day for Vilda and DAPA are given in Table 3. All the %RSD values was inside the range, demonstrating the precise method.

As per ICH, three criteria were used to evaluate the proposed method’s accuracy. The outcomes demonstrate the accuracy of the proposed method. It was discovered that Vilda’s percentage recovery fell between 100.56-102.28%, whereas DAPA’s percentage recovery fell between 100.05-102.87%. Table 4 represents the results for accuracy.

For VILDA and DAPA, the limit of detection was determined to be 1.0419 μg/mL and 0.2435 μg/mL, respectively. The limit of quantification was determined to be 3.1574 μg/mL and 0.7381 μg/mL for VILDA and DAPA respectively.

The robustness study was done by modifying the flow rate, 0.9 mL/min (-) and 1.1 mL/min (+); mobile phase,77+23(-) and 79+21(+), detection wavelength, 233 nm (-) and 235 nm (+). The %RSD values were found to be less than 2, therefore the method is regarded as robust (Table 5).

The percentage purity of VILDA and DAPA was found to be 100.93% and 100.35%, respectively.

According to degradation studies in exposed acidic, alkaline, peroxide and neutral environments, neither Vildagliptin nor Dapagliflozin underwent significant degradation. In a photolytic environment, DAPA shows significant degradation. Table 6 presents the force degradation results, whereas Figure 5a-e displays the chromatograms.

Figure 5a:
Peroxide degradation.

Figure 5b:
Acid degradation.

Figure 5c:
Neutral degradation.

Figure 5d:
Peroxide degradation.

Figure 5e:
Photolytic Degradation.

Extensive experimentation was conducted by changing the chromatographic parameters like buffer pH, mobile phase and flow rate in order to create a simple UHPLC method for the simultaneous estimation of Vildagliptin and Dapagliflozin. Initially, using mobile phase methanol and 0.1% OPA in the ratio 80:20 and flow rate of 0.7 mL, failed to achieve satisfactory separation. The best chromatographic results were obtained by applying a mobile phase consisting of methanol and 0.1% OPA in a 78:22 ratio and flow rate of 1.0 mLmin. It was discovered that both drugs had an excellent resolution, and retention time of less than 4 min. The stability studies and validation of the method were conducted in compliance with ICH guidelines. The validation parameters, which include accuracy and precision, were determined to be within the acceptable range, suggesting that the approach is precise and accurate. A method’s robustness research revealed that both intentional and minor alterations had no effect on the system suitability parameters’ results. The degradation analysis revealed that, with the exception of photolytic degradation, there was a lower proportion of degradation under other conditions. All the parameters related to system suitability did not change throughout the forced degradation study.

In order to estimate Dapagliflozin and Vildagliptin simultaneously in pharmaceutical formulations, the current stability-indicating UHPLC method was developed and validated. The proposed method is easy to use, quick, affordable, precise, accurate and still stability indicating. The established UHPLC method can be successfully used to analyze dapagliflozin and vildagliptin in pharmaceutical formulations on a regular basis.

The authors are thankful to the Principal, Pravara Rural College of Pharmacy, Loni, Maharashtra, India, for providing the necessary facilities to perform this research work.