Development and Validation of a Stability-Indicating RP-HPLC Method for the Quantification of Sitagliptin and Dapagliflozin in Bulk Drug and Commercial Formulation

Dapagliflozin

Dapagliflozin, a drug in the gliflozin family, is used for the treatment of type 2 diabetes ^[1]. Dapagliflozin restricts the sodium-glucose transport proteins (SGLT2) class 2, which are responsible for around 90% of the kidney's glucose reconsumption ^[2]. In the renal proximal tubule, sodium-glucose co-transporter 2 (SGLT2) reabsorbs both glucose and sodium chloride. Dapagliflozin is an SGLT2 restrictor that increases renal glucose elimination in order to treat hyperglycemia in type 2 diabetes. When this transporter activity is blocked, blood glucose is eliminated through urine ^[3]. The Molecular formula of dapagliflozin is C₂₁H₂₅ClO_6. A white to off-white crystalline powder, dapagliflozin dissolves in Methanol, Acetonitrile (ACN), ethanol and other organic solvents ^[4,5].

Dapagliflozin chemical Name: (2S,3R,4R,5S,6R)-2-{4-chloro-3-[(4- ethoxy phenyl) methyl] phenyl}-6-(hydroxymethyl) oxane-3,4,5-triol. ^[6]

Figure.1: Structure of Dapagliflozin

1. Sitagliptin Phosphate

Sitagliptin phosphate is an oral blocker of dipeptidyl peptidase-4 (DPP-4). This enzyme-inhibiting drug is used individually or in combination with other oral antihyperglycemic drugs to treat type 2 diabetes. Sitagliptin restricts the DPP-4 by a competitive mechanism. This enzyme breaks down incretins, which are digestive hormones released after a meal and are also known as GLP-1 and GIP ^[7]. The pancreas is able to limit the release of glucagon and boost the production of insulin by blocking Gastric Inhibitory Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) inactivation ^[8]. Blood glucose levels proceed near to normal as a result. The Molecular formula of Sitagliptin Phosphate is Molecular C₁₆H₁₅F₆N₅O. It is slightly soluble in acetone and soluble in water, methanol, ethanol, and other solvents. ^[9,10]

Sitagliptin chemical Name: 7 - [(3R) – 3 – amino – 1 – oxo – 4 - (2, 4, 5-trifluorophenyl) butyl] -5, 6, 7, 8 tetrahydro – 3 (trifluoromethyl)-1, 2, 4 – triazolo [4, 3-a] pyrazine phosphate ^[11]

Figure.2: Structure of Sitagliptin

The review of the literature demonstrated that UV spectroscopy and RP-HPLC were used to quantify the drug from bulk ^[12]. The recommended study demonstrates a simple to operate, quick, and affordable RP-HPLC technique for Quantify Dapagliflozin and Sitagliptin in Formulation and API ^[13,14,15]. In accordance with ICH Standards, the Established technique was verified for Linearity, Accuracy, Precision, Limit of Detection (LOD), Limit of Quantification (LOQ), Robustness, Ruggedness, and sensitivity. Additionally, stress testing was done in a variety of Environments, including temperature, light, oxidation, humidity, and pH (acid/base). ^[16]

MATERIAL AND METHOD:

Chemical and Reagents:

Table.1: Chemical and Reagents

Instruments:

Table.2: Instruments used for Experiment

ANALYTICAL METHOD DEVELOPMENT BY HPLC METHOD

1. Dapagliflozin:

Determination of solubility for HPLC analysis

A specific quantity of the standard medication was dissolved in a variety of solvents, including acetonitrile, methanol, and water. The UV technique was used to estimate solubility quantitatively.
Selecting an appropriate solvent: A 50:50 v/v composition of water to methanol was used as the solvent. Based on studies on solubility choice solvents and solubility experiments conducted on a number of solvents, the solvent was chosen.

Formation of stock solution for standard

10 mg of dapagliflozin were carefully measured and pour into a 10-milliliter volumetric flask, diluted with 50:50 water and methanol, and then sonicated to the appropriate concentration of 1000 µg/mL to create the standard stock solution. A 1 mL aliquot of the previously mentioned standard stock solution was put into a 10 mL volumetric flask. It was then diluted with 50:50 water and methanol to achieve the required concentration, which resulted in a sub-stock solution of 100 µg/mL.

Formation of stock solution for sample

To make the sample stock solution, five pills were accurately weighed and crushed into powder. A volume equal to 10 mg were weighted and pour into a 10-milliliter volumetric flask, diluted with 50:50 water and methanol, and sonicated for 15 minutes. 1 mL of the previously stated sample stock solution, diluted with water: methanol (50:50), was added to a 10-milliliter volumetric flask to create the solution with a concentration of 100 µg/mL.

Detection of Wavelength for Dapagliflozin

The standard solution of 10 µg/mL of Dapagliflozin was prepared and scanned over the 400-200nm range. After the scan was completed, it showed maximum absorbance at 224 nm, so the detection wavelength was selected as 224 nm.

Figure.3: Spectrum of Dapagliflozin.

Determination of solubility for HPLC analysis

A specific quantity of the standard medication was dissolved in a variety of solvents, including acetonitrile, methanol, and water. The UV technique was used to estimate solubility quantitatively.
Selecting an appropriate solvent: A 50:50 v/v composition of water to methanol was used as the solvent. Based on studies on solubility choice solvents and solubility experiments conducted on a number of solvents, the solvent was chosen.

Formation of stock solution for standard

10 mg of Sitagliptin Phosphate were carefully measured and pour into a 10-milliliter volumetric flask, diluted with 50:50 water and methanol, and then sonicated to the appropriate concentration of 1000 µg/mL to create the standard stock solution.

Formation of stock solution for sample

To make the sample stock solution, five pills were accurately weighed and crushed into powder. A volume equal to 100 mg were weighted and pour into a 100-milliliter volumetric flask, diluted with 50:50 water and methanol, and sonicated for 15 minutes.

Detection of wavelength for Sitagliptin Phosphate

The standard solution of 100 µg/mL of Sitagliptin Phosphate was prepared and scanned over the 400-200nm range. After the scan was completed, it showed maximum absorbance at 265 nm, so the detection wavelength was selected as 265 nm.

Table.3: Chromatographic Conditions

ANALYTICAL METHOD VALIDATION BY HPLC METHOD

1. Specificity study

To illustrate the specificity of the described approach, the following solutions were prepared and injected.

Diluent is used as a blank solution. (Methanol: Water 50:50)

Standard solution: Standard solution was produced, in accord with the ICH Guidelines.
Sample solution: Sample solution was produced, in accord with the ICH Guidelines.

Verify the drug's peak and interference study identification.

2. Linearity

10 ml volumetric flask was filled with 10 mg of the drug, which had been carefully weighed. The solvent was then added to dilute the drug to the required concentration of 1000 µg/mL. Using the previously described solution, the drug's linear response was determined for Dapagliflozin (0–25 µg/mL) and Sitagliptin Phosphate (0–125 µg/mL), respectively. The calibration curve indicated peak area as a function of concentration. We calculated the correlation coefficient and Y-intercept of the linearity curve.

3. Accuracy

According to the analytical method, a known quantity of drug X was spiked into sample solutions in triplicate to produce drug concentrations that were proportional to the working concentration, 80%, 100%, and 120%. This data was then used to evaluate the procedure's accuracy. One estimated the percentage of recovery.

4. Precision

A standard solution comprising 125 ?g/mL of sitagliptin phosphate and 25 ?g/mL of dapagliflozin was used for six injections into the system. The chromatogram was recorded, and the % RSD of the Peak Area was then calculated.

1. Intraday precision:

A standard peak's responses were measured on the same day with the same concentration solution in order to determine the Intraday precision.

2. Interday Precision:

A standard peak's responses were measured on a different day with the same concentration solution in order to determine the Interday precision.

5. Limit of Detection (LOD) & Limit of Quantification (LOQ)

Six sets of linearity concentrations were assessed based on the response and slope of a regression equation, and LOD and LOQ were calculated using the following calculations in accordance with ICH guidelines.

6. Robustness

The following parameters and their effect on the system suitability test have been changed one by one. In accordance with ICH Guidelines, change in flowrate (±0.2 ml/min); Change in temperature (±5°C); Change in wavelength (±2 nm).

RESULT AND DISCUSSION

Specificity

Standard Peaks of Dapagliflozin and Sitagliptin Phosphate

The linearity of the drug Dapagliflozin was assessed by dilutions produced using the standard stock solution. The concentration range spanned by the dilutions was 0–25 ?g/mL.

Table.4: Linearity for Dapagliflozin

Figure.7: Calibration curve of Dapagliflozin by HPLC

The linearity of the drug Sitagliptin Phosphate was assessed by dilutions produced using the standard stock     solution. The concentration range spanned by the dilutions was 0–125 ?g/mL.

Table.5: Linearity for Sitagliptin Phosphate

Figure.8: Calibration curve of Sitagliptin Phosphate by HPLC

80%, 100%, and 120% of three distinct concentrations' percentage drug accuracy. Using the information gathered from the recovery experiments, the mean recovery % was then computed.

Table.6: Accuracy for Dapagliflozin

Accuracy for Sitagliptin Phosphate

80%, 100%, and 120% of three distinct concentrations' percentage drug accuracy. Using the information gathered from the recovery experiments, the mean recovery % was then computed.

Table.7: Accuracy for Sitagliptin Phosphate

Precision for Dapagliflozin:

Both intraday (repeatability assessed by analysing the standard solution on the same day) and interday methods (repeatability conducted by analysing the standard solution on different day) were used to analyse precision. Injecting the standard solution six times was the method used for this precision investigation. The slope is used to express the results.

Intraday Precision

Table.8: Intraday Precision for Dapagliflozin

Figure.15: Chromatogram of Intraday Precision for Dapagliflozin

Table.9: Interday Precision for Dapagliflozin

Figure.16: Chromatogram of Interday Precision for Dapagliflozin

In this level of accuracy investigation, the standard solution was injected six times; the results were expressed using the slope. Both intraday (repeatability evaluated by analyzing the standard solution on the same day) and interday methods (repeatability carried out by analyzing the standard solution on Different day) were employed to analyze precision.

Intraday Precision

Table 10 Intraday Precision for Sitagliptin Phosphate

Figure.17: Chromatogram of Intraday Precision for Sitagliptin Phosphate

Table.11: Interday Precision for Sitagliptin Phosphate

Figure.18: Chromatogram of Interday Precision for Sitagliptin Phosphate

The system suitability test has been affected by the gradual changes made to the following parameters.

1. Change in flowrate ±0.2 ml/min (0.6mL and 1mL)
2. Change in temperature ±5°C (20^oC and 30^oC)
3. Change in wavelength ±2 nm [Dapagliflozin (222nm and 226nm)]

       [Sitagliptin Phosphate (263nm and 267nm)]

1. Dapagliflozin

Table.12: Robustness Data of Dapagliflozin

2. Sitagliptin Phosphate

Table.13: Robustness Data of Sitagliptin Phosphate

Forced Degradation

Research on forced degradation can be used to investigate the quality of drug substance and drug product changes over time and in response to various climatic variables. They may be helpful in identifying the drug molecule's inherent stability, the degradation route, the degradants, and proven stability-indicating analytical procedures. In accordance with ICH Guidelines, which are ICH Q1A, Q1B, and Q2B represent forced degradation. ^[17,18]

Dapagliflozin

Table.14: Degradation Data of Dapagliflozin

Sitagliptin Phosphate

Table.15: Degradation Data of Sitagliptin Phosphate