Diabetes mellitus, a widespread medical condition, is characterized by increasing rates of obesity, physical inactivity, and urbanization. In July 2009, the US Food and Drug Administration (FDA) approved saxagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, for treating type 2 diabetes mellitus (T2DM).

Saxagliptin monohydrate, a non-hygroscopic crystalline powder, inhibits DPP4, thereby enhancing incretin hormone concentrations and reducing glucose levels in a glucose-dependent manner. Simultaneously, dapagliflozin—an orally active sodium-glucose cotransporter 2 (SGLT2) inhibitor—improves glycemic control by promoting glucose elimination via the kidneys. Its insulin-independent mechanism complements existing antidiabetic drugs.

Saxagliptin belongs to the dipeptidyl peptidase-4 (DPP-4) inhibitor class. It prolongs the action of incretin hormones (GLP-1 and GIP) by inhibiting their degradation. This results in enhanced insulin secretion and reduced glucagon release.

Clinical Use: Saxagliptin helps improve glycemic control without causing significant weight gain. It is commonly prescribed as part of combination therapy for T2DM. Saxagliptin helps improve glycemic control without causing significant weight gain. It is commonly prescribed as part of combination therapy for T2DM. Dapagliflozin is effective in lowering blood glucose levels, promoting weight loss, and reducing cardiovascular risks. It is often used as an adjunct to diet and exercise in patients with type 2 diabetes. Dapagliflozin inhibits the sodium-glucose co-transporter 2 (SGLT2) in the renal tubules. By doing so, it reduces glucose reabsorption, leading to increased urinary glucose excretion. This study explores analytical methods for quantifying saxagliptin and dapagliflozin, both individually and in combination. Additionally, we discuss the FDA-approved fixed-dose combination of these agents known as Qtern. Furthermore, we highlight the need for robust detection methods, including RP-HPLC and mass spectrometry, for assessing pharmaceutical dosage forms.

The proposed method, validated according to ICH guidelines, holds promise for stability studies, impurity profiling, and pharmacokinetic investigations.

MATERIALS AND METHODS

Chemicals and Reagents: Dapagliflozin and pioglitazone hydrochloride were sourced from Sinochem, China. We used HPLC-grade orthophosphoric acid (85%) from Fluka chemicals, acetonitrile from Fisher chemicals, potassium dihydrogen orthophosphate from EL Nasr Pharmaceuticals Chemicals, and HPLC-grade hydrochloric acid (37%) from Merck. Distilled water was used throughout the experiments. Chromatographic System: We utilized an Agilent 1200 HPLC system equipped with a diode array (DA) detector. The separation and quantitation were performed on a Luna C18 column (150 mm × 4.6 mm, 5 µm particle size). The mobile phase consisted of a mixture of Phosphate Buffer pH 3.0 and acetonitrile in a ratio of 45:55 (v/v). The buffer solution was prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate in 900 mL of water, adjusting the pH to 3.0 using orthophosphoric acid, and completing the volume to 1000 mL with water. The mobile phase was filtered using a 0.45 µm nylon membrane filter (Chrom Tech).

For the preparation of standard solutions:

1. Pioglitazone Working Standard (S1 Solution):

• Accurately weigh 30 mg of pioglitazone working standard and transfer it into a 100 ml volumetric flask.
• Add 75 ml of diluent, sonicate for 5 minutes, then cool.
• Complete the volume with diluent to obtain the desired concentration (S1 solution).

2. Dapagliflozin Working Standard (S2 Solution):

• Weigh 40 mg of dapagliflozin working standard and transfer it into a 100 ml volumetric flask.
• Add 75 ml of diluent, sonicate for 5 minutes, then cool.
• Complete the volume with diluent to obtain the desired concentration (S2 solution).

3. Sample Solution Preparation:

• Weigh and grind not less than 10 tablets.
• Transfer an accurately weighted portion of the powder equivalent to one tablet into a 100 ml volumetric flask.
• Add 75 ml of diluent, sonicate for 20 minutes, then cool at room temperature.
• Complete the volume with diluent.
• Dilute 5 ml of the resulting solution to a 25 ml volumetric flask, then complete to volume with mobile phase.
• Filter the solution through a 0.45 µm syringe filter into HPLC vials, rejecting the first portion of the filtrate, and inject it into the HPLC system.

4. Diluent Composition:

• The diluent consists of a 0.1 N concentration of hydrochloric acid in acetonitrile (1:9).

Linearity:

Linearity of an analytical procedure refers to its ability to produce test results that are directly proportional to the concentration of the analyte in the sample. For single-point standardization, linearity should extend at least 20?yond the specification range and include the target concentration. We evaluated linearity by preparing a minimum of five different concentrations (50%, 80%, 100%, 120%, and 150%) of the working standard solution and making three replicates of each concentration.

Accuracy:

Accuracy was assessed by spiking the standard solution. We measured concentrations of Dapagliflozin and Saxagliptin around the target concentration. Specifically, we spiked the placebo (except for the active ingredient) with known quantities of Dapagliflozin and Saxagliptin working standard. Accuracy was evaluated using four determinations over three concentration levels, covering the specified range (i.e., three concentrations and three replicates).

Specificity:

1. Specificity provides an indication of the selectivity and specificity of the procedure. The method is considered selective if the main peak is well resolved from any other peak by a minimum resolution.
2. We achieved this by injecting placebo and comparing it with the standard and placebo spiked with the standard and sample. Then, we ascertained peak purity using a photodiode array (PDA).

System Suitability:

System suitability was verified by injecting six replicates of the standard solution at 100% of the test condition to assess the precision of the chromatographic system. The proposed HPLC method allows the concurrent determination of Dapagliflozin and Saxagliptin in the sample drug, as they exhibit different retention times. System suitability data are provided in Table 2.

       
           
       
Table 2 System suitability parameters for Dapagliflozin and pioglitazone HCl

Method development

Linear calibration plots for the proposed method were obtained in concentration ranges of 4.00-12.00 µg mL^-1 (4.00, 6.40, 8.00, 9.60 and 12.00 µg mL^-1) for Dapagliflozin and as shown in Fig. 5 and data are shown in Table 3 and 30.00-90.00 µg mL^-1 Saxagliptin (30.00, 48.00, 60.00, 72.00 and 90.00 µg mL^-1) as shown in Fig. 6 and data are shown in Table 4.

       
           
       
Figure 6 Calibration Curve of Dapagliflozin

       
           
       
Table 5 Statistical data for calibration curves of Dapagliflozin

       
           
       
Table 6 Statistical data of calibration curves of Saxagliptin

Y _{Dapagliflozin} = 38.3465x +3.7698, r² = 0.9998

Y _Saxagliptin = 44.32317x +0.05454, r² = 0.9999

Slopes and intercepts were obtained by using regression equation (Y = mx + c) and least square treatment of the results used to confirm linearity of the method developed.

The limit of detection (LOD) and quantitation (LOQ)

The LOD and LOQ were established through serial dilutions. For Dapagliflozin and Saxagliptin:

LOD:

Dapagliflozin: 0.171 µg/mL (signal-to-noise ratio of 3:1)

Saxagliptin: 0.267 µg/mL (signal-to-noise ratio of 3:1)

LOQ:

Dapagliflozin: 0.519 µg/mL (signal-to-noise ratio of 10:1)

Saxagliptin: 0.808 µg/mL (signal-to-noise ratio of 10:1)

Accuracy

Accuracy Assessment: Recovery Study

To evaluate accuracy, we conducted a recovery study by adding standard drugs to reanalyzed samples at three different concentration levels. The percentage recoveries were then calculated. According to ICH guidelines, the standard limit for percentage recovery should fall within the range of 98% to 102%.

Our findings indicate that the recovery study for Dapagliflozin and Saxagliptin aligns with the standard ICH guideline. The accuracy results are supported by the data presented in Tables 7 and 8.

In both intra-day and inter-day variability testing for precision data, the method demonstrated high accuracy across the tested ranges for Dapagliflozin and Saxagliptin. The peak area of the sample solution closely matched that of the standard solution in both cases, with a relative standard deviation (RSD) of less than 2%. Specifically, Dapagliflozin had an RSD ranging from 0.02% to 1.22% in intra-day studies, while Saxagliptin had an RSD ranging from 0.04% to 0.48%. In inter-day studies, Dapagliflozin exhibited an RSD of 0.02% to 0.76%, and Saxagliptin had an RSD of 0.07% to 1.42%. These results indicate high precision and minimal variation.

Conc., Concentration; RSD, relative standard deviation

Conc., Concentration; RSD, relative standard deviation

       
           
       
Table 12 Precision data of inter-day variability Saxagliptin