Instrumentation
The HPLC system (Shimadzu Corporation, Japan) was connected to the LC solution software, consisting of binary pump (LC-20AD), Rheodyne syringe sample injector (20 μL), and an UV detector (SPD-20A). Type-II (paddle) dissolution apparatus (Electro lab TDT-08L), ultra-sonicator (RK 106, Spincotech), millipore (0.45 μm) filters, and digital pH meter (LI-120, Elico) were utilized for this work. Design-Expert version 11.0.5.0 software (Stat-Ease Inc. Minneapolis) was employed for designing of LC experiments and response-modeling to generate design space for optimized robust analytical method.
Chemicals and reagents
The reference standards of saxagliptin and dapagliflozin were obtained from Hetero Laboratories, Hyderabad, India. Marketed formulation (QTERN) with label claim 10 mg of dapagliflozin and 5 mg of saxagliptin per tablet was procured from local pharmacy. All solvents used for mobile phase were of HPLC grade and obtained from Merck, Mumbai, India.
Chromatographic conditions
Chromatographic separation of analytes was achieved on SPOLAR C18 (250 × 4.6 mm, 5μ) column at ambient temperature with isocratic elution. The mobile phase consisting of acetonitrile: phosphate buffer, pH 5.8 (26:74% v/v) at flow rate of 0.96 mL/min. Injection volume was 20 μL and UV detection at 236 nm.
Preparation of standard solution
In order to prepare binary standard solution, dapagliflozin (20 mg), and saxagliptin (10 mg) were weighed, decamped into volumetric flask of 10 mL and dissolved in diluent by sonication. Volume was contrived up to the mark with diluent solution and the flask was shaken well (solution A). Further 0.1 mL of this solution was diluted to 10 mL with diluent so as to get binary working standard solution concentration of 20 μg/mL and 10 μg/mL for dapagliflozin and saxagliptin, respectively.
Method development using AQbD approach
Method optimization was premeditated with 20 experimental runs under (8 factorial points, 6 center points, and 6 axial points with α= 0.6073) central composite design (CCD) to study the interactions of the three critical method parameters (CMPs), namely [S1], % acetonitrile (X1), aqueous phase pH (X2), and flow rate (X3). The chromatographic responses (resolution, capacity factor) were obtained through experimentation on HPLC instrument using working standard solution of dapagliflozin (20 μg/mL) and saxagliptin (10 μg/mL). Statistical analysis was performed together with CCD in the Design-Expert software. The significance of factors was calculated using analysis of variance (ANOVA). Design space was generated by numerical optimization where Derringer’s desirability function was used to attain high method performance criteria.
Method validation
The validation parameters like precision, linearity, accuracy, system suitability, sensitivity, and robustness were premeditated in accordance to ICH Q2(R1) guidelines.
Linearity
Aliquots of 0.002, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL were withdrawn from mixed standard stock and diluted to 10 mL with mobile phase such that the final concentration of dapagliflozin and saxagliptin and in the range of 0.2-300 μg/mL and 0.1-150 μg/mL was obtained respectively. Calibration curve was generated by plotting the peak area against the concentration of drug.
System suitability
The efficiency of optimized method was monitored by system suitability test. It was carried out via injection of freshly prepared working standard solution into HPLC under optimized conditions for six times. The chromatographic responses (analytical attributes) studied were retention time, resolution, capacity factor, theoretical plate number, peak area, selectivity factor, and tailing factor of analyte peaks.
Precision
Repeatability of proposed method was verified by analyzing 6 replicate injections of freshly prepared working standard solution of dapagliflozin (100 μg/mL) and saxagliptin (50 μg/mL) in mobile phase on the same day. Intermediate precision was performed by analyzing replicates of same concentration solution prepared in three consecutive days. The peak area of the analytes was determined and %RSD was calculated.
Accuracy
Recovery of standard that spiked to target concentration of sample at three levels (80, 100, 120%) was studied for method accuracy. The % recovery of drug was calculated by measurement of its peak area in the chromatogram.
Specificity
Specificity of the method was established by comparing the chromatogram of blank (mobile phase), placebo solution, and matrix (degradants) with test solution (analytes in mobile phase). The placebo solution comprises all the commonly used excipients for manufacturing of tablet dosage form. Stress degraded samples were prepared as mentioned in the forced degradation studies where the drugs were exposed to various stress conditions such as acid, base, peroxide, heat, and light to generate their degradation products.
Sensitivity
Limit of detection (LOD) and limit of quantification (LOQ) were premeditated from the regression analysis data of linearity studies (LOD = 3.3 σ/s, LOQ = 10 σ/s; where σ, standard deviation of response; s, slope of calibration curve).
Robustness
In order to assess the robustness of the method, deliberate changes in method, critical parameters were made within the design space. The variations in the parameters include pH (± 0.1), organic phase (± 1 %), and flow rate (± 0.04 mL/min) of mobile phase. The % RSD of theoretical plate number and retention time of chromatogram obtained for every variation was calculated.
Assay of marketed formulation
Twenty tablets of marketed formulation (QTERN), each containing 10 mg of dapagliflozin and 5 mg of saxagliptin were taken, average weight was measured and the fine powder was crushed. An accurately weighed amount of powder equal to 10 mg of dapagliflozin and 5 mg of saxagliptin was transferred to volumetric flask of 10 mL capacity containing methanol and sonicated for 15 min. The flask was shaken and volume was produced with methanol till the mark and filtered via Whatman filter paper (no: 41). From the filtrate, 0.2 mL was transferred into 10 mL volumetric flask and the volume was leveled to the mark with mobile phase. The amount of saxagliptin and dapagliflozin present in sample solution was determined.
Dissolution studies
Dissolution testing of marketed formulation was carried out in FDA-recommended dissolution media, i.e., acetate buffer pH 4.5 (1000 mL) for dapagliflozin and 0.1 N HCl with pH 1.2 (900 mL) for saxagliptin using paddle apparatus (USP apparatus 2) at 50 rpm and 37 ± 0.5 °C for 45 min. Sampling aliquots of 5 mL were withdrawn at 10, 15, 20, 25, 30, and 45 min interval and replacing the fresh medium with an equal amount. After the end of each test time, sample aliquots were filtered. Filtrate of 0.1 mL was diluted 10 mL with mobile phase (phosphate buffer pH 5.8: acetonitrile, 74:26) and analyzed by the contemplated RP-HPLC method. Amount of drug dissolved (saxagliptin/dapagliflozin) was calculated using their respective calibration curves. The cumulative percentage of drug dissolved was plotted against the time.
Forced degradation studies
Stress studies were executed on saxagliptin and dapagliflozin standards under acid, base, oxidative, thermal, and UV light conditions. Acid degradation was carried out with 5 mL of mixed standard stock solution, to this 5 mL of 1 N hydrochloric acid was added and kept at 60 °C for 60 min, then neutralized with 1 N sodium hydroxide. From this, 0.2 mL was diluted to 10 mL with mobile phase and injected into the HPLC system. Similarly, 1 N sodium hydroxide for base degradation and hydrogen peroxide (6%) for oxidative conditions were used. Dry heat degradation (105 °C, 6 h) and light degradation (UV Chamber, 200-Watt h/m2, 48 h) were performed on selected drugs in solid state. After degradation, the solid samples (20 mg dapagliflozin/saxagliptin 10 mg) were dissolved in 10 mL methanol and 0.1 mL of this solution was diluted to 10 mL with mobile phase. These solutions were injected into the HPLC system and sample stability assessment chromatograms were documented.