Ertugliflozin and Metformin were procured as gift samples from Startech laboratories.
Segluromet formulation combination of Metformin and Ertugliflozin was obtained from local pharmacy store.
Acetonitrile, Methanol of HPLC grade, and O-phosphoric acid of analytical grade were obtained from Rankem laboratories.
Chromatographic conditions
Equipment
Waters HPLC 2695 chromatographic System set with quaternary pumps, Photo Diode Array detector and Auto sampler. Chromatograms were recorded using Empower 2 Software.
Chromatographic conditions
Mobile phase: 0.1%OPA buffer: Acetonitrile (60:40% v/v)
Column: Phenomenex (4.6 mm × 150 mm, 5 μm)
Flow rate: 1.0 ml min−1
Column temperature: 30 °C
Programming: Isocratic
Wavelength: 220 nm
Run time: 10 min
Sample volume: 10 μl
Solvent
Acetonitrile (ACN) and water in the ratio 50:50% v/v is used as solvent.
Both standard and sample solutions were filtered through 0.45 μm before injecting into the HPLC.
Preparation of standard solutions
Standard stock solution of 15 μg ml−1 of Ertugliflozin and 1000 μg ml−1 of metformin were prepared by weighing accurately 0.75 mg of Ertugliflozin and 50 mg of Metformin and transferring to 50 ml volumetric flask. Three-fourth volume of diluent was added and kept for sonication for 25 min and finally they were made up to the mark using the diluent. Working standard solution of conc 100 μg/ml of Metformin and 1.5 μg/ml of Ertugliflozin was obtained by pipetting out 1 ml of stock solution and transferring it to 10 ml volumetric flask and making up to the mark with the diluent.
Preparation of sample solutions
Weigh 20 tablets, calculate the average weight. Weight equivalent to one tablet was transferred to volumetric flask of 500 ml, next 50 ml of diluent was applied and sonicated for 25 min, further volume was made up to the mark using diluent to obtain concentration of 1000 μg/ml of metformin and 15 μg/ml of Ertugliflozin. Then, 1 ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent to obtain concentration of 100 μg/ml of Metformin and 1.5 μg/ml of Ertugliflozin.
Buffer
0.1%OPA buffer: 0.1 ml orthophosphoric acid diluted to 100 ml using water of HPLC grade.
Validation
System suitability
System suitability parameters were evaluated and analyzed to check system performance using standard solutions of Ertugliflozin (1.5 ppm) and Metformin (100 ppm).
Linearity
Linearity is to produce test results that are exactly proportional to the analyte concentration in the sample. It is obtained with in the concentration range of 0.375–2.25 μg/ml for Ertugliflozin and 25–150 μg/ml for metformin. The calibration graph is plotted with X-axis concentration and Y-axis peak areas and seen in Figs. 3 and 4.
Specificity
The interference in the optimized method is verified. There is no evidence of interference of peaks in blank and placebo at retention times of these drugs in this method. So, the developed method is specific.
Accuracy
Accuracy is a measure of how close is the experimental value to the true value. Accuracy is determined by calculating the recovery values accuracy is done at intervals of 50%, 100%, and 150% levels. % recovery and % RSD were taken into consideration.
Precision
Precision is defined as the closeness of agreement between a series of measurements obtained from multiple sampling of the homogeneous sample. It is estimated by injecting six replicates of Ertugliflozin of conc 1.5 ppm and metformin of conc 100 ppm.
LOD and LOQ
Limit of detection (LOD) and limit of quantification (LOQ) of Ertugliflozin and Metformin were determined from the calibration curve method using the following equations:
$$ \mathrm{LOD}=3.3\times \upalpha /\mathrm{s} $$
$$ \mathrm{LOQ}=10\times \upalpha /\mathrm{s} $$
Where α is standard deviation, s is the slope.
Robustness
Even though there are small deliberate changes in method like flow rate, mobile phase ratio, and temperature there is no change in the result and the results are within range as per ICH Guide lines.
Assay
Segluromet bearing the label claim Metformin 500 mg and Ertugliflozin 7.5 mg. Assay was performed with the above formulation. Average % Assay for Metformin and Ertugliflozin obtained was found to be 101.32 and 99.46%, respectively.
Forced degradation studies
This was achieved by following recommendations of an international conference on harmonization [11,12,13].
Standard samples of Ertugliflozin and Metformin were degraded under different stress conditions like acidic, alkali, oxidative, thermal, photostability, and neutral conditions.
Samples for acidic and alkali degradation were refluxed at 60 0C for 30 min with 2N HCl and 2N NaOH. Oxidative degradation is carried out by using 20% H2O2 and kept aside for 30 min at 60 0C. Thermal degradation is done by placing the sample in an oven at 105 °C for 6 h. For photostability, studies the sample solutions were subjected to UV light by placing the sample in photo stability chamber for 7 days or 200 W h/m2 .The sample solutions were refluxed in water for 6 h at 60 0C .All the samples were finally diluted to obtain concentration of 1.5 μg/ml of Ertugliflozin and 100 μg/ml of Metformin. Ten microliters was injected into the system and the chromatogram is recorded to assess the stability of sample. Chromatograms of acid, alkali,peroxide,thermal,U.V and water stress conditions were depicted as Figs. 5, 6, 7, 8, 9 and 10