A standard sample of SIT and ERT was procured from Fortune Pharma Pvt., Ltd., Hyderabad. HPLC grade acetonitrile, water, and methanol were procured from Merck Specialties Private Limited, Mumbai, India.
UPLC instrument
Acquity UPLC SYSTEM was equipped with quaternary pumps, ACQUITY TUV detector, and autosampler integrated with Empower 2 software.
Preparation of standard solution
100mg of SIT and 15mg of ERT were weighed accurately into a 100-ml volumetric flask and diluted with 50ml diluent and sonicated for 10min and finally diluted up to the mark with diluent. Calibration standards were prepared by serial dilution of stock solution of SIT and ERT 0.25ml, 0.5ml, 0.75ml, 1ml, 1.25ml, and 1.5ml with diluent in a 10-ml volumetric flask to produce a calibration concentration about 25, 50, 75, 100, 125, and 150μg/ml of SIT and 3.75, 7.5, 11.25, 15, 18.75, and 22.5 μg/ml. The solution with100μg/ml of SIT and15μg/ml of ERT was considered as a standard solution, and it was scanned in the UV wavelength region to determine the absorption maximum of 218nm.
Preparation of sample solution
In commercially available Steglujan (15mg Ertugliflozin and 100mg Sitagliptin), ten tablets were weighed accurately and triturated in a mortar and pestle into a fine powder. Accurately weighed powder equivalent to 100mg of SIT and 15mg of ERT were transferred into a 100-ml volumetric flask. Approximately 70ml of diluent was added and sonicated for 10min, to completely dissolve SIT and ERT in the presence of other excipients in the formulation. Finally, the volume was made up to the mark with diluent. An aliquot of 1ml of the above solution was filtered through ALL PURE hydrophilic PVDF filter membrane of 0.45μm and was further diluted to 10ml with diluent to produce a concentration of 100μg/ml and 15μg /ml of SIT and ERT, respectively.
Method development
Complete scrutinization of chromatographic parameters like column chemistry, mobile phase, column temperature, and flow rate aids in optimization of chromatographic conditions and to accomplish symmetric peak shape and better resolution of drugs. Mobile phase optimization was done with various combinations of suitable solvents in different ratios and finalized that acetonitrile: water (pH 3.5) (50:50%, v/v) as mobile phase with flow rate 0.2ml/min. Various trials were listed in Table 1. Optimized chromatogram as shown in Fig. 3. The marketed formulation chromatogram was shown in Fig. 4.
Method validation
The proposed method was validated as per ICH Q2R1 guidelines, and the method validation parameters include system suitability, linearity, precision, accuracy, robustness, and specificity.
Linearity
As per ICH, linearity is the ability of the analytical procedure to obtain test results that are directly proportional to the concentration of an analyte in the sample. The range is the interval from the upper concentration to the lower concentration of an analyte in the sample, which indicates a suitable level of precision, accuracy, and linearity in the analytical procedure. Linearity is determined by preparing aliquots at six different levels of calibration curve over the concentration range of 25–150μg/ml for sitagliptin and 3.75–22.5μg/ml for ertugliflozin and was analyzed in triplicate. The correlation coefficient with the regression line equation was determined from the calibration curve. Linearity data was shown in Figs. 5 and 6.
Precision
The precision of analysis was performed in terms of intra-day precision and inter-day precision. Precision is expressed in terms of relative standard deviation.
Accuracy
Accuracy is expressed in terms of recovery. It is determined by spiking a known amount of standard SIT and ERT to pre-analyzed samples at three different levels such as 50%, 100%, and 150%, and the percentage recovery was determined.
LOD and LOQ
As per ICH, the limit of detection is the lowest amount of analyte that can be detected but not necessarily quantitated. The limit of quantification is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. LOD and LOQ were calculated using the following formula.
$$ \mathrm{LOD}=3.3\sigma /S $$
$$ \mathrm{LOQ}=10\sigma /S $$
S is the mean of the slope, and σ is the standard deviation of the intercept.
LOD and LOQ were estimated, using the calculations from the calibration curve based on the standard deviation of response and slope of the calibration curve.
Forced degradation studies
Forced degradation studies were conducted to assess the stability-indicating property of the proposed method [20]. Various stress studies conducted were acid hydrolysis (0.5N HCl/60°C/1h), base hydrolysis (0.5N NaOH/60°C/1h), oxidation (10%H2O2/60°C/1h), hydrolytic degradation (water/60°C/1h), photolysis (UV energy-254nm/3 days/dark control), and thermal degradation (105°C/75%RH/24h). Forced degradation studies conducted at distinct stress conditions to assess the stability of drug products at various stress conditions provide information about the stability of SIT and ERT.