The developed RP-HPLC method was validated as per ICH guidelines. The parameters validated are Specificity, Forced degradation studies, Accuracy, Precision (Intra-day precision, Inter-day precision), Linearity, Limit of Detection (LOD), Limit of quantitation (LOQ), Solution stability, Robustness, and System suitability parameters.
Specificity
Specificity of the developed RP-HPLC method was established by injecting 10 μl each of the blank, working standard, and sample solutions.
Forced degradation studies [13,14,15]
The stability of the developed method was established by performing forced degradation studies of the drug in the presence of acid, alkali, H2O2, temperature, UV light, and HPLC grade water.
Acid degradation
Degradation under acidic condition was evaluated by treating 1 ml of standard stock solution of ERTU and MET with 1 ml of 2N HCl and refluxed for 30 min at 60 ± 2 °C. The resulting solution was diluted to 10 ml with the diluent.
Alkali degradation
Under alkaline conditions, degradation was studied by refluxing 1 ml of standard stock solution of ERTU and MET with 1 ml of 2N NaOH for 30 min at 60 ± 2 °C. The stressed solution was made up to 10 ml with the diluent.
Oxidative degradation
About 1 ml of standard stock solution of ERTU and MET was subjected to oxidative degradation by refluxing with 20% v/v H2O2 in a 10-ml volumetric flask for 30 min at 60 ± 2 °C and made up with the diluent.
Thermal degradation
Thermal stability of the drugs was evaluated by placing the standard stock solution in the oven at 105 ± 2 °C for 6 h. About 1 ml of the stressed solution was diluted to 10 ml with the diluent.
Photolytic degradation
Photolytic degradation was studied by exposing the standard solution of ERTU and MET to UV light in the UV chamber for 7 days. The resulting stressed solution was diluted to 10 ml with the diluent.
Neutral degradation
Neutral degradation was carried out by refluxing 1 ml of standard stock solution with 1 ml of HPLC grade water in a 10-ml volumetric flask at 60 ± 2 °C for 6 h. The volume was made up with the diluent.
About 10 μl of each of the solutions exposed to different stress conditions were injected separately into the column, and the chromatograms were recorded to evaluate the stability of the drugs.
Accuracy
Accuracy was established by injecting about 10 μl of ERTU and MET at 80, 100, and 120% levels into the column, and the procedure was repeated thrice. Mean percent recovery of three levels was determined using the peak areas at each level.
Precision
Precision of the optimized method was determined by injecting six samples of working standard solution of ERTU and MET into the column on the same day for intra-day precision and on two continuous days for inter-day precision; % RSD was calculated.
Linearity
Linearity of the developed method was evaluated by injecting ERTU in the concentration range of 0.9375–5.625 μg/ml and MET in the range of 62.5–375 μg/ml. Calibration curve was constructed by plotting peak area on the y-axis against concentration (μg/ml) on the x-axis. The correlation coefficient of the calibration curve was calculated by using the method of least squares in MS Office Excel 2007.
LOD and LOQ
LOD and LOQ were calculated using the formulae based on the standard deviation of the y-intercept of regression lines and the slope of the calibration curve.
$$ \mathbf{LOD}=\mathbf{3.3}\ \boldsymbol{X}\ \frac{\boldsymbol{\sigma}}{\boldsymbol{S}}\kern3.75em \mathbf{LOQ}=\mathbf{10}\ \boldsymbol{X}\ \frac{\boldsymbol{\sigma}}{\boldsymbol{S}} $$
where σ is the standard deviation and S is the slope of the curve.
Stability of standard solution of ERTU and MET
Stability of the standard solution of ERTU and MET was conducted by placing the solution in a volumetric flask at 30 ± 2 °C for 24 h. At three time points 0, 12, and 24 h, about 10 μl of the stored solution was injected into the column to calculate the percent (%) assay difference of the drug.
Robustness
Robustness of the developed RP-HPLC method was evaluated by making minor changes in the flow rate (0.9 to 1.1 ml/min), percentage of acetonitrile in the mobile phase (30 to 40%) and temperature (25 to 35 °C). The parameters evaluated were % RSD of peak areas, theoretical plates, tailing factor, and resolution.
System suitability testing
Suitability of the system was evaluated by injecting working standard solution into the column to evaluate parameters like % RSD of peak areas, theoretical plates, tailing factor, and resolution in the optimized chromatographic conditions.
Assay
The working sample solution was injected six times into the column, and % assay was calculated by using the formula:
$$ \%\mathrm{Assay}=\frac{\mathrm{Sample}\ \mathrm{area}}{\mathrm{Standard}\ \mathrm{area}}\times \frac{\mathrm{Dilution}\ \mathrm{of}\ \mathrm{standard}}{\mathrm{Dilution}\ \mathrm{of}\ \mathrm{sample}}\times \frac{P}{100}\times \frac{\mathrm{Avg}.\mathrm{wt}}{\mathrm{LC}}\times 100 $$
where Avg. wt is the average weight of tablets, P is the percentage purity of working standard, and LC is the label claim of the drugs