The established chromatographic methods were absolutely validated followed by ICH guidelines Q2 (R1) and Q1A (R2) for the validation of analytical methods and Stability testing of new substance and product, respectively [16, 17].
From standard stock solution, six sets of series of concentration ranging from 03 to 18 µg mL−1 were obtained by diluted 0.3–1.8 mL concentration from 100 µg mL−1 with methanol in 10 mL volumetric flask and analyzed it. Linear curve equation was made by plotting the peak area versus drug concentration.
Precision studies were carried out by using drug standard solution in concentration taken in the calibration range. The precision of the developed method by means of intra-day variation (% RSD) was observed by analyzing standard drug solution in three sets of concentration of Mifepristone, i.e., 6 µg mL−1, 9 µg mL−1, and 12 µg mL−1 on same day. Inter-day precision (% RSD) was studied by analyzing the drug solution in same concentration used for intraday three times on three days in one week period.
Repeatability studies were performed by analyzing six replicates of same concentrations (9 µg mL−1) on same day.
Accuracy study of established method was determined by percent recovery studies. To the pre-tested sample, standards drug concentrations were added at three different points (80%, 100%, and 120%). At each level of the amount, three estimations were evaluated and the percentage recovery and percentage mean recovery were calculated.
Ruggedness study of established method was estimated by analysis of same aliquots 9 µg mL−1of pure mifepristone at same operational and environmental conditions by two different analysts.
Detection and quantitation limit
Detection limit and quantification limit were determined by analysis of lower concentration of the linear range of the linear curve equation which were calculated using formulae “L.O.D = 3.3 × ASD/M” and “L.O.Q = 10 × ASD/M,” where “ASD” is average standard deviation of the peak areas of the mifepristone (n = 3), taken as a measure of noise, and “M” is the slope related to linear curve equation.
Analysis of tablet formulation
Twenty tablets of mifepristone (RELEZED having label claim 200 mg) were accurately weighed, average weight determined then transferred to a clean waterless mortar and crushed into fine powder by pestle. Above fine powder equivalent to 10 mg mifepristone was transferred to volumetric flask having capacity 100 mL containing 70 mL of methanol; this mixture subjected to sonication for 10 min after that volume was made by methanol up to the mark and filtered through 0.45 µm Whatman filter paper its give 100 µg mL−1 concentration. From this stock solution, sets of same concentration (i.e., 1.5 mL) separately transferred to volumetric flask having capacity 10 mL and volume was made by methanol up to the mark to get final concentration of 15 µg mL−1 and this solution was injected into column with Hamilton syringe. The peak area recorded and drug concentration in sample were estimated from linear curve equation.
System suitability study was done to confirm that the HPLC system is working correctly and can provide accurate and precise results. It was evaluated by injecting 10 µg mL−1 solution of mifepristone six times. Solution of concentration of 10 µg mL−1of mifepristone was prepared by pipette out 1.0 mL solution from the standard stock solution into volumetric flask having capacity up to 10 mL and diluted with methanol up to the mark. The following parameters of system suitability like theoretical plates, tailing factor, retention time and capacity factor were evaluated.
Robustness study was performed for established RP-HPLC method by change in the chromatographic conditions, To study the impact of flow rate on the resolution, the flow rate was changed by 0.2 units, i.e., 0.4 and 0.8 mL min−1 from the actual flow rate 0.6 mL min−1. The impact of temperature of column on resolution was studied at 30 °C and 34 °C instead of 32 °C. The impact of change in the composition of mobile phase was observed by changing the % of acetonitrile in gradient by 2%. The impact of pH was studied by changing pH by 0.2 units from the actual value 3.0 keeping remaining method conditions were kept constant.