Methyl Prednisolone reference standard (MP), Methyl Prednisolone hemisuccinate reference standard (MPHS), and Methylprednisolone sodium succinate working standard (MPSS) working standard was supplied as a gift sample from UP pharma (Assuit, Egypt). Acetonitrile HPLC-grade, disodium hydrogen phosphate, sodium dihydrogen phosphate, glacial acetic acid 99%, hydrochloric acid 37 %, sodium hydroxide, and Hydrogen peroxide 30 % (Scharlau, Spain). Water for injection (WFI) was used in the analysis and passed through a 0.45 μm nylon membrane filter before use. Phosphate solution (1) was prepared by weighing 1.6g of disodium hydrogen phosphate in 1000 mL WFI. Phosphate solution (2) was prepared by weighing about 0.3g of sodium dihydrogen phosphate in 1000 mLWFI.
2.1 Chromatographic system configuration
Compared with the previously conducted HPLC methods and the current analysis method, we did not use a high percentage of the organic modifier of acetonitrile, dedicated pH solution adjustment, or special chemical reagent to realize the optimum separation for the ideal system suitability achievement.
MP, 17-MPHS, and MPHS assay determination were conducted using the HPLC model HP 1100 series with variable wavelength. The current method was conducted with the RP-BDS column (250 mm x 4.6 mm x 5 μm) (Thermo Scientific). The mobile phase was prepared as WFI: glacial acetic acid: acetonitrile in a volume ratio (63:2:35) at a flow rate of 2.0 mL/min with detection wavelength at 254 nm at room temperature and injection volume 20 μL.
2.2 Parameters of method validation
The HPLC validation method was performed according to the International Conference on Harmonization (ICH) guidelines concerning parameters including system suitability, Range of linearity, the limit of detection (LOD), the limit of quantification (LOQ), repeatability (precision), recovery and accuracy, robustness, ruggedness, the stability of the solution, specificity, and selectivity 9-11,31,32.
2.2.1 System suitability check
System Suitability was performed by injecting six replicate injections of the same sample solution which was prepared by dissolving a quantity of MP reference standard equivalent to 5 mg/100mL of mobile phase and mixing 10 mL of this solution with a weight of MPSS working standard equivalent to 65 mg and 1 mL of each phosphate buffer solutions in 100 mL volumetric flask and complete with mobile phase to obtained a concentration about 500 µg/mL of total MP.
2.2.2 Range & linearity
The analytical approach is deemed to be linear if there is a substantial portion between the response and claimed working concentration starting at the lowest point in the tested range and increasing to the highest point with R2 ≥ 0.999 3,4,9-12,31,32.
Regression linearity equation:
Y = a X ± b (1)
Where Y represents the response of the average peak area, X represents the claimed working concentration in (%), a represents the slope and b is the intercept of the calibration curve.
The linearity parameter was submitted using different five concentrations in the range (50%-150%) of the MP working standard. The stock solutions were prepared as a quantity of MP reference standard 48.9 mg in 100 mL mobile phase and complete with WFI to 1000 mL and MPSS working standard equivalent to 640 mg/100 mL in the mobile phase. Then make serial dilutions to obtain concentrations (50%, 70%, 100%, 120%, and 150%) by taking (5mL, 7mL, 10mL, 12mL, and 15mL) from each solution of the stoke solutions and complete to 100 mL with mobile phase and inject 2 replicates of each concentration.
2.2.3 Limit of detection (LOD)
It was defined as the lowest specified analyte concentration in the matrix that could be identified using the detection of the instrument. Furthermore, it should not be included in the accuracy, precision, and linearity ranges 9-11,31,32.
2.2.4 Limit of quantitation (LOQ)
It was defined as the lowest specified analyte concentration in the matrix that could be identified using the detection of the instrument. Furthermore, it must be included in the accuracy, precision, and linearity ranges 9-11,31,32.
LOD and LOQ could be calculated according to the slope and standard error data from the linearity of the calibration as the following:
LOD = 3.3 σ / S (2)
LOQ = 10 σ / S (3)
Where σ: is the standard error of X & Y arrays and S: represents the slope of the linearity calibration curve.
2.2.5 Accuracy and recovery
Both recovery and accuracy are used alternatively. The measurement's accuracy is defined as the proximity of the actual concentration (measured value) to the theoretical concentration (true value) 9-11,31,32.
Accuracy was implemented by the preparation of three different stock solutions of MP reference standard at 3.74, 5.49, and 6.64 mg in 100 mL mobile phase individually. Then 10 mL of each 45.7 mg, 64.8mg, 77.4mg/ 100mL WFI of MPSS working standard individually respectively and 1 mL of each phosphate buffer solution were mixed with MP concentrations. Then injected three replicates of each concentration were to make 70%, 100%, and 120% concentrations of total MP.
Accuracy % could be estimated using the linearity equation:
Accuracy%= Actual Conc.% / Theoretical Conc.% x 100 (4)
2.2.6 Repeatability and precision
Repeatability was conducted using six different determinations of the 100% test concentration by dissolving about a quantity of MP reference standard equivalent to 5 mg/100mL of mobile phase and mixing 10 mL of this solution with a weight of MPSS working standard equivalent to 65 mg and 1 mL of each phosphate buffer solutions in 100 mL volumetric flask and complete with mobile phase to obtained a concentration about 500 µg/mL of total MP 9-11,31,32.
2.2.7 Robustness
Robustness was submitted using designed small changes including slight changes in the temperature, composition of the mobile phase, etc.
The designed small changes were conducted in a different organic solvent ratio (Acetonitrile) at (± 1%) and a flow rate (± 0.005mL/min).
2.2.8 Ruggedness
Ruggedness was submitted using designed and major observable changes including analyst- analyst, column- column, and day- day with maintaining all of the analysis method parameters and conditions as it is without changes.
2.2.9 Specificity and selectivity
The following solutions were injected individually for selectivity confirmation:
- Phosphate buffer.
- Mobile phase.
- MP reference standard.
- MP + MPHS standard.
- MP reference standard + MPHS reference standard + Phosphate buffer.
- MPSS working standard.
- MP reference standard + MPSS working standard.
- MP reference standard + MPSS working standard + Phosphate buffer.
- Forced degradation studies were performed to indicate the stability indicating properties, selectivity, and specificity of the procedure using acid hydrolysis, and base H2O2 oxidation hydrolysis.
- Acid hydrolysis for MP was performed as a test under recovery test at 100% and in the final step add 10 mL of HCl [0.1 M], and it left for 30 min then complete with WFI to 100 mL.
- Base hydrolysis for MP was performed as a test under recovery test at 100% and in the final step add 10 mL of NaOH [0.1 M], and it left for 30 min then complete with WFI to 100 mL.
- H2O2 hydrolysis for MP was performed as a test under recovery test at 100% and in the final step add 10 mL of H2O2[3.0%], and it left for 30 min then complete with WFI to 100 mL.
2.3 Test of the validated method of the local market product of UP Pharma in Egypt
2.3.1 Methylprednisolone 1.0 g vials batch number (221160) after the constitution stability studies
The after-constitution stability study was conducted using the supplied solvent WFI at zero time, 24 hours in the refrigerator at a temperature of 5±3 ◦C.
The constituted vial was performed using 16 mL of the WFI then all of the content of the vial was transferred into a 200 mL volumetric flask. Then a dilution of 10 mL of the constituted solution (1mg/mL) in a 100 mL volumetric flask using WFI was conducted and introduced to the HPLC for assay in a final theoretical concentration (0.5mg/mL of MP).