Preparation of solution A
Accurately weighed 5 mg each MTF working standard, melamine impurity and cyanoguanidine impurity was transferred into a 50-mL volumetric flask. About 30 mL diluent was added, and the contents were sonicated to dissolve completely. The volume was made up to the mark with the diluent and sonicated for 5 min to mix, before use.
Preparation of solution B
Accurately weighed 4 mg of each TNG working standard, impurity A and impurity B was transferred all of them into a 100-mL volumetric flask. About 70 mL diluent was added and sonicated to dissolve the contents completely. The volume was made up to the mark with the diluent and sonicated for 5 min to mix, prior to use.
Further, 5 mL of solution A and solution B was transferred in a 100-mL volumetric flask and diluted with diluent up to the mark, mixed and used as a system suitability solution.
Preparation of standard solution 1
About 5 mg of MTF working standard was transferred into a 50-mL volumetric flask dissolved and diluted to volume with diluent (standard stock solution A). Accurately weighed 4 mg of TNG working standard was transferred into a 100-mL volumetric flask. About 70 mL of diluent was added and sonicated to dissolve completely. The volume was adjusted up to the mark with diluent and mixed properly (standard stock solution B).
Further 5 mL of each standard stock solutions A and B was transferred in a 100-mL volumetric flask. The volume was adjusted up to the mark with diluent and used as a standard solution 1. The standard solution 2 was similarly prepared for the calculation of similarity factor.
Preparation of standard solution 2
The standard solution 2 was prepared as per standard solution 1.
Ten tablets of IH combination product were crushed to a fine powder. Powder equivalent to 500 mg of MTF was accurately weighed and transferred into a 100-mL volumetric flask. About 70 mL of the diluent was added, and the contents were sonicated for 20 min. The volume was made up to the mark with the diluent and mixed well. The sample was filtered through 0.45 µm filter and used for HPLC analysis.
Placebo powder was weighed equivalent to sample weight and further processed as per sample preparation.
System suitability criteria
For development of a robust analytical method, we have defined the system suitability parameters like resolution, capacity factor, signal-to-noise ratio, theoretical plates, etc. Considering the long run time and solution stability issue, similarity factor was estimated by injecting the two standard solutions (standard solutions 1 and 2). The limit of similarity factor was set at 95–105%. The resolution between each impurity and principle analyte peak was set NLT 2, the theoretical plate limit was kept at NLT 2000, and tailing factor limit was set NMT 2.0 as per USP system suitability criteria.
All the known impurities eluted in the sample chromatogram were calculated against the respective principle analyte peak in the standard solution 1. The quantification of the unknown degradants peaks was done by degrading the individual API and specifying the relative retention time with respect to the principle analyte peak. The unknown impurities were calculated against the analyte having a low concentration (TNG) in the combination product.
The developed method was validated as per ICH Q2 guidelines. The developed HPLC method was validated in terms of the following parameters: system suitability, specificity (selectivity and forced degradation), sensitivity (LOD and LOQ), linearity, precision, accuracy, filter study, stability of analytical solutions and robustness studies.
Accuracy study was carried out by spiking all the known impurities as well as MTF and TNG working standard at LOQ level, 50%, 100% and 150% level of impurity limit in the placebo. Six preparations at LOQ level and three preparations each at 50%, 100% and 150% level were injected into the system. The % recovered amount was calculated for each known impurity, and principle analyte peak against the spiked amount was assessed. The limit of recovery was set at 80% to 120% at LOQ level and 85% to 115% at 50% to 150% level.
Separately prepared standard solutions 1 and 2 were injected into the HPLC system. The % similarity factor was calculated for standard solutions 1 and 2 and monitored as per system suitability criteria. The % similarity factor limit was set at 95–105% for MTF and TNG.
Six sample solutions were prepared as described in the method and injected into the HPLC system. The % RSD was calculated for all the known impurities, individual unspecified impurity and total impurities. The % RSD limit was set at NMT 15%.
Intermediate precision study was carried out by changing the analyst and HPLC system on different days. All the samples were prepared as per the method of analysis and injected into the HPLC system. The % RSD was calculated for all the known impurities, individual unspecified impurity and total impurities.
To prove the specificity of the developed method, the diluent as a blank solution, placebo solution, cyanoguanidine impurity solution, melamine impurity solution, TNG impurity A solution, TNG impurity B solution, MTF and TNG standard solutions were injected separately. The sample solution and impurity spiked solutions were prepared and injected into the HPLC system. The response of the individual analytes and peak purity was recorded by using the PDA (photodiode array) detector.
Forced degradation study
Forced degradation study of sample and placebo was carried out to prove the selectivity of the method and to evaluate the stability indicating nature of the method. Sample and placebo were exposed under relevant stress conditions like heat, acid, base and oxidation. These stressed samples were then analyzed by HPLC. The acid degradation was carried out by using 0.5 N hydrochloric acid for 2 h at 60 °C and alkali degradation by using 1 N sodium hydroxide for 5 h at room temperature. For thermal degradation, the sample and placebo were kept at 80 °C for 2 days. The oxidative stress study was carried out by using 0.3% hydrogen peroxide for 12 h. The photolytic degradation was carried out by exposing the drug sample for not less than 1.2 million lux h at 200 Wh/sq. meter near ultraviolet energy by using the Sun test apparatus (Atlas, Germany).
LOD and LOQ determination
Series of known impurity solutions were prepared over a range starting from 1 to 50% of the working concentration and injected in triplicates. The linearity graph was plotted for average area at each level against the concentration in PPM. The correlation coefficient, slope and intercept were determined for each known impurity.
Precision at LOQ Level
Six solutions were prepared by spiking the MTF and TNG working standard along with all the known impurities at LOQ level in the placebo. The % RSD of peak area and % results were calculated.
The samples containing all the known impurities along with MTF and TNG working standard solutions were prepared from 150% to LOQ level (150%, 120%, 100%, 80%, 50%, 20% and LOQ) and injected in triplicate. The linearity graph was plotted for average area of individual analyte against the concentration in µg/mL at each level. The correlation coefficient, slope and intercept were calculated.
The range of concentration of analyte and impurity solutions was determined by evaluating the area under the curve and concentration for linear response.
Stability of analytical solutions
The MTF and TNG working standard solution and the impurity spiked sample solution were prepared. The solutions were stored at room temperature and evaluated against the freshly prepared standard solution on day 0, day 1, day 2 and day 3. The % known impurity and total impurity were calculated for the stored samples at each time point and compared against results of freshly prepared sample.
Filter validation study
Filter validation study was conducted to prove the compatibility of the molecule and the impurities with the filter used during the sample and standard preparation. Sometimes the analyte may get adsorbed on the filter paper resulting in inconsistent results. The study was conducted by filtering the sample solution through filters like Nylon filter (MDI, India) and PVDF (Merck Millipore, Germany) filter having 0.45µ porosity and discarding the initial 2 mL, 3 mL and 5 mL of filtrate to optimize the sample filtration study for precise and accurate results.
This study was conducted by intentionally altering the critical method parameters like flow rate (± 0.1 mL), pH of mobile phase (± 0.2), change in column temperature (± 5 °C), change in wavelength (± 2 nm) and change in gradient composition. The diluent, standard solution, sample solution and impurity spiked sample solution were injected to check the robustness of analytical method.