Analytical method development finalization for Drug Substance or Drug Product is dependent on finalization of Forced degradation studies or Stress studies as it indicates a clear scenario of potential degradant impurities which may arise during the course of study and we can be prepared for further preparation, isolation and characterization of Impurities for Regulatory Requirement.
To investigate possible degradation products, arising due to degradation mechanisms carried out on either drug substance or drug product which further helps to establish the degradation pathways and the intrinsic stability of the drug molecule.
To provide a foundation for developing suitable stability indicating method
Forced degradation studies should be carried out on drug substances, drug products and placebo.
Stress study shows difference in degradation products arising either from drug substance, drug product or placebo.
Completion of study shows discrimination between; Synthetic process impurities, excipients, degradation products derived from excipients, product and due to drug excipient interaction.
The degradation products generated during stress study are termed as “potential” degradation products that may or may not be formed under relevant storage conditions.
Study should be carried out using Photo-diode array detector or suitable mass compatible method to gain information on potential degradant arising from stress study and for further preparation and qualification of relevant impurities.
Analyte having melting point less than 150°C, stress study should be carried out at 70°C or about 40°C below the melting point.
Analyte having melting point greater than 150°C, stress study should be carried out at 105°C.
Stress study should be done at relevant temperature conditions and exposure time can be varied to achieve degradation between 5 to 20%.
If no degradation is achieved even after harsher stress, justification can be provided that the molecule is stable.
This involves treatment of analyte using water.
Hydrolytic forced degradation studies involve treatment with dilute acid and alkali at different exposure time to achieve target degradation
Estimate solubility of analyte in water as hydrolytic stress studies are to be conducted in aqueous solutions. If drug is hydrophobic & found to be insoluble in water, use a co-solvent to dissolve the required quantity.
Commonly used co-solvents are Acetonitrile and Methanol.
# Methanol has the potential of participating in the degradation mechanism; it should be used with caution especially under acidic conditions when the compound being tested contains a carboxylic acid, ester or amide.
# Acetonitrile is not completely inert and can participate in the degradation reactions. For such as it Acetonitrile can contribute to base to catalyse oxidation reactions in the presence of peroxides. Acetonitrile will also degrade in presence of base (at pH 13)and /or acid (at pH1) under elevated temperatures to detectable levels of Acetamide or Acetic acid which can show up as early eluting peaks in RP-HPLC when monitored at lower wavelengths. The size of the HPLC peaks from these two products is relatively small and use of stressed blank solutions permits ready identification of these peaks.
# Hydrolytic reflux degradation study should be performed in fume hood at a temperature of about 70°C using a reflux condenser with few glass beads or porcelain pieces to avoid bumping and further loss due to evaporation.
# Typical conditions are, Reflux using water/ 0.1M HCl/ 0.1M NaOH for stress testing with or without co-solvent at a temperature of about 70°C.Reflux for about 12 hours or until about 5 to 20% degradation is achieved or whichever is earlier. Neutralize the stressed solutions before injection. Prepare a stressed solution at a higher concentration than that of test concentration. After the stress, dilute with diluent to achieve required test concentration, so that peak shapes are good.
# Evaluate the peak purity of the Analyte peak and the major degradant peaks having peak height less than 1000 m AU.
If more than 1000 mAU, solutions should be diluted to check for Peak Purity as per the Software being used.
If any of the major degradant is not passing peak purity, modify the stress study conditions and check that the peak purity is passing.
# For GC methods and methods where RI, ELSD and FLD detectors are used, peak homogeneity needs to be established by doing Mass spectral study and evaluate the peak purity of the Analyte peak and the major degradant. Ensure that the method is Mass compatible.
# If any known impurity is observed to be increasing in stress study, check if it is process impurity or a possible degradant. In case a known compound is a process impurity and the peak is found to be increasing in forced degradation, it indicates that degradant peak is eluting at the same RT as that of the process impurity or there could be a secondary pathway of formation of process impurity via some other degradation route
# Based on the structure of Analyte, check for possible formation of degradant
# If any of the Peaks are found to be not separating, Optimise the separation using the forced degradation samples
# Degradation products are not eluted from the HPLC column or are not detected by detector used for study
# Loss in degradation products or analyte due to extraction from sample placebo, due to poor solubility, volatility or adsorption losses
# Co-elution of degradation products or impurity with parent Analyte peak
# Issues in integration of peaks due to poor chromatography
# In-accurate quantification due to differences in response factors
After separation of all impurities from Analyte peak check overlaid spectra and finalize the wavelength
Perform the robustness of the method for the following parameters;
Mobile phase composition (organic component) change by (±10%),
pH of mobile phase change by (± 0.2) units
Gradient composition change (± 0.2 % per min)
Verify in different brand of HPLC’s columns
Flow rate (± 0.2 ml/min)
Temperature (± 5°C)
Fine-tune the method in the range where it is most robust.
In case any parameter is sensitive, specify the same in the test method so that it will be monitored.