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
- Samples used for the study:
Forced degradation studies should be carried out on drug substances, products, and placebo.
- Information explored from forced degradation studies:
Stress study shows differences in degradation products arising either from drug substance, drug product, or placebo.
Completion of the study shows discrimination between; Synthetic process impurities, excipients, degradation products derived from excipients, products, 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.
- Major forced degradation studies: Thermal Degradation, Hydrolytic degradation, Oxidative degradation, Photolytic degradation.
The study should be carried out using a Photo-diode array detector or a suitable mass-compatible method to gain information on potential degradant arising from stress study and for further preparation and qualification of relevant impurities.
- Thermal degradation:
To analyze having a melting point of less than 150°C, a 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, a stress study should be carried out at 105°C.
A 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.
- Hydrolytic degradation:
This involves the treatment of the analyte using water.
Hydrolytic forced degradation studies to involve treatment with dilute acid and alkali at different exposure times to achieve target degradation
Estimate solubility of analyte in water as hydrolytic stress studies are to be conducted in aqueous solutions. If the 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 Acetonitrile can contribute to the base to catalyze oxidation reactions in the presence of peroxides. Acetonitrile will also degrade in presence of a 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 the use of stressed blank solutions permits ready identification of these peaks.
A hydrolytic reflux degradation study should be performed in a 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 the test concentration. After the stress, dilute with diluent to achieve the required test concentration, so that peak shapes are good.
- Humidity-treated samples: Stress the samples to 90% Humidity for 1 week to gain information on the influence of humidity on the drug.
- Oxidative degradation: Stress with 3% hydrogen peroxide in dark at room temperature for 24 hours or until about 5 to 20% degradation is achieved
- Photolytic degradation: Degradation resulting from exposure to UV or visible light is termed Photolytic degradation. Samples should be exposed to 1.2 million lux-hr visible and200 W-hr/m2 UV.
- Multi-component Stress Study:
Stress testing of placebo with each active separately shall be performed in order to know the information about which degradant is from which active. Alternatively, these can be identified by the UV spectra. Stress testing of placebo with other actives excluding the one at a time shall be performed, in order to know the non-interference from each other. Alternatively, these can be identified by Peak purity. If methods are different, the placebo shall include the other actives.
- Evaluation of results:
Evaluate the peak purity of the Analyte peak and the major degradant peaks having peak heights 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 significant 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 a Mass spectral study and evaluating 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 the stress study, check if it is a 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 the 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 the possible formation of degradant
If any of the Peaks are found to be not separating, Optimise the separation using the forced degradation samples
- Mass balance study:
The process of adding together the assay value and levels of degradation products to see how closely these add up to 100% of the initial value. Estimate the assay of the final force degradation samples and assess the mass balance. Mass balance is to be achieved at least up to 95% level. If the mass balance is less than the required criteria investigation is to be done and justified.
- Reasons for not achieving the mass balance:
- Degradation products are not eluted from the HPLC column or are not detected by the detector used for the 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 the integration of cliffs due to poor chromatography
- In-accurate quantification due to differences in response factors
- Finalization of detector wavelengths:
After separation of all impurities from the Analyte, peak check overlaid spectra and finalize the wavelength
- Finalization of Chromatographic conditions & System suitability:
- Perform the robustness of the method for the following parameters;
- Mobile phase composition (organic component) changes by (±10%),
- the pH of the mobile phase changes by (± 0.2) units
- Gradient composition change (± 0.2 % per min)
- Verify in a 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.