The identification of degradation products can provide an understanding of impurity formation and define degradation mechanisms. If the identification process is performed at an early stage of drug development, there is adequate time for improvements in the drug substance process and drug product formulation to prevent these impurities and degradants.
Impurity and degradant structure elucidation is a collaborative effort involving the analytical chemist, process chemist, and/or formulator as well as the degradation, mass spectrometry, and NMR experts.
It is imperative that all those who are familiar with the project of interest are involved. The group meets to assess the timelines for completion and to gather all pertinent information. A few questions that need to be answered at this early stage are:
- Is this an impurity or degradant problem?
- At what level is the impurity/degradant present?
- Is it a process-related impurity, and, if so, at what step of the process is it formed?
- Is it a degradant, and, if so, under what degradation condition is it formed?
- Are enriched samples with the unknown impurity/degradant available?
By gathering all relevant information, the most efficient method of isolation and identification can be selected.
The first step of the process is to determine the level of the unknown compound. According to the ICH Guidelines on Impurities in New Drug Substances or New Drug Products. Identification of impurities below the 0.1% level is generally not considered to be necessary unless the potential impurities are expected to be unusually potent or toxic.
Therefore, it is imperative to determine the level of the unknown impurity and/or degradant early in the process. If the level is below the 0.1% threshold, then the project team members will need to discuss whether isolation and identification are necessary. However, if the level is at or above the 0.1% limit, then effort should be put forth to identify it.
Once a decision has been made to identify an unknown compound, the next logical step is to evaluate all known process-related impurities, precursors, intermediates, and degradation products. By observing the relative retention times (HPLC) of all known process-related impurities, precursors, and intermediates (if available), one can quickly determine whether or not the impurity of interest is truly unknown.
If the relative retention time of the unknown impurity matches that of a standard, then it can be identified using HPLC (with UV photodiode array detection) and LC–MS. The identity is confirmed by correlating the retention time, ultraviolet spectra, and mass spectra of the unknown impurity with those of the standard.
Identifying an unknown impurity by using a standard, as described in the above paragraph, is a quick and easy process. What happens when the relative retention time does not match that of a standard? The next step is to obtain molecular mass and fragmentation data via HPLC–MS. It is essential to determine the molecular mass of the unknown impurity. Not only does the molecular mass help in the identification of the unknown impurity, but it also enables one to track the correct peak by HPLC if isolation becomes necessary. To run LC–MS, a mass spectrometry-compatible HPLC method must be available.
A number of methods can be used for isolating impurities and/or degradants. Three of the most utilized techniques are TLC, flash chromatography (column chromatography), and preparative HPLC. The actual technique used depends upon the nature of the impurity and/or degradant, including the amount present in the original material from which it must be isolated.