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The Role of Separation Science in Forced Degradation Studies of Pharmaceuticals

by Sean McCrossen, IEXA100 Consulting Ltd, UK.

The stability of a new pharmaceutical is established as part of its development. To determine suitable analytical methods that monitor drug stability forced degradation (stressing) studies are performed on drug substance and drug product. Stressing experiments provide important information that helps support formulation activities, establish degradation pathways, identify storage conditions and assist in shelf-life setting.

Separation techniques are crucial in studying degradation behaviour of new chemical entities and revisiting stability methods of marketed drugs. The sensitivity and selectivity of chromatography techniques are used to the full in supporting forced degradation. With the advance of MS capability its use to support the development of stability-indicating methods is booming. This short overview highlights how chromatography is used in degradation studies and stability testing for small molecules.

Early development phase
With the well-known high attrition rate of drug candidates in drug development the prevailing aim of the business is to provide minimal, but fit-for-purpose, data early in development; i.e., preclinical to Phase II clinical trials, before the more rigorous demands of ICH stability [1,2].
The primary motivation for initial forced degradation studies is to ensure the analytical impurities and assay methods are stability-indicating; i.e., that any potential degradants are chromatographically resolved and thus the methods are suitable to monitor long-term stability studies on drug substance and drug product.

   Forced degradation samples are generated by acid and base hydrolysis, thermolysis, photolysis and oxidation of the drug substance/drug product (Table 1). These experiments typically produce crude and complex samples.

   The harshness and duration of degradation is adjusted during stress testing to give an appropriate (and arbitrary) level of degradation, e.g., 10%, and is a compromise between generating significant impurities, from expected degradation routes, whilst minimizing the chance of degrading primary degradants to secondary ones. Some drugs are inherently more stable than others and the use of too severe forcing conditions needs to be avoided. The good practice of combining analytical and chemistry knowledge of the molecule at this stage helps the rational choice of stressing conditions.

   HPLC reversed-phase (RP) is the workhorse analytical technique used in examining forced degradation samples; e.g., [3,4]. The samples are typically studied as part of the method development process (screen) and assessed with process impurities derived from drug substance [5]. Many pharma companies have developed automated method development screens and some platforms are capable of performing the stressing studies automatically [6,7]. Most degradation studies employ UV detection by first intent; however, degradants with low molecular weight and weak chromophores can be hard to detect. Most UV work is diode array-based to facilitate method development and peak purity measurements [3,4]. Where UV is precluded some experimenters have used ELSD [8] and CAD [9] as alternative detectors for stability methods. The final stability-indicating method should be specific, sensitive with a low quantitation limit and be validated. Not surprisingly, with the advent of ultra-high pressure LC its utility in degradation studies and stability methods is increasing [10,11]. By comparison with RP, there are relatively few examples of normal phase HPLC used in degradation studies and stability methods. The determination of chiral and achiral compounds has been achieved in a normal phase system [12]. Potential stereochemical changes to a molecule; e.g., racemization of enantiomers, caused by drug degradation need to be monitored with chiral LC methods [13].

   GC is used in studying stressing samples and developing stability-indicating methods for weakly chromophoric compounds [14]. Volatile degradants are amenable to GC analysis and often in combination with MS for structural identification [15,16]. GC still plays an important role in some pharmacopoeial monographs for measuring degradant impurities.

   Ideally, MS-compatible methods are sought to profile forced degradation samples for LC method development and validation activities in LC work. MS provides valuable information of co-eluting components to support DAD work. Of particular concern is the peak purity of the main component and to assist the demonstration of a method’s specificity. Analysis of
LC/MS data and the ion profiling of the significant masses observed provides a good degree of confidence that co-eluting species are absent; however, isomers cannot be distinguished unless unambiguous fragmentation occurs.

   The control of trace level genotoxic impurities is a major concern in the quality of pharmaceuticals so it is important to consider whether any genotoxic degradants are likely to be formed on stability. Whilst any of the forced degradation pathways can give rise to potentially genotoxic degradants, it is photolysis and oxidation that provide the most frequent source of impurities with genotoxic structural alerts. LC/MS is a key investigative tool to determine such impurities and genotoxic liability.

Late development phase
To fulfil regulatory requirements in later clinical development (Phase III) detailed degradation studies are required by regulatory authorities. These studies confirm drug degradation pathways and involve formal identification of degradants. Whilst most degradants can be at least tentatively assigned by accurate mass data they will generally need isolation to provide samples for unambiguous structure assignment. Formal identification of degradation products is a resource intensive activity. To meet the demand for compound isolation solid phase extraction procedures are gaining prominence. General preparative (and semi-prep) HPLC are used routinely to isolate sufficient quantities of degradants for structural analysis [17,18] and MS has afforded mass-directed purification possibilities. More advanced hyphenated technologies; e.g., LC/SPE/NMR, have been used to isolate and identify drug degradants [16].

   LC/MS is the key analytical tool in structural elucidation. There are many examples in the scientific literature using single stage and tandem MS to identify degradants and pathways [19,20]. Particularly so for generic drugs where improved analytical capability has provided more thorough degradation pathway data, a means of identifying new degradants and elucidating new mechanisms [21,22].

   GC/MS is used in a similar fashion to LC/MS; however, its use is not so widespread. An example of its utility has been the identification of unknown degradants in the analysis of anthelmintic drugs [23].

   Mass-balance studies are an important aspect of degradation work - the loss of degraded drug has to be accounted for in terms of weight per cent degradation products [24]. MS detection is useful in determining non-chromophoric analytes that are missed in HPLC-UV analysis though MS is not typically used quantitatively. One of the major difficulties with degradation studies is ensuring that all major degradants are detected within the chromatogram(s) so orthogonal approaches are used such as HILIC (cf. RP). LC and GC are the key techniques in mass-balance work; however, size-exclusion chromatography has been used to assist in mass-balance studies [25] and other chromatographic techniques e.g. TLC, are useful in troubleshooting.

Forced degradation studies are a vital component of drug development. The major chromatography techniques play an essential role in supporting these studies with RP-HPLC and GC dominant. The prominence of structural elucidation techniques (MS and NMR) combined with LC is increasing in degradation work. We see MS playing an indispensable role in degradation studies; its resolving power and sensitivity are exploited through an array of activities like peak purity determination, genotoxin risk assessment, degradant characterisation, establishment of degradation pathways and degradant isolation.

1. ICH Q1A(R2) Feb 2003 Stability testing of new drug substance and products
2. ICH Q1B Nov 1996, Stability testing: Photostability testing of new drug substances and products
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Sean McCrossen is part of Iexa100 working in pharmaceutical analysis and separation science. He specialises in the application of chromatography in R&D drug development and the manufacturing environment within pharma. His current focus is trace analysis for genotoxic impurities and stability testing.