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U. A. Deokate, A. M. Gorde*
Dept. Of Pharmaceutical Chemistry,
Government College of Pharmacy, Hotel Vedant Road, Osmanpura,
Aurangabad, Maharashtra, India 431005

This review discusses the regulatory aspects of forced degradation and methodology aspects for degradant investigations. It also focuses on the prediction of degradation products and pathways and development of stability indicating assay method. While reviewing the analytical perspectives various conventional and hyphenated techniques for degradant separation and characterization are described in detail.


PharmaTutor (ISSN: 2347 - 7881)

Volume 2, Issue 6

Received On: 07/04/2014; Accepted On: 15/04/2014; Published On: 01/06/2014

How to cite this article: UA Deokate, AM Gorde; Forced degradation and Stability Testing: Strategies and Analytical Perspectives; PharmaTutor; 2014; 2(6); 61-74

Chemical stability of pharmaceutical molecules is a matter of great concern as it affects the safety and efficacy of the drug product. Forced degradation studies provide data to support identification of possible degradants; degradation pathways and intrinsic stability of the drug molecule and validation of stability indicating analytical procedures. A draft guidance document suggests that results of one- time forced degradation studies should be included in Phase 3 INDs (Investigational New Drugs). NDA (New Drug Application) registration requires data of forced degradation studies as forced degradation products, degradation reaction kinetics, structure, mass balance, drug peak purity, etc. This forced degradation study provides information about degradation pathways of API, alone and in drug product, any possible polymorphic or enantiomeric substances and difference between drug related degradation and excipient interferences[1,2].

Controlling degradation related impurities involves identifying which of the potential degradation products found during forced degradation testing actually form in either the drug substance or product under long term or accelerated storage conditions and then selecting the appropriate counter measures to minimize the impurities or degradants. An impurity profiling study of forced degradation samples of drug substance illustrates the identification process and its potential impact on pharmaceutical development.

In the exercise of controlling impurities/degradants, their identification and characterization are the two key steps. These are required to be done when impurities/degradants are present at the prescribed stringent limits of 0.1%, or even lower for those genotoxic in nature. The conventional approach encompasses separation of impurities/degradants by a suitable method and their identification with the help of standard material. Alternatively, they are either enriched or isolated, followed by characterization through spectral analysis. The more modern concepts their characterization through the use of hyphenated tools. [3, 4]

Knowledge of the stability of molecule helps in selecting proper formulation and package as well as providing proper storage conditions and shelf life, which is essential for regulatory documentation. Forced degradation is a process that involves degradation of drug products and drug substances at conditions more severe than accelerated conditions and thus generates degradation products that can be studied to determine the stability of the molecule.[5]

1. Regulatory perspectives of forced degradation
A. From a regulatory perspective, forced degradation studies provide data to support the following:
• Identification of possible degradants
• Degradation pathways and intrinsic stability of the drug molecule
• Validation of stability indicating analytical procedures.

B. Issues addressed in regulatory guidances include:
• Forced degradation studies are typically carried out using one batch of material.
• Forced degradation conditions are more severe than accelerated stability testing such as
>50 °C; ≥75% relative humidity; in excess of ICH light conditions; high and low pH, oxidation, etc.
• Photostability should be an integral part of forced degradation study design.
• Degradation products that do not form in accelerated or long term stability may not have to be isolated or have their structure determined.
• Mass balance should be considered.

C. Issues not specifically addressed in regulatory guidance:
• Exact experimental conditions for forced degradation studies (temperatures, duration, and extent of degradation, etc.) are not specified.
• Experimental design is left to the applicant's discretion.[6, 7]

2. How much degradation is enough?
The question of how much stressing is enough has been the subject of much discussion amongst pharmaceutical scientists. In general, values anywhere between 5% to 20% degradation of the drug substance have been considered as reasonable and acceptable for validation of chromatographic assays.[8,9] However, for small pharmaceutical molecules for which acceptable stability limits of 90% of label claim is common, pharmaceutical scientists have agreed that approximately 10% degradation is optimal for use in analytical validation.[10] In the event that the experimental conditions generate little or no degradants due to the exceptional stability of the molecule, an evaluation should be made to verify if the drug substance has been exposed to energy in excess of the energy provided by accelerated storage (i.e., 40°C for 6 months). If the answer is yes, then the experiment can be stopped and a note of the stability of the drug substance can be made. Unduly overstressing the drug substance may produce aberrant results.[11]

3. Strategies for selection of forced degradation conditions [12]

Table 1. Strategies of selection of forced degradation conditions.

A.   Hydrolytic degradation:
Hydrolysis is a chemical process that includes decomposition of a chemical compound by reaction with water. Hydrolytic study under acidic and basic condition involves catalysis of ionizable functional groups present in the molecule. Acid or base stress testing involves forced degradation of a drug substance by exposure to acidic or basic conditions which generates primary degradants in desirable range. The selection of the type and concentrations of acid or base depends on the stability of the drug substance. Hydrochloric acid or sulfuric acids (0.1–1 M) for acid hydrolysis and sodium hydroxide or potassium hydroxide (0.1–1M) for base hydrolysis are suggested as suitable reagents for hydrolysis.[13, 14]

Hydrolysis of most of the drugs is dependent upon the relative concentration of hydronium and hydroxyl ions such as i) Anastrozole, significantly degraded in basic conditions as compared to acidic conditions and two new degradation products were formed under basic pH, ii)Doxofylline, a bronchodilator drug that show degradation more in acidic condition. Hence pH at which each drug is optimaly stable can be determined. [15, 16]

B. Photo degradation:
According to ICH Q1B guideline for photo degradation, samples should be exposed to light providing an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 watt hours/square meter with spectral distribution of 320-400nm to allow direct comparisons to be made between the drug substance and drug product. Samples may be exposed side-by-side with a validated chemical actinometric system to ensure the specified light exposure is obtained, or for the appropriate duration of time when conditions have been monitored using calibrated radiometers/lux meters.[17]

The photolytic degradation can occur through nonoxidative or oxidative photolytic reaction. The nonoxidative photolytic reactioninclude isomerization,dimerization, cyclization, rearrangements, decarboxylation and hemolytic cleavage of X-C hetero bonds, N-alkyl bond (dealkylation and deamination), SO2-C bonds etc and while oxidative photolytic reactionoccur through either singlet oxygen(1O2) or triplet oxygen(3O2) mechanism. The singlet oxygenreacts with the unsaturated bonds, such as alkenes, dienes, polynuclear aromatic hydrocarbon to form photoxidative degradation products whereas triplet oxygenreact with free radical of the drug molecule, which than react with a triplet oxygen molecule to form peroxide. Hence, light can also act as a catalyst to oxidation reactions.[18,19]

C. Oxidative degradation:
Many drug substances undergo autoxidation i.e., oxidation under normal storage condition and involving ground state elemental oxygen. Therefore it is an important degradation pathway of many drugs. Autoxidation is a free radical reaction that requires free radical initiator to begin the chain reaction. Hydrogen peroxide, metal ions, or trace level of impurities in a drug substance act as initiators for autoxidation. [20]

Selection of an oxidizing agent, its concentration, and conditions depends on the drug substance. It is reported that subjecting the solutions to 0.1–3% hydrogen peroxide at neutral pH and room temperature for seven days or upto a maximum 20%degradation could potentially generate relevant degradation products.[14]

The mechanism of oxidative degradation of drug substance involves an electron transfer mechanism to form reactive anions and cations. Amines, sulphides and phenols are susceptible to electron transfer oxidation to give N-oxides, hydroxylamine, sulphones and sulphoxide.[18]The functional group with labile hydrogen like benzylic, carbon, allylic carbon, and tertiary carbon or α – positions with respect to hetero atom is susceptible to oxidation to form hydroperoxides, hydroxide or ketone.[21,22]

D. Thermal degradation:
The thermal degradation studies carried out in dry or moist environment with temperature range of 40-700 C and moisture of 60-75%RH.[23] Thermal degradation study is carried out at 40°C to 80°C. The most widely accepted temperature is 70°C at low and high humidity for 1-2 months. High temperature (>80°C) may not produce predictive degradation pathway.[24] The use of high-temperatures in predictive degradation studies assumes that the drug molecule will follow the same pathway of decomposition at all temperatures.[19]

Effect of temperature on thermal degradation of a drug is studied through Arrhenius equation:
K= Ae-Ea/RT
Where k is specific reaction rate, A is frequency factor, Ea is energy of activation, R is gas constant (1.987 cal/deg mole) and T is absolute temperature. [20, 25]



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