**4. Stability-indicating method**

*Analytical Chemistry - Advancement, Perspectives and Applications*

mine system suitability of final method.

2.It is nothing but the part of method validation. The experience and information obtained at the time of method development, which is helpful to deter-

3.At every time when system is used for performing the assay there is necessary to use system suitability test. For longer period if it is continuously in use then

there is need to reevaluate the system suitability at proper intervals.

which can explain the system are working properly [2].

in methods again requires validation or method transfer treatment.

one of the key points of method development and final validation [3].

performance can obtain with these supportive methods.

4.The system suitability means criteria and parameters obtained collectively

The important aspects of pharmaceutical development program are analytical method development, validation method transfer but it is fact that they are less considered in sense of total contribution in development process, time and economy. At the time of drug development phases, all the analytical method related activities are interrelated. In early development stages they are related and occur one after another in coming phases of development. During drug development process the changes may require to be performed in current methods and these changes

If one's objective of method development and validation is achieved, then it can prove that the laboratory facilities are accurate and fit for further development process that is one can say optimized. Method validation is the "process of demonstrating that analytical procedures are suitable for their intended use." Both method validation and methods transfer have important share in drug development and further changes in methods. To generate supportive data during manufacturing and quality control, these methods provides a valuable data by comparing with specifications including all types stability study, Safety, characterization and drug

Method development is the simultaneous process as the gradual development of drug product continues. The system suitability parameters are set of tests to checks the proper working of the system. After performing robustness with proper statistical data collection one can set the criteria for final system suitability of the method. These methods focus on active pharmaceutical ingredient (API) behavior. As the knowledge about API and drug product goes on progress the analytical methods become more refined. The important aspects of analytical method are that should be robust, simple and meeting the regulatory guidelines. Various trial an error experiments are to be carried out to develop the method. The performance criterion's to be finalized before the final validation of method. Forced degradation study which is integral part of stability-indicating method and system suitability tests are

The International Conference for Harmonization (ICH) guidances are available that are related to the qualification of impurities in new drug substances that are produced by chemical synthesis. These impurities can be addressed in two perspectives that are chemistry aspects and safety aspects. The chemistry aspects explain the identification and classification of impurities, the various analytical procedures and setting of specifications. In the safety aspects explains the qualification of impurities which are not addressed in clinical trials. In this aspect the threshold limits are defined. This ICH guidance classifies impurities in three classes as Organic, Inorganic and Solvent. Each class of impurities should be properly reported, with all aspects, developed during synthesis, storage of the new drug product. The

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**3.3 Impurity profiling**

To ensure safety, efficacy and quality of drug product there are need of stability indicating methods. The Food and Drug Administration (FDA) defines the stability indicating method as a validated analytical procedure that accurate and precisely measure active ingredients (drug substance or drug product) free from process impurities, excipients and degradation products [6].

To obtain forced degraded samples for assessing selectivity of method, method development and method validation are three important steps of stability indicating methods.

#### **4.1 Importance of forced degradation**

As per guidance document available for stability indicating method does not contain any explanation about extent up to which the degradation should be carried out. There are no certain guidelines regarding stress degradation. Therefore, it is always necessary to keep all experimental conditions of degradation with more reality and deliberate degradation.

The main purpose of forced degradation is to obtain stability of drug. It should provide information about route of degradation and utility towards the stability indicating [6].

Forced degradation can be able to judge excipients or non-drug substances. It also provides information useful for structure elucidation of degradation products. Importantly it gives data related to thermal, hydrolysis, and oxidation and photo degradation behavior of drug substance and drug product. It is very important to know about chemical behavior of drug product and drug substance in formulation development, manufacturing and packaging. The data helps in quality improvement [7].

#### **4.2 Acceptance criteria for forced degradation**

There are vast discussions among the various scientists that what should be the limits of stress testing? Generally, values in between 5–20% are proper and acceptable for chromatographic assays. The acceptable stability limit for small molecules are 90% of label claim and generally employed by pharmaceutical scientist that is 10% degradation is optimum for use in analytical validation. There are some experiments in which very little or no degradants are obtained due to exceptional stability of molecule under study in such case accelerated storage 400 c for 6 months should be carried out. If positive result is obtained, then the stability of drug is noted. But overstressing the drug substance may produce false results [7].

According to the recent recommendations of Food and Drug administration (FDA) and ICH guidelines, the stability indicating property of analytical method can be obtained by carrying out forced degradation study. The pathway of decomposition from API, solution and formulation also be determined. The structural information, their characterization and isolation of major degradants are the important part of the new drug approval (NDA). The use of forced degradation study is primary to understand molecular chemistry of drugs, its stability indicating properties and its degradation products and their pathways. In most of the cases the Hydrolysis, Oxidation, Photolysis, Racemization, and Decarboxylation are the type of reactions that are responsible for decomposition of most the drugs. However, the regulatory guidelines do not define the procedures to carry out degradation study. Therefore, there are various approaches to carry out forced degradation study [8].

#### **4.3 Stress conditions**

In pharmaceutical industry thermal, hydrolysis, oxidation and photo degradation are generally employed. If one must serve the purpose of degradation, the expected degradation should be achieved. The optimum percentage of degradation should be obtained in all types of conditions or in minimum of one according to FDA guidelines. If no degradation is achieved, then in that case, all reports related degradation experiment carried out should be produced. It is important fact to obtain the degradation as per expected level. The degradation in between 5 and 20% is recommended [9].

### **4.4 Hydrolysis**

By using acid and base the hydrolysis studies are performed. Generally, a chemical reaction is carried out with water to obtain decomposed analyte. The wide pH range that is from 2 to 12 is used for acid and base, which is to be used for hydrolysis purpose. For acid hydrolysis generally Hydrochloric acid (HCl) or Sulfuric acid (H2SO4) is used and for base hydrolysis Sodium or Potassium hydroxide is used. According to stability of molecule, the concentration of acid or base is decided. One can used more than one stress conditions to obtain desired degradation. If the desired degradation is not achieved at room temperature, then higher temperature is used. After the degradation process completed the degraded samples are neutralized by same acid or base so as it can easily injected in HPLC column without any harm to silica stationary phase. For water insoluble samples alcoholic acid or base are used for obtaining degradation [9].

#### **4.5 Oxidation**

To carry out oxidation degradation of drug substance or drug product, generally hydrogen peroxide is used. Apart from hydrogen peroxide metal ions, oxygen and radical initiators can also be employed. The oxidizing agent, its quantity requirement, properties are depending on the drug substance under study. If hydrogen peroxide is used as a degradant, in that case combination of stress should not be employed. If elevated temperature is used in case of Hydrogen Peroxide, it leads to hydrolysis instead of oxidation because in Hydrogen Peroxide O-O are not stable and they may decompose at ambient temperature also. Due to heating, these bonds break faster and oxidation occurs. Sodium metabisulfite solution is used for neutralization of oxidation degraded samples [9].

#### **4.6 Heat**

Active pharmaceutical ingredients, Dosage form with or without humidity can be undergoing thermal degradation. The sample is exposed to heat. (weather there is

**157**

**Figure 2.**

*Analytical, Bioanalytical, Stability-Indicating Methods: Key Part of Regulatory Submissions*

humidity or absence of humidity as mentioned earlier) In case of liquid, humidity is completely avoided. While applying stress to liquid samples especially for injections, oral solutions, and syrups as further diluting the samples, the precaution should be taken because these types of samples may loss water and concentration of actual sample. By obtaining multiple time results, the detail information about primary and secondary degradation can collected. If any molecule is so stable that it cannot generate degradation, in such situation the energy analogous to the accelerated stress condition is to be applied to express efforts taken for obtaining degradation [9].

The exposure to light is one of the important degradation steps to obtain degradation caused by light. This degradation is evaluated by obtaining any unacceptable change due to light. The recommendations related to photo stability are described in ICH guidelines Q1B.The UV–VIS light exposure with not less than 1.2 million hours to achieve degradation of sample. The samples are preferably exposed to cool white fluorescent light and near ultraviolet lamp. The natural light can be used, if specific instrument is not available, but there will be intensity problem as it is varying with time, weather conditions, pollution etc. due to which natural light becomes not

After generating forced degradation samples obtained by accelerated stress conditions, their evaluation is the important task. For evaluation purpose, each sample should be studied individually. The Chromatographic techniques including High performance liquid chromatography (HPLC), Ultra-performance liquid chromatography (UPLC), UHPLC and Capillary Electrophoresis are commonly used techniques for this important task. The most important work is development and validation of stability-indicating method which can be able to separate every degradation product from each other and from drug. Therefore, peak purity is important in sense of selectivity determinations of the method. One more important parameter is sensitivity, which can be helpful to asses' impurities at lower level. There are chances that the impurity peak may get depressed at the time of method development which is co-eluted. Many times, it may happen that two unknown impurity get merged due to which false results are appear for stability, therefore it is important to implement such analytical method that can have capacity to resolve each unknown impurity and that is helpful to control out of specification results [9] (**Figure 2**).

*Important parameters and acceptance criteria for impurity profiling and stability indicating methods.*

*DOI: http://dx.doi.org/10.5772/intechopen.93566*

**4.7 Photo stability**

suitable for degradation [9].

**4.8 Evaluation of results**

*Analytical, Bioanalytical, Stability-Indicating Methods: Key Part of Regulatory Submissions DOI: http://dx.doi.org/10.5772/intechopen.93566*

humidity or absence of humidity as mentioned earlier) In case of liquid, humidity is completely avoided. While applying stress to liquid samples especially for injections, oral solutions, and syrups as further diluting the samples, the precaution should be taken because these types of samples may loss water and concentration of actual sample. By obtaining multiple time results, the detail information about primary and secondary degradation can collected. If any molecule is so stable that it cannot generate degradation, in such situation the energy analogous to the accelerated stress condition is to be applied to express efforts taken for obtaining degradation [9].

#### **4.7 Photo stability**

*Analytical Chemistry - Advancement, Perspectives and Applications*

**4.3 Stress conditions**

is recommended [9].

are used for obtaining degradation [9].

tralization of oxidation degraded samples [9].

**4.4 Hydrolysis**

**4.5 Oxidation**

of decomposition from API, solution and formulation also be determined. The structural information, their characterization and isolation of major degradants are the important part of the new drug approval (NDA). The use of forced degradation study is primary to understand molecular chemistry of drugs, its stability indicating properties and its degradation products and their pathways. In most of the cases the Hydrolysis, Oxidation, Photolysis, Racemization, and Decarboxylation are the type of reactions that are responsible for decomposition of most the drugs. However, the regulatory guidelines do not define the procedures to carry out degradation study. Therefore, there are various approaches to carry out forced degradation study [8].

In pharmaceutical industry thermal, hydrolysis, oxidation and photo degradation are generally employed. If one must serve the purpose of degradation, the expected degradation should be achieved. The optimum percentage of degradation should be obtained in all types of conditions or in minimum of one according to FDA guidelines. If no degradation is achieved, then in that case, all reports related degradation experiment carried out should be produced. It is important fact to obtain the degradation as per expected level. The degradation in between 5 and 20%

By using acid and base the hydrolysis studies are performed. Generally, a chemical reaction is carried out with water to obtain decomposed analyte. The wide pH range that is from 2 to 12 is used for acid and base, which is to be used for hydrolysis purpose. For acid hydrolysis generally Hydrochloric acid (HCl) or Sulfuric acid (H2SO4) is used and for base hydrolysis Sodium or Potassium hydroxide is used. According to stability of molecule, the concentration of acid or base is decided. One can used more than one stress conditions to obtain desired degradation. If the desired degradation is not achieved at room temperature, then higher temperature is used. After the degradation process completed the degraded samples are neutralized by same acid or base so as it can easily injected in HPLC column without any harm to silica stationary phase. For water insoluble samples alcoholic acid or base

To carry out oxidation degradation of drug substance or drug product, generally hydrogen peroxide is used. Apart from hydrogen peroxide metal ions, oxygen and radical initiators can also be employed. The oxidizing agent, its quantity requirement, properties are depending on the drug substance under study. If hydrogen peroxide is used as a degradant, in that case combination of stress should not be employed. If elevated temperature is used in case of Hydrogen Peroxide, it leads to hydrolysis instead of oxidation because in Hydrogen Peroxide O-O are not stable and they may decompose at ambient temperature also. Due to heating, these bonds break faster and oxidation occurs. Sodium metabisulfite solution is used for neu-

Active pharmaceutical ingredients, Dosage form with or without humidity can be undergoing thermal degradation. The sample is exposed to heat. (weather there is

**156**

**4.6 Heat**

The exposure to light is one of the important degradation steps to obtain degradation caused by light. This degradation is evaluated by obtaining any unacceptable change due to light. The recommendations related to photo stability are described in ICH guidelines Q1B.The UV–VIS light exposure with not less than 1.2 million hours to achieve degradation of sample. The samples are preferably exposed to cool white fluorescent light and near ultraviolet lamp. The natural light can be used, if specific instrument is not available, but there will be intensity problem as it is varying with time, weather conditions, pollution etc. due to which natural light becomes not suitable for degradation [9].

#### **4.8 Evaluation of results**

After generating forced degradation samples obtained by accelerated stress conditions, their evaluation is the important task. For evaluation purpose, each sample should be studied individually. The Chromatographic techniques including High performance liquid chromatography (HPLC), Ultra-performance liquid chromatography (UPLC), UHPLC and Capillary Electrophoresis are commonly used techniques for this important task. The most important work is development and validation of stability-indicating method which can be able to separate every degradation product from each other and from drug. Therefore, peak purity is important in sense of selectivity determinations of the method. One more important parameter is sensitivity, which can be helpful to asses' impurities at lower level. There are chances that the impurity peak may get depressed at the time of method development which is co-eluted. Many times, it may happen that two unknown impurity get merged due to which false results are appear for stability, therefore it is important to implement such analytical method that can have capacity to resolve each unknown impurity and that is helpful to control out of specification results [9] (**Figure 2**).

**Figure 2.**

*Important parameters and acceptance criteria for impurity profiling and stability indicating methods.*
