Analytical quality by design [AQbD] and CCD help in regulatory compliance for RP-HPLC method development, stress testing or stability indicating methods^10-15.

2. OBJECTIVE OF STABILITY STUDIES¹⁶:

1. To establish shelf life and storage conditions of API and products.

2. To provide evidence on how much quality of API varies with time under influence of various environmental factors such as temperature, humidity, and light.

3. To study chemical properties of molecules and to establish degradation pathways of drug substances and drug products.

4. To generate stable formulations by solving stability relating problems.

5. Forced degradation conditions, stress agent concentration and time of stress are to be established in such a way that they effect degradation, preferably 5–30% of the parent constituent

3. SHELF LIFE OF DRUG AND ITS IMPORTANCE¹⁷:

Prescription and over the counter (OTC) medicines are labelled according to the product's ability to stay functional under normal storage circumstances before the specified date on a prescription or OTC medicine label. The effectiveness of a medication when administered as a tablet, capsule, syrup, or injection depends on its pharmacological qualities, such as dissolving, disintegration, hardness, etc.

4. LIMITS FOR DEGRADATION^18-21

Drug ingredient degradation between 5%-30% has been acknowledged as appropriate for the purpose of validating chromatographic tests. Some pharmaceutical professionals believe 10% degradation is ideal. If there is no degradation after the drug substance or drug product has been subjected to stress conditions other than those listed in an accelerated stability protocol, the study may be greater exaggerated to higher stress condition or greater time.

Figure 2. Flow diagram of acceptable criteria for degradation

5. SCOPE AND REGULATORY STATUS OF STABILITY-INDICATING METHODS^22-25:

These are the following regulatory guideline for stability indicating methods:

Table 1: Regulatory guidelines

1. International Stability Guidelines

2. ICH Guidelines

3. FDA (Food Drug Administration)

4. WHO (World Health Organization)

5. ASEAN Guidelines

6. US Pharmacopoeia

The ICH guidelines are put into practice by three countries: Japan, Europe, and the United States, while the WHO guidelines are related to the development of products worldwide.

FDA published a final new guidance document, published in draft form since 1998, replacing the first guidance issued in 1987. The new guidance incorporates the requirements for stability testing established by ICH. None of the ICH guidelines provides exact definition of a stability-indicating method. In 1937, ethylene glycol was used as a vehicle for an elixir of sulfanilamide, which caused more than 100 deaths. Thereafter the Food, Drug and Cosmetic Act was revised requiring advance proof of safety and various other controls for a new drug.

Table 2: ICH guidelines for SIAMS

Type	Clause
1. Stability testing of new substance and products	Q1A
2. Photostability testing of new substance and products	Q1B
3. Text on validation of analytical procedure	Q2A
4. Extension of ICH text ‘‘validation of analytical procedure’’	Q2B
5. Impurity in drug substances	Q3A
6. Impurity in drug products	Q3B
7. Residual solvents	Q3C
8. Specification, test procedure and acceptance criteria for new drug substances and new drug products: chemical substances	Q6A

6. STEPS INVOLDED IN DEVELOPMENT OF STABILITY INDICATING ASSAY METHODS^26-33

Ø Following steps are involved in stability-indicating assay method development

6.1 Critical study of the drug structure to assess the likely decomposition routes.

6.2 Collection of information on physicochemical properties.

6.3 forced degradation studies

6.4 Preliminary separation studies on stressed samples

6.5 Final method development and optimization

6.6 Identification and characterization of degradation products and preparation of standards

6.7 Validation of SIAMs

6.1 Critical study of the drug structure to assess the likely decomposition routes:

Information on degradation can be easily gained from the structure by the study of functional groups and other key components.

Figure 3: Functional group and their degradation

6.2 Collection of information on physicochemical properties:

Various physicochemical parameters such as p ka, log P, Solubility, absorptivity and absorption maxima of drug are to be studied.

IMPORTANCE OF VARIOUS PARAMETERS:

· pKa - pH related changes in retention occur at pH values within 1.5 units of pka values. Ionization values - helps in selecting pH of buffer to be used in mobile phase.

· Log P - identification of log p values for drug and degraded products provides much information about the separation behavior likely to be obtained in particular stationary phase.

· Solubility data - if solubility profile of drug in aqueous and organic solvents is known it will be useful in selection of sample solvents and mobile phase in HPLC.

· Absorption maxima - HPLC and UV detector is widely used absorption maxima should be known due to extinction of drug and degradation products in different solvents.

6.3 Forced Degradation Studies:

Figure 4: Force degradation study

The ICH guideline Q1A on Stability Testing of New Drug Substances and Products gives indications for the testing of parameters which may be susceptible to change during long storage. It is mentioned that forced degradation studies or stress testing at temperatures in 10 °C increments above the accelerated temperatures, extremes pH and under oxidative and photolytic conditions have to be carried out on the drug substance.

6.6 Identification and characterization of degradation products and preparation of standards:

The drug degradation products were identified and arranged for their standards. By this specificity /selectivity of the method can be established. Generally, to identify the formed products, they are isolated and then their structure was determined through spectral and elemental analysis, but this approach was tedious and time consuming when the degradants are produced.

Modern approach was using hyphenated LC techniques coupled with MS. This also includes instrumental approach such as HPLC, UV detection, MS and tandem mass spectroscopy. If the products identity was already established through sophisticated LCMS or LC-NMR studies, the molecules can be synthesized, characterized and the presence can be confirmed through spiking in degraded samples.

6.7 Validation of SIAMs³⁴:

Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for its intended use. All analytical methods that are intended to be used for routine analysis will need to be validated.

Table 5: Validation Parameter and acceptance limit

Characteristics	Proposed use acceptance criteria
Linearity	r² ≥ 0.99, similar response ratios
Precision system	RSD < 2%
Precision Method	RSD < 2%
Repeatability/ Reproducibility	RSD ≤ 2%
Accuracy	FDA 98-102 %, EPA 50-150 %
Specificity	No interference
Detection Limit	>2 times base line
Quantitative Limit	Signal to Noise ratio = 10:1
Range	Concentration where data can be reliably detected

7. DEGRADATION PREDICTION TOOLS³⁵:

Degradation prediction tools are computer-based programs that can predict the potential degradation pathways of a drug substance or drug product based on its chemical structure and environmental conditions. These tools can be used to identify the factors that may contribute to degradation, such as pH, temperature, and light exposure, and to determine the likely degradation products that may form. They can also be used to design stability-indicating methods and to generate stability profiles for drug substances and drug products. Some common degradation prediction tools include ChemYO, ADMET Predictor, and PASS Prediction. These tools can significantly reduce the time and cost associated with stability testing, as well as provide valuable insights into the stability of a drug and how to improve it. However, it is important to validate the predictions made by these tools through experimental testing.

ChemYO, ADMET Predictor, and other computer-based software programs are used for degradation prediction in pharmaceuticals.

1. ChemYO: ChemYO is a computational chemistry software program that can be used to predict the stability and degradation of drug substances and drug products. It can be used to simulate the impact of various environmental conditions, such as pH, temperature, and light exposure, on the stability of a drug.

2. ADMET Predictor: ADMET Predictor is a software program that uses pharmacokinetic and pharmacodynamic data to predict the in vitro and in vivo degradation of drugs. It can also be used to determine the factors that may contribute to degradation, such as pH and temperature, and to predict the likely degradation products that may form.

3. ChemDraw: This is a chemical drawing software that can be used to predict the potential degradation pathways of drugs based on their chemical structure.

4. ACD/Labs: This is a suite of software tools that includes prediction tools for degradation, solubility, and stability.

5. StabPharm: This is a tool that predicts the stability of drugs based on their chemical structure and the conditions they are exposed to.

6. M-Box: This is a tool that predicts the stability of drugs based on the molecular mechanics calculations.

7. SciQSAR: This is a tool that predicts the stability of drugs based on their molecular structure and the environmental conditions they are exposed to.

8. DREAM: This is a tool that predicts the stability of drugs based on the molecular dynamic simulations.

These tools can provide valuable insights into the stability of a drug and help to design and optimize stability testing programs. However, it is important to validate the predictions made by these tools through experimental testing.

8. CO-SOLVENT³⁶:

Cosolvents can play a role in forced degradation studies by modifying the chemical, physical, or biological properties of a drug substance or product and potentially accelerating its degradation. In forced degradation studies, cosolvents can be added to the drug sample to simulate real-life scenarios, such as exposure to high humidity, and to assess the stability of the drug under these conditions. The choice of cosolvent and the concentration used can have a significant impact on the stability of the drug and the results of the forced degradation study. Therefore, it is important to carefully consider the selection of cosolvents and their concentration in forced degradation studies to ensure that the results accurately reflect the real-life stability of the drug. Additionally, the use of cosolvents in forced degradation studies should be validated to ensure that they do not interfere with the analysis of the drug or the identification of degradation products.

When a drug is not soluble in hydrochloric acid or sodium hydroxide, it may pose a challenge in forced degradation studies. These two solvents are commonly used in forced degradation studies to simulate acidic and basic stress conditions, respectively. If a drug is not soluble in these solvents, it may not be possible to accurately assess its stability under these conditions. In such cases, alternative solvents or methods may need to be used to achieve the desired degradation conditions.

For example, if a drug is not soluble in hydrochloric acid, a different acid such as sulfuric acid or acetic acid can be used instead. The choice of alternative solvent should be carefully considered to ensure that it accurately simulates the intended degradation condition and does not interfere with the analysis of the drug or the identification of degradation products.

In some cases, a combination of solvents or the use of a cosolvent can help to improve the solubility of the drug in the intended degradation solvent. However, it is important to validate any modifications made to the forced degradation conditions to ensure that they are reliable, accurate, and specific for the drug in question.

In conclusion, if a drug is not soluble in hydrochloric acid or sodium hydroxide, alternative solvents or methods may need to be used in forced degradation studies to accurately assess its stability under acidic and basic stress conditions. The choice of solvent and any modifications made to the forced degradation conditions should be carefully considered and validated to ensure reliable and accurate results.

9. SEVERAL IN SILICO TOXICITY PREDICTION TOOLS ARE AVAILABLE FOR THE ASSESSMENT OF CHEMICAL TOXICITY. SOME OF THE COMMONLY USED TOOLS INCLUDE³⁷:

1. Toxtree: This is a decision tree-based tool that predicts the toxicity of chemicals based on their structural characteristics.

2. OSIRIS: This is a rule-based system that predicts the toxicity of chemicals based on the presence of specific functional groups.

3. VEGA: This is a virtual screening tool that predicts the toxicity of chemicals based on their molecular structure and properties.

4. LeadScope: This is a software platform that uses computational chemistry and machine learning algorithms to predict the toxicity of chemicals.

5. eTOX: This is a European-wide platform that integrates multiple in silico toxicity prediction tools and databases.

6. Toxtron: This is a neural network-based tool that predicts the toxicity of chemicals based on their molecular structure and properties.

7. ToxPi: This is a tool that combines multiple in silico toxicity prediction tools into a single platform and allows for the comparison and ranking of chemicals based on their predicted toxicity.

These in silico toxicity prediction tools can provide valuable information on the potential toxicity of chemicals and can be used to prioritize chemicals for further testing and assessment. However, it is important to note that these tools are not perfect and should be used with experimental data to make informed decisions about chemical safety.

10. MASS BALANCE³⁸:

Mass balance in forced degradation study refers to the extent to which the total amount of drug substance and degradation products present in a sample after undergoing a specific degradation process can be accounted for and reconciled with the initial amount of drug substance present. The goal of mass balance in a forced degradation study is to confirm that the degradation of the drug substance is complete and to determine the chemical nature and quantity of the degradation products formed. The result of mass balance provides important information about the stability of the drug substance and helps ensure the quality and safety of the final product.

The acceptable criteria for mass balance in a forced degradation study depend on the specific regulations and guidelines for the pharmaceutical industry and the type of drug substance being tested. Generally, a mass balance is considered acceptable if the initial amount of drug substance and the recovered amount of degradation products after the degradation process equals at least 95-98% of the total amount of material. Additionally, the identity, purity and quantity of the degradation products should also be confirmed through analytical methods such as HPLC, GC, LC-MS, etc. The criteria for mass balance may vary based on the individual study design, regulatory requirements, and the intended use of the drug substance.

11. CONCLUSION:

A stability-indicating method is an analytical procedure capable of distinguishing between the principal active (intact) pharmaceutical ingredients (API) and any degradation (decomposition) product(s) created throughout the stability evaluation period under stipulated storage conditions. As part of a validation protocol, forced degradation studies are required for the development of stability-indicating and degradant-monitoring technologies. Forced degradation experiments can also be used to investigate degradation products. A correctly conceived and executed forced degradation study will yield a representative sample, which will aid in the development of a stability-indicating method.

11. REFERENCES:

1. ICH. International Conference on Harmonization (ICH). Guidance for industry: Q1A(R2) Stability testing of new drug substances and products. ICH Harmon Tripart Guidel. 2003; 4(February):24.

2. Rathod N, Prajapati PAI. Analytical Method Development and Validation of Stability Indicating Method and Related Substance by Using RP-HPLC of Drug Substance. J Pharm Anal. 2018; 8(5):367–72.

3. Vyas AJ, Visana NM, Patel AI, Patel AB, Patel NK, Shah SR. Analytical quality by design in stress testing or stability-indicating method. Asian J Pharm Anal. 2021; 11(2):170–8.

4. Patel AB, Asnani AH, Vyas AJ, Patel NK, Patel AI, Lumbhani AN. A Brief Review on Genotoxic impurities in Pharmaceuticals. Asian J Pharm Res. 2021 [cited 2023 Feb 16]; 11(3):187–93

5. B. Patel A, K. Jinja B, Kumar J. Vyas A, K. Patel N, I. Patel A, B. Sheth D, et al. A Retrospective Study of Warning letters Issued by US FDA Over 2019-2021. Asian J Pharm Res. 2022; (483):295–301.

6. Patel AB, Bundheliya AR, Vyas AJ, Patel NK, Patel AI, Lumbhani AN. A review on metal impurities in pharmaceuticals. Asian J Pharm Anal. 2021; 11(3):212–22.

7. Ajay I. Patel, Krupa B.Prajapati, Amit J. Vyas, Ashok B. Patel, Nilesh K. Patel. Determination and validation of phthalate impurities in milk by uv-spectrophotometry method. Monitor PS. 2019; 10(4):49–58.

8. Rajput M, Patel N, Chotaliya U, Patel A, Patel A, Vyas A. Determination of genotoxic impurity by chromatographic method. Pharma Sci Monit. 2017; 8(2):24–31.

9. Vyas AJ, Patel SM, Patel AB, Patel AI, Patel NK, Shah S, et al. Stability testing: An Essential study for Vaccine Formulation Development. Asian J Pharm Res. 2022; 12(1):29–36.

10. Vyas AJ, Gol DA, Patel AI, Patel AB, Patel NK, Chavda JR, et al. Implementing analytical quality by design (AQbD) approach for simultaneous estimation of tadalafil and macitentan by RP-HPLC asian. Anal Chem Lett. 2021; 11(4):539–52.

11. Patel AI, Prajapati KB, Jolapara SH, Vyas AJ, Patel AB, Patel NK, et al. RP-HPLC method for determination of Gemfibrozil using central composite design (CCD). Res J Pharm Technol. 2021; 14(6):3009–14.

12. Shinde NG, Bangar BN, Deshmukh SM, Sulake SP, Sherekar DP. Pharmaceutical Forced Degradation Studies with Regulatory Consideration. Asian J Res Pharm Sci. 2013; 3(4):178–88.

13. Singh Chauhan Y, Nex R, Sevak G, Singh Rathore M. Stability Testing of Pharmaceutical Products. Res J Pharm Dos Forms Technol. 2021; 13(04):317–28.

14. Roge AB, Tarte PS, Kumare MM, Shendarkar GR, Vadvalkar SM. Forced Degradation Study: An Important Tool in Drug Development. Pharm Res. 2013; 3(January):99–102.

15. Sutar S V., Yeligar VC, Patil SS. A review: Stability indicating forced degradation studies. Res J Pharm Technol. 2019; 12(2):885–90.

16. Sherawat R, Maithani M, Singh R, Regulatory aspects in development of stability indicating method: A review. Chromatographia 2010; 72:1-6

17. Analytical procedures and methods validation for drugs and biologics guidance for industry. Fda.gov. 2015

18. Szepesi G, Gazdag M, Mihályfi K. Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. III. Method validation. J Chromatogr. 1989; 464(2):265–78.

19. Carr GP, Wahlich JC. A practical approach to method validation in pharmaceutical analysis. J Pharm Biomed Anal. 1990; 8(8–12):613–8.

20. Jenke DR. Chromatographic method validation: A review of current practices and procedures. I. general concepts and guidelines. J Liq Chromatogr Relat Technol. 1996; 19(5):719–36.

21. International Conference on Harmonization. Q3B (R2): Impurities in New Drug Products. London, UK: European Medicines Agency; 2003; 68(220):64628–64629

22. FDA, Guidelines for Submitting Documentation for Stability of Human Drugs and Biologics Food and Drug Administration, Rockville 1998.

23. Guidance for Industry: Submitting Documentation for the Stability of Human Drugs and Biologics Food and Drug Administration, Rockville, 1987.

24. Guidance for Industry (Draft): Stability Testing of Drug Substances and Drug products Food and Drug Administration, Rockville, 1998.

25. Michael Kats. Forced Degradation Studies: Regulatory Considerations and Implementation. BioPharm International, 2005: 1-08

26. Hovorka S, Schöneich C. Oxidative degradation of pharmaceuticals: theory, mechanisms and inhibition. J Pharm Sci. 2001; 90(3):253–69.

27. Anderson NH, Johnston D, McLelland MA, Munden P. Photostability testing of drug substances and drug products in UK pharmaceutical laboratories. J Pharm Biomed Anal. 1991; 9(6):443–9.

28. Bakshi M, Singh S., Development of Validated StabilityIndicating Assay Methods: Critical Review. J. Pharm. Biomed. Anal. 28; 2002: 1011–1040

29. Singh S, Bakshi M. Guidance on conduct of stress tests to determine inherent stability of drugs. Phrama Tech 24, 2000: 1- 14.

30. Skoog, DA, West DM. Principles of Instrumental Analysis. Saunderg Golden, Japan. 1980: 2-3.

31. Brummer H., How to approach a forced degradation study. Life science technical bulletin issue. 2011: 3.

32. Alsante KM, Martin L, Baertschi SW., A Stress Testing Benchmarking Study. Pharm. Technol. 27 (2); 2003: 60–72

33. Baertschi SW, Thatcher SR. Sample presentation for photostability studies: problems and solutions, pp: 445, In; Pharmaceutical Photostability and Stabilization Technology. Piechocki J, Editor, Taylor & Francis, New York. 2006.

34. ICH Harmonised Tripartite Guideline Q2 (R1), Validation of Analytical Procedures: Text and Methodology, November 2005.

35. Patel JR, Joshi HV, Shah UA, Patel JK. A Review on Computational Software Tools for Drug Design and Discovery. Indo Global J. Pharm. Sci. 2022 Jan. 29; 12:53-81.

36. Clarke CJ, Tu W-C, Levers O, Bröhl A, Hallett JP. Green and sustainable solvents in chemical processes. Chem Rev. 2018; 118(2):747–800.

37. Singh AK, Bilal M, Iqbal HMN, Raj A. In silico analytical toolset for predictive degradation and toxicity of hazardous pollutants in water sources. Chemosphere. 2022; 292:133-250.

38. Baertschi SW, Pack BW, Hoaglund Hyzer CS, Nussbaum MA. Assessing mass balance in pharmaceutical drug products: new insights into an old topic. Trends Analyt Chem. 2013; 49:126–36.

Received on 10.02.2023 Modified on 01.03.2023

Asian J. Pharm. Ana. 2023; 13(2):131-139.

DOI: 10.52711/2231-5675.2023.00022

Degradation type	Experimental	conditions Storage	conditions Sampling time (Days)
Hydrolysis	Control (no acid or base)	40°C, 60°C	1,3,5
	Hydrochloric acid	40°C, 60°C	1,3,5
	Sodium Hydroxide	40°C, 60°C	1,3,5
	Acid control (no API)	40°C, 60°C	1,3,5
	Base control (no API)	40°C, 60°C	1,3,5
Oxidation	3% H2 O2	25°C, 60°C	1,3,5
	Peroxide control	25°C, 60°C	1,3,5
	Azobisisobutyronitrile (AIBN)	40°C, 60°C	1,3,5
	AIBN control	40°C, 60°C	1,3,5
Photolytic	Light 1 × ICH	Not Applicable	1,3,5
	Light 3 × ICH	Not Applicable	1,3,5
	Light control	Not Applicable	1,3,5
Thermal	Heat chamber	60°C	1,3,5
	Heat chamber	60°C/75% RH	1,3,5
	Heat chamber	80°C	1,3,5
	Heat chamber	80°C/75% RH	1,3,5
	Heat control	Room Temperature	1,3,5