3.
1 Pre-formulation studies-Solubility
and stability Analysis -II
Dr. Ruchi Chawla
Department of Pharmaceutical Engg. & Technology,
IIT(BHU), Varanasi
Learning objectives
Pre-formulation studies
• Stability studies
• Factors affecting stability
• pH
• Light
• Solvent
• Air
• Excipients
• Compatibility studies
• Conclusion
Preformulation stability studies
⚫The active ingredient in drugs must remain stable to ensure patients
receive accurate dosages. For drugs prone to degradation that may release
toxic byproducts, identifying conditions for prevention or stabilization is
key to safety.
⚫Beyond the drug itself, excipients (inactive ingredients) also play a vital
role. They must remain stable, both individually and in combination with
drugs or other excipients,
⚫Preformulation stability to createserve
studies a viable and market-ready product.
as the
first quantitative evaluation of a new drug's
chemical stability. These studies include
experiments in both solution and solid-state
forms under typical conditions such as
handling, formulation, storage, and
administration.
⚫Stability studies are essential for
understanding a drug's behavior in solid and
liquid states. They play a crucial role in drug
Objectives of performing Preformulation
stability studies are:
• The primary objectives of preformulation stability studies are to
identify environmental conditions such as light, heat, and moisture
that can affect the compound and to analyze the degradation profile
under these conditions.
• Degradation can occur through a number of
chemical/physiochemical and biological pathways.
e.g.
⮚ Hydrolysis
⮚ Isomerisation
⮚ Oxidation
⮚ Polymerisation
⮚ Solid-state phase transformation
⮚ Dehydration or desolvation
⮚ Cyclization
⮚ Photolytic degradation
⮚ Microbial attack
Types of Stability
Figure 1: Types of stability
⚫ICH (International Conference on harmonization of regulatory
requirements) Drug stability test guideline Q1A (R2) requires that the drug
substance be tested under different stress conditions.
⚫It is suggested that stress testing include the effect of:
⮚ pH
⮚ Temperature
⮚ humidity
⮚ Light
⮚ Oxidizing agents
Chemical Degradation in Solution
⚫The chemical stability of a drug substance in the solid state can be evaluated under
various temperature and humidity stress conditions.
⚫Stability testing involves storing pre-weighed samples in stability cabinets under
conditions such as:
⚫40°C, 60°C, or 80°C.
⚫25°C with 85% relative humidity (RH).
⚫40°C with 75% RH.
⚫Samples are observed over periods of up to 8 weeks, with pre-determined
intervals (e.g., 2, 4, and 8 weeks).
⚫The samples are dissolved in suitable solvents and assessed using stability-
indicating assays, primarily reverse-phase high-performance liquid
chromatography (HPLC). This method is effective for direct injection and
analyzing stability over time. The chosen stress conditions must align with the
chemical/biological characteristics of the substance and regulatory guidelines.
⚫Ideally, assays should detect degradation peaks as small as 0.1% of the main
compound, though this can be challenging during the early discovery phase.
⚫Some methods utilize 96-well formats, enabling the analysis of multiple
degradation experiments under diverse stress conditions efficiently.
Hydrolytic Degradation
⚫Hydrolysis is common in many molecular species, especially
carboxylic acid derivatives or substances with functional groups
based on carboxylic acid (e.g., ester, amide, lactone, lactam,
imide, carbamate).
⚫To identify and quantify degradation through hydrolysis,
stress testing should be conducted in acidic and alkaline
conditions.
⚫This can involve refluxing the compound in 0.1 N HCl and 0.1
N NaOH Hydrolytic
for 8–12 hours.
degradation
Ester Amide
Hydrolysis Hydrolysis
Table 1: Types and reactions involved in Hydrolysis
Type of Reaction Chemical
Products Catalysts
Hydrolysis Characteristics reaction
Involves breaking a
covalent bond
Mineral between carbon
R¹-COOR + H⁺
Ester Acid + acids, and oxygen;
+ OH⁻ → R¹-
Hydrolysis Alcohol alkalies, catalyzed by polar
COOH + HOR
enzymes substances
supplying hydrogen
or hydroxyl ions
Similar to ester
hydrolysis but
Mineral R-CONR¹ + H⁺
slower due to the
Amide Acid + acids, + OH⁻ → R-
greater stability of
Hydrolysis Amine alkalies, COOH + H₂N-
amides; less kinetic
enzymes R¹
data available in
literature
Dimerization and Polymerisation
Similar molecules may interact to produce complex structures—including
dimers and polymers of various lengths and orientations. The potential for
such interactions should be evaluated by examining the polymeric state of
samples during stress testing for heat, light and solution stability.
Photostability
⚫Substances prone to degradation under light exposure can benefit from specialized
coatings or packaging to prevent or reduce instability. The evaluation of a
compound's tendency to degrade in light should occur early in the process.
⚫Solid-state photostability testing involves exposing thin sample layers to high-
intensity light or UV conditions, starting at 25°C and progressing to higher
temperatures, using a photostability chamber.
⚫International Conference on Harmonisation (ICH) guidelines recommend exposure
at 1.2 million lux hours for visible light and 200 W hours m⁻² for UV to replicate
light radiation in diverse regions.
⚫Photostability should also be assessed in both aqueous and non-aqueous solutions if
the drug is intended for oral, parenteral, or topical solutions.
⚫Samples protected from light serve as controls and are stored under the same
conditions for comparison during solid and solution photostability studies.
pH-dependent Stability
⚫Stability tests should be conducted under multiple physiological and
formulation pH conditions to analyze the characteristics of the drug
candidate in physiological environments.
⚫These tests also provide vital information for the formulation of
solution dosage forms.
⚫Stability is typically assessed at 37°C in various buffer solutions, such
as pH 1, pH 4, pH 7, and pH 9.
⚫Intervals for testing may range from 1 day to 1 month.
⚫The studies should use appropriate concentrations of drug or
excipient to detect even minor decomposition products within the
detection range.
Oxidative Stability
Drugs and excipients can degrade due to oxidation reactions, which are
classified into two distinct types:
⚫Oxidation via direct reaction with atmospheric oxygen.
⚫Oxidation through a chain reaction, involving the formation of peroxy free
radicals.
⚫The second type of degradation is more prevalent in compounds with
double carbon bonds, particularly in long-chain unsaturated fatty acids
and oils.
⚫If a drug or excipient's chemical structure suggests susceptibility to
oxidation, preformulation studies should expose samples to elevated
temperatures in the presence of oxygen.
⚫Oxidation pathways may vary with temperature, leading to different
energetics and sites of peroxidation at low versus high temperatures,
resulting in distinct reaction orders.
⚫Elevated temperature tests might not represent reactions at ambient
conditions.
Solid-state Stability
• The primary objectives are:
✔ Identifying stable storage conditions for the drug in solid-state
form.
✔ Identifying compatible excipients for formulation.
• Solid-state studies differ from solution stability profiles, as they
may be significantly affected by:
• Changes in purity.
• Variations in crystallinity, often due to process improvements.
• Key procedures include:
• Repetitive testing of the initial bulk lot alongside newer bulk lots.
• Setting aside adequate material specifically for these studies.
• Solid-state reactions are generally:
• Slower and more complex to interpret than solution-state
reactions.
• Affected by reduced molecular interactions between drug and
•Challenges in kinetic analysis:
•Retention of intact drug may fail to accurately estimate shelf-life.
•Assay variations at low temperatures may equal or surpass observed
degradation.
•Solutions to these challenges:
•Analyzing the appearance of decay products (typically 1-5% of the sample)
can provide better clarity.
•Additional studies using techniques like TLC, fluorescence, or UV/Visible
spectroscopy may be necessary to determine decay product kinetics.
Stability–Drug Excipient Compatibility
• The development of a stable and effective dosage form relies not
only on the active pharmaceutical ingredient but also on the
thoughtful and precise selection of excipients.
• Drug-excipient compatibility studies are primarily conducted to
identify possible incompatibilities and to justify the selection of
excipients in a formulation, aligning with the requirements for
regulatory filings.
• Changes in organoleptic
• An incompatibility in the properties
dosage form can result in any of the
following changes:
Changes in dissolution performance
Physical form conversion
A decrease in potency
An increase in degradation products.
Figure 2: Approach for Determining Chemically
Compatible Excipients
Liquid Compatibilities
⚫Studies are conducted by dissolving the drug in a solution containing
the additive. Amber and flint vials are often utilized, with autoclaved
conditions frequently included. For emulsions, preformulation
assessments involve determining the critical micelle concentration of
the formulations.
⚫For oral preparations, compatibility tests with ethanol, glycerine, syrup,
sucrose, buffers, and preservatives are conducted to evaluate the
activation energy of the primary reaction occurring in the solution.
Conclusion
⚫Formulation stable formulation is the ultimate goal of all the pre-
formulation and formulation development studies
⚫Identification of the factors helps us in modifying the process or
taking necessary precautions.
⚫There are regulatory guidelines as well for these studies
Important guidelines for stability studies
Thank You
