The impact of humidity on the stability of l-alanyl-l-tyrosine (Ala-Cys₂, a disulfide-containing dipeptide) is primarily manifested through water-mediated chemical reactions and physical hygroscopicity, with specific mechanisms and manifestations as follows:
1. Humidity-Induced Chemical Degradation
1.1 Hydrolysis (Primary Pathway)
Mechanism:
In high-humidity environments, water molecules can penetrate the surface of crystalline or amorphous solids, causing hydrolysis of the peptide bonds or disulfide bonds in l-alanyl-l-tyrosine.
Peptide bond hydrolysis: Generates alanine (Ala), cysteine (Cys) monomers, and oligopeptides, leading to a decrease in content.
Disulfide bond hydrolysis (secondary): Under acidic or alkaline conditions, disulfide bonds may be attacked by water molecules, forming thiol groups (-SH) and sulfonic acid groups, thereby destroying structural integrity.
Humidity threshold:
When the environmental relative humidity (RH) exceeds its critical relative humidity (CRH), the hydrolysis rate increases significantly. For example, the CRH of pure l-alanyl-l-tyrosine may be below 60% RH, with specific values depending on crystal morphology and purity.
1.2 Alteration of Oxidation-Reduction Equilibrium
Indirect effects of humidity:
High humidity may promote contact between the system and air, enhancing oxygen dissolution or the migration of metal ions (such as Fe²⁺, Cu²⁺), which catalyze the oxidative cleavage of disulfide bonds or further oxidation of thiol groups (e.g., formation of sulfonic acids).
Outcome:
Increased humidity may accelerate the conversion of l-alanyl-l-tyrosine to cystine (Cys-Cys) or alanine-cysteine mixed products, leading to loss of active ingredients.
2. Effects of Physical Hygroscopicity
2.1 Moisture Absorption and Caking
Phenomenon:
l-alanyl-l-tyrosine is hygroscopic due to its polar amino, carboxyl, and amide groups. When exposed to high humidity, water films form on the surface of solid particles, causing interparticle adhesion, caking, and even liquefaction (deliquescence).
Cascade effects on stability:
Caking increases the specific surface area of the sample, further accelerating hydrolysis and oxidation.
The liquid environment provides conditions for microbial contamination (e.g., bacterial and mold growth), exacerbating degradation.
2.2 Crystal Form Transformation
Humidity-induced phase transition:
Some crystal forms of l-alanyl-l-tyrosine may transform into hydrates (such as monohydrates) or amorphous states under high humidity.
Consequences:
Amorphous structures have higher molecular mobility, with chemical degradation rates typically 10–100 times faster than crystalline forms.
Crystal form transformation may alter physical properties such as powder flowability and compressibility, affecting formulation processes (e.g., tablet formability).
3. Key Factors Influencing Stability
3.1 Synergistic Effect of Humidity and Temperature
Extension of the Arrhenius equation:
Humidity changes are often accompanied by temperature fluctuations (e.g., high-temperature and high-humidity environments), and the two synergistically accelerate chemical reactions. For example, the hydrolysis rate at RH 75% and 40°C may be 5–10 times higher than at RH 30% and 25°C.
3.2 Regulatory Effect of pH
Coupling of humidity and pH:
The microenvironmental pH of the sample may change after moisture absorption (e.g., local acidity due to carboxyl ionization of alanine), affecting the kinetics of hydrolysis reactions.
Optimal pH range:
l-alanyl-l-tyrosine exhibits higher stability in neutral (pH 6–8) environments, while high-humidity acidic (pH < 4) or alkaline (pH > 9) conditions significantly accelerate peptide bond cleavage.
3.3 Influence of Formulation Processes
Moisture buffering effect of excipients:
Adding low-hygroscopic excipients (such as mannitol or lactose) to formulations can reduce the system’s sensitivity to humidity; conversely, highly hygroscopic excipients (such as polyethylene glycol) exacerbate degradation.
Packaging selection:
Moisture-proof packaging materials like aluminum foil bags and glass bottles effectively block moisture, while polyethylene bags have high moisture permeability in high-humidity environments, potentially causing sample deterioration.
4. Stability Control Strategies
4.1 Optimization of Storage Conditions
Low-humidity environment: Store in a dry environment with RH < 40% (e.g., using desiccants) to avoid exposure to moist air.
Temperature control: Low temperatures (e.g., 2–8°C) can reduce the rate of hydrolysis and oxidation reactions, especially for liquid formulations (such as injections).
4.2 Chemical Modification and Formulation Design
Salt formation or esterification: Convert l-alanyl-l-tyrosine into hydrochloride or methyl ester derivatives to reduce the hydration capacity of polar groups and improve moisture resistance.
Solid dispersion or microencapsulation: Encapsulate the drug in hydrophobic polymers (such as ethyl cellulose) via spray drying or solvent evaporation to isolate moisture.
4.3 Stability Monitoring Methods
Accelerated testing: Accelerate degradation under high-temperature and high-humidity conditions (e.g., 60°C/RH 90%) to predict shelf life.
Analytical techniques: Use high-performance liquid chromatography (HPLC) to monitor changes in the main peak area, and mass spectrometry (MS) to identify hydrolysis products (such as alanine and cysteine).
Conclusion:
The impact of humidity on the stability of l-alanyl-l-tyrosine is centered on hydrolysis and physical hygroscopicity, with high humidity significantly accelerating peptide bond cleavage, disulfide bond oxidation, and crystal form transformation. In practical applications, moisture-proof packaging selection, excipient compatibility optimization, and storage condition control—combined with its CRH and pH stability characteristics—are essential to ensure the shelf-life quality of drugs or biomaterials. Future research may explore supramolecular inclusion technologies (such as cyclodextrin complexation) to further enhance moisture resistance.
