Qualification of an indicative stability method for the detection of impurities in Histidine at a concentration of 117,20 mg / 3mL AP-HP

The Histidine-Copper kit, developed by the General Agency for Health Equipment and Products (AGEPS), is a hospital preparation used for the treatment of Menkes disease. While there exists in internal and literature-based knowledge regarding histidine stability within the ampoules, the necessity to conduct enforced degradation studies arises to comprehend the product’s attributes in accordance with ICH Q8 guidelines. These studies aim to facilitate subcontracting by effectively transferring analytical methodologies and crucial manufacturing processes.

The objective of this research is to elucidate histidine degradation pathways, generate degradation products (DPs) to reevaluate the specificity and validation of the SIAM character, and indicate the stability of the methodology for identifying DPs used in stability assessments.

Enforced degradation studies were carried out by subjecting histidine dissolved in solution to various stressors, mimicking the composition found in hospital preparations. These stressors included oxidative conditions, achieved through oxygenation with hydrogen peroxide (H2O2), and initiation of radical reactions using magnesium monoperoxyphthalate (MMPP), catalyzed by metal species (Fe II and Fe III), or unassisted, along with exposure to light. Hydrolytic stressors were also utilized, involving pH modulation with acid (HCl) and alkaline (NaOH) solutions. Furthermore, thermal stressors were applied, subjecting histidine to temperatures above 50 °C. Impurity detection and identification were carried out using reverse-phase high-performance liquid chromatography (HPLC) with a photodiode array (PDA) detector operating in the ultraviolet-visible spectral range.

Forced degradation of histidine led to observable color changes (ranging from colorless to yellow to brown). HPLC-PDA analysis revealed distinct chromatographic peaks, each corresponding to unique DPs. Four of these DPs exceeded the defined 0.1% threshold in accordance with ICH Q3B standards. These compounds were observed under oxidative conditions (MMPP and H2O2), with their formation being enhanced under light exposure. Additionally, the presence of an alkaline environment amplified the generation of these DPs due to the involvement of hydroxyl groups, thus supporting the oxidative production pathway. Among the DPs identified in existing literature, oxo-histidine, urea, and asparagine were prominent. The less-known 5-imidazole-carboxaldehyde displayed a retention time similar to the second most predominant peak among the four identified DPs. The identities of the remaining three DP peaks were undisclosed in the present study. Lastly, the validation of 5-imidazole-carboxaldehyde identification was confirmed through chromatography coupled with mass spectrometric analysis.

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