A Novel Method for Development and Validation of the Degradation Products Analysis of N-Carbamylglutamate with UHPLC by Using Design of Experiment Approach

Abstract

Carglumic acid, also known as N-carbamyl-L-glutamic acid, is a medication used in the treatment of a rare genetic disorder called N-acetylglutamate synthase (NAGS) deficiency. To the authors' knowledge, there was no method reported in the literature for the determination of degradation products suitable for quality control analyses of carglumic acid. Thus, the aim of the presented study is to develop an impurity method with a UHPLC/DAD detector configuration compatible with industrial standards from the European Pharmacopeia and the United States Pharmacopeia, making the drug more accessible for developing and underdeveloped countries through its precise evaluation. The method involved the separation of carglumic acid and its degradation products using a reverse-phase C18 column (Waters, BEH 150 mm × 2.1 mm, 1.7 μm) at a flow rate of 0.39 mL/min with a stop time of 10 min. To separate all unknown and known impurities, a gradient elution (phosphate buffer, pH 2.4, and acetonitrile) system was used. The detection was performed at 214 nm. Forced degradation studies were conducted under different stress conditions, including acidic, basic, oxidative, thermal, and photolytic stress. Placket−Burman statistical experimental design was used to demonstrate the robustness of this method, and the suitability of the method was confirmed under the applied conditions. Box− Behnken design was used to provide the optimum resolution between the peaks determined to be critical during the optimization. The developed method was validated according to ICH guidelines for specificity, linearity, accuracy, precision, and robustness. The limit of detection and limit of quantification were 0.7 and 0.15 μg/mL for carglumic acid, respectively.