Identification and Structural Elucidation of a Potential Citric Acid and Caprolactam Adduct Impurity in Aminocaproic Acid Injection Using Preparative Chromatography, LC–MS, and NMR

ABSTRACT Aminocaproic acid (ACA) is a pharmacological substance possessing antifibrinolytic characteristics that improves hemostasis by diminishing excessive bleeding caused by fibrinolysis. The medication is presently available in tablet, capsule, and oral solution forms. The analysis was carried out on Waters Alliance 2695 HPLC system separation module connected to a 2996 Photodiode Array Detector. The separation was achieved using Eclipse XDB‐C18, 4.6 × 250 mm 2 column, and a 5 µm analytical column. In the course of developing a new injectable dosage form, a substantial unknown impurity was identified above its threshold during the determination of its shelf life, with a retention time of 57.5 min after 6 months under accelerated stability conditions of 40°C temperature and 75% relative humidity. Due to the critical significance of pharmaceutical quality and purity, any impurities identified during manufacturing, storage, or transportation may lead to significant adverse or undesirable effects. Should any unknown impurity surpass its threshold limits during the product's stability testing, it is imperative to elucidate its structure to evaluate the potential concerns it may present. This work seeks to elucidate the methodology for identifying isolating and characterizing the unknown impurity detected during the stability testing of ACA injection. The impurity was isolated using preparative chromatography, and its mass was ascertained using high‐resolution mass spectrometry (HRMS). Nuclear magnetic resonance (NMR) spectroscopy was employed for structural elucidation. The research indicates that impurity is a new adduct formed from citric acid and ACA with a molecular mass of 287.10 with a chemical name of 2‐hydroxy‐2‐(2‐oxo‐2‐(2‐oxoazepan‐1‐yl)ethyl)succinic acid.