Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research-Ahmedabad (Ministry of Chemicals and Fertilizers, Government of India), Gandhinagar, India.
Rapid Commun Mass Spectrom. 2024 Mar 15;38(5):e9696. doi: 10.1002/rcm.9696.
Enasidenib (EDB) is an orally active selective mutant isocitrate dehydrogenase-2 enzyme inhibitor approved by the U.S. Food and Drug Administration to treat acute myeloid leukemia. It lacks a reported forced degradation study and a stability-indicating assay method (SIAM). This study addresses this gap by establishing a degradation profile in accordance with the International Council for Harmonisation Q1A and Q1B (R2) guidelines and developing a validated SIAM for EDB.
EDB was exposed to forced degradation under various conditions (hydrolytic, photolytic, oxidative, and thermal stress). Degradation samples were analyzed using high-performance liquid chromatography on an Agilent ZORBAX Eclipse Plus C18 column with a mobile phase consisting of 0.1% formic acid in Milli-Q water and acetonitrile at a flow rate of 1 mL/min and detection at 270 nm. Liquid chromatography-quadrupole time-of-flight-high-resolution mass spectrometry (LC/Q-TOF HRMS) was used for the identification and characterization of degradation products. Nitrosamine risk assessment was conducted using a modified nitrosation assay procedure (NAP) test due to the presence of a secondary amine group in the drug, which is liable to forming nitrosamine drug substance-related impurities (NDSRI).
The drug exhibited significant degradation under acidic, basic, photolytic, and oxidative conditions in the solution state. A total of nine degradation products (DP) were formed (DP-I, DP-III, and DP-IV: acidic conditions; DP-I and DP-III: basic conditions; DP-II, DP-V, DP-VI, and DP-VII: oxidative stress; and DP-VII, DP-VIII, and DP-IX: photolytic conditions), which were separated and identified using reversed-phase high-performance liquid chromatography and characterized using liquid chromatography-tandem mass spectrometry. The mechanism behind the formation of EDB degradation products has been discussed, and this study was the first to develop a degradation pathway for EDB. In addition, the possibilities of NDSRI formation for EDB were studied using a modified NAP test, which can contribute to the risk assessment of the drug.
Forced degradation studies were conducted by establishing a SIAM for EDB. All the degradation products were characterized by mass spectral data obtained using LC/Q-TOF-HRMS.
依尼司他（EDB）是一种口服活性选择性突变型异柠檬酸脱氢酶-2 酶抑制剂，已被美国食品和药物管理局批准用于治疗急性髓系白血病。它缺乏已报道的强制降解研究和稳定性指示分析方法（SIAM）。本研究按照国际协调委员会 Q1A 和 Q1B（R2）指南建立了降解谱，并开发了 EDB 的验证后的 SIAM，以此填补了这一空白。
EDB 在各种条件下（水解、光解、氧化和热应力）进行强制降解。使用高效液相色谱法在 Agilent ZORBAX Eclipse Plus C18 柱上分析降解样品，流动相由 0.1%甲酸在 Milli-Q 水中和乙腈组成，流速为 1 mL/min，检测波长为 270nm。液相色谱-四极杆飞行时间-高分辨率质谱联用（LC/Q-TOF HRMS）用于降解产物的鉴定和表征。由于药物中存在仲胺基团，容易形成与亚硝胺有关的药物杂质（NDSRI），因此使用改良的亚硝化试验程序（NAP）试验进行亚硝胺风险评估。
在溶液状态下，药物在酸性、碱性、光解和氧化条件下均表现出明显的降解。共形成了 9 个降解产物（DP-I、DP-III 和 DP-IV：酸性条件；DP-I 和 DP-III：碱性条件；DP-II、DP-V、DP-VI 和 DP-VII：氧化应激；DP-VII、DP-VIII 和 DP-IX：光解条件），使用反相高效液相色谱法分离和鉴定，并使用液相色谱-串联质谱法进行了表征。讨论了 EDB 降解产物形成的机制，本研究首次建立了 EDB 的降解途径。此外，使用改良的 NAP 试验研究了 EDB 形成 NDSRI 的可能性，这有助于对药物进行风险评估。
通过建立 EDB 的 SIAM 进行了强制降解研究。所有降解产物均通过 LC/Q-TOF-HRMS 获得的质谱数据进行了表征。
