Department of Pharmaceutical Analysis, GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India.
Rapid Commun Mass Spectrom. 2024 Oct 30;38(20):e9896. doi: 10.1002/rcm.9896.
Nirmatrelvir is a protease inhibitor that is essential for virus replication. Nirmatrelvir is indicated for the management of mild to severe cases of COVID-19 in individuals who are 12 years of age or older. Forced degradation studies of nirmatrelvir were carried out on the drug substance in solid and solution forms, subjecting it to various stress conditions according to International Conference on Harmonisation (ICH) Q1A(R2) and Q1B guidelines. The analytical method was validated as per the ICH Q2(R1) guidelines.
The drug substance (nirmatrelvir) was subjected to hydrolysis (acidic, alkaline, and neutral), thermal, photolytic, and oxidative stress conditions. Five degradation products (DPs) of nirmatrelvir formed under hydrolytic (acidic and alkaline) and oxidative (2,2-azobisisobutyronitrile) stress conditions. These degradation products were identified and separated using reverse-phase HPLC on a phenomenex kinetex C8 column (250 mm × 4.6 mm × 5 μm) with gradient elution. The mobile phase consisted of 0.1% formic acid and acetonitrile, and detection was carried out at a wavelength of 210 nm.
Nirmatrelvir and its five DPs were efficiently separated using reverse phase-HPLC. These five DPs were identified and characterized using LC-electrospray ionization (ESI)-Q-TOF-coupled mass spectrometry analysis in the ESI-positive ionization mode. The formation mechanisms of the DPs and the most probable mass fragmentation pathways for both nirmatrelvir and its DPs were elucidated. The developed method demonstrated selectivity, accuracy, linearity, and reproducibility, making it appropriate for quality control of nirmatrelvir and future research studies. Additionally, the physicochemical and Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties of nirmatrelvir and its DPs were predicted using ADMET predictor software. The toxicity profile revealed that DP2 and DP3 have teratogenic effects while DP1 and DP3 caused phospholipidosis.
奈玛特韦是一种蛋白酶抑制剂，对病毒复制至关重要。奈玛特韦适用于治疗 12 岁及以上个体的轻至重度 COVID-19 病例。根据国际人用药品注册技术协调会（ICH）Q1A（R2）和 Q1B 指南，对奈玛特韦原料药的固体和溶液形式进行了强制降解研究。根据 ICH Q2（R1）指南对分析方法进行了验证。
将原料药（奈玛特韦）置于水解（酸、碱和中性）、热、光解和氧化应激条件下。在水解（酸和碱）和氧化（2,2-偶氮二异丁腈）应激条件下，奈玛特韦形成了 5 种降解产物（DP）。使用 Phenomenex Kinetex C8 柱（250mm×4.6mm×5μm）在反相 HPLC 上，采用梯度洗脱法对这些降解产物进行分离和鉴定。流动相由 0.1%甲酸和乙腈组成，检测波长为 210nm。
使用反相 HPLC 可以有效地分离奈玛特韦及其 5 种 DP。使用 LC-电喷雾电离（ESI）-Q-TOF 耦合质谱分析，在 ESI 正离子化模式下对这 5 种 DP 进行了鉴定和表征。阐明了 DP 的形成机制以及奈玛特韦及其 DP 的最可能的质量断裂途径。所建立的方法表现出选择性、准确性、线性和重现性，适用于奈玛特韦的质量控制和未来的研究。此外，使用 ADMET 预测软件预测了奈玛特韦及其 DP 的物理化学和吸收、分布、代谢、排泄和毒性（ADMET）特性。毒性概况显示 DP2 和 DP3 具有致畸作用，而 DP1 和 DP3 引起磷脂沉积症。
