Development and Validation of a UPLC-MS/MS Method for the Quantification of Amantadine in Rat Plasma: Application to a Pharmacokinetic Study Under High-Altitude Hypoxia and Mechanistic Insights

<b>Background/Objectives</b>: This study aimed to develop an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantifying amantadine (AMA) in rat plasma and to investigate its pharmacokinetics under simulated high-altitude hypoxia, contrasting its behavior with that of its structural analog memantine (MEM). <b>Methods</b>: The method entailed using memantine (MEM) as an internal standard. Sample preparation involved protein precipitation, followed by gradient elution with detection via positive electrospray ionization and selective reaction monitoring (SRM). The method validation complied with the International Conference on Harmonization (ICH) M10 guidelines. Pharmacokinetic studies were conducted in rats exposed to either low altitude (1500 m) or simulated high altitude (6500 m) after a single oral dose of AMA (10 mg/kg). <b>Results</b>: The assay demonstrated linearity from 5 to 1000 µg/L, with accuracy, precision, recovery, and stability all meeting the respective acceptance criteria. Hypoxia did not significantly alter systemic exposure to AMA, as measured by parameters such as the area under the concentration-time curve (AUC), maximum concentration (Cmax), and apparent clearance (CLz/F). However, hypoxia prolonged the elimination half-life by 55% and increased the variance in the mean residence time. This finding contrasts sharply with our previous results on MEM under identical hypoxic conditions, which showed a 72.15% increase in AUC and a 41.99% decrease in CLz/F. <b>Conclusions</b>: A robust UPLC-MS/MS method for quantifying AMA was successfully established. AMA exhibits unique pharmacokinetic resilience to acute hypoxia, characterized by increased variability in elimination without changes in overall exposure. This profile starkly differs from the heightened exposure and reduced clearance observed for drugs like MEM, which are predominantly cleared by hepatic metabolism (under the studied conditions). These findings are consistent with the concept that a drug's primary elimination pathway (renal excretion vs. hepatic metabolism) critically determines its pharmacokinetic susceptibility to hypoxic stress.