Quantification of Methylene Blue and Evaluation of Its Pharmacokinetics in ICR Mice by Liquid Chromatography‐Quadrupole Time‐of‐Flight Mass Spectrometry Using Difluoroacetic Acid

Methylene blue (MB), a phenothiazine derivative, is currently under clinical trials for Alzheimer's disease (AD) due to its potential to inhibit tau aggregation, a key pathological process in AD. In this study, we developed and qualified a rapid and reliable liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) method for the quantification of MB in mouse plasma and brain samples. Chromatographic separation was achieved using a PolymerX RP-1 100 Å (50 × 2 mm, 5 μm) column with a mobile phase consisting of water and methanol containing 0.5% difluoroacetic acid, delivered at a flow rate of 0.5 mL/min. Calibration curves were constructed using quadratic regression (weighted 1/concentration²) over a range of 3.05-2222.22 ng/mL in both matrices. The method was successfully applied to characterize the pharmacokinetics of MB in male ICR mice, revealing a high systemic clearance (65.64 mL/min/kg) and substantial brain penetration, as indicated by a brain-to-plasma partition coefficient (K_p,brain) of 23.50 following single intravenous bolus administration. These findings provide critical insights into MB's in vivo behavior and demonstrate the utility of this bioanalytical method for evaluating MB in preclinical studies.