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 (Kp,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.