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

Abstract

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.

Keywords: LC‐qTOF‐MS; brain penetration; difluoroacetic acid; methylene blue; pharmacokinetics.

FIGURE 1

MS/MS spectrum of MB with observed mass‐to‐charge ratio and its predicted fragment ions with theoretical mass‐to‐charge ratio.

FIGURE 2

Extracted ion chromatograms of MB (m/z 284.1 to 268.1) under various conditions following protein precipitation from mouse plasma spiked with MB (1818.18 ng/mL).

FIGURE 3

Chromatograms of MB spiked into mouse blank plasma at concentrations of 9.15 ng/mL, using different concentrations of DFA as mobile phase additive in DW and MeOH (mobile phase flow rate: 0.5 mL/min). Panels (a) 0.1% DFA, (b) 0.25% DFA, and (c) 0.5% DFA.

FIGURE 4

Representative calibration curve for the quantification of MB in blank mouse plasma (range = 3.05–2222.22 ng/mL and r value = 0.99532). The curve range and r value in blank mouse brain homogenate were 3.05–2222.22 ng/mL and 0.99643, respectively (data not shown).

FIGURE 5

(a) Chromatogram of MB at the LLOQ (3.05 ng/mL) spiked in blank mouse plasma and (b) chromatogram of MB at the LLOQ (3.05 ng/mL) in blank mouse brain homogenate.

FIGURE 6

The time‐plasma concentration pharmacokinetic profile of MB following a single IV bolus injection at 2 mg/kg in male ICR mice (n = 4).

FIGURE 7

The time‐concentration profile of MB in brain and plasma following a single IV bolus injection at 2 mg/kg in male ICR mice (n = 3).