Simultaneous quantification of methylene blue and its major metabolite, azure B, in plasma by LC-MS/MS and its application for a pharmacokinetic study.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of methylene blue (MB) and its major metabolite, azure B (AZB), in rat plasma. A simple protein precipitation using acetonitrile was followed by injection of the supernatant on to a Zorbax HILIC Plus column (3.5 µm, 2.1 × 100 mm) with isocratic mobile phase consisting of 5 mM ammonium acetate in 10:90 (v/v) water:methanol at a flow rate of 0.3 mL/min and detection in positive ionization mode. The standard curve was linear over the concentration range from 1 to 1000 ng/mL for MB and AZB with coefficient of determination above 0.9930. The lower limit of quantification was 1 ng/mL using 20 μL of rat plasma sample. The intra- and inter-assay precision and accuracy were <12%. The developed analytical method was successfully applied to the pharmacokinetic study of MB and AZB in rats.