Determination of methylene blue and detection of its demethylated metabolites in blood plasma using micellar electrokinetic chromatography with UV detection.

A new approach to the direct determination of the drug methylene blue (MB) in blood plasma was developed based on MEKC. We used liquid-phase extraction with acetonitrile and dichloromethane and solid-phase extraction on a strong cation exchange sorbent to extract the analyte from the matrix. Using UV detection (292 nm) and a 50 μm internal diameter silica capillary with a total length of 38 cm (31.5 cm effective) filled with 40 mM phosphate Na, 150 mM SDS, 5 % (v/v) isopropanol, and 12 % (v/v) n-butanol (pH 2.06), an LOD of 5.8 ng/mL for MB and a total analysis time of 16.5 min were obtained. The accuracy was 97.4-103 %, and intra- and interday imprecision were 5.7 and 2.5 %, respectively. This approach was tested on blood samples from rats that were injected with MB. The developed approach also allows for the detection of demethylated MB metabolites (azure A and azure B) and is suitable for the analysis of organs (the brain, liver, and kidneys).