Analysis of methylene blue in human urine by capillary electrophoresis

A capillary electrophoresis method for the determination of the dye methylene blue (tetramethylthionine, MB) in human urine depending on liquid/liquid-extraction and diode array detection has been developed, validated, and applied to samples of healthy individuals, who had been dosed with methylene blue within clinical studies. After extraction with dichloromethane and sodium hexanesulfonate, sample extracts were measured on an extended light path capillary. The dye was detected simultaneously at 292 and 592 nm using methylene violet 3 RAX as internal standard. The limit of quantification was 1.0 microg/ml. The accuracy of the method varied between -15.2 and +0.8% and the precision ranged from 2.0 to 12.0%. The method was linear at least within 1.0 and 60 microg/ml. In contrast to earlier indirect determinations no leuco methylene blue (LMB) was directly detected in urine, whereas in aqueous test solutions containing surplus amounts of ascorbic acid leuco methylene blue was well separated from MB in a single run.