Liquid Chromatographic Determination of Methylene Blue and Its Metabolites in Milk

Abstract

Abstract A method is described for the liquid chromatographic (LC) determination of trace levels of methylene blue (MB) and its metabolites in milk. The cleanup involves protein precipitation with acetonitrile, extraction into chloroform of MB and its metabolites (azure A [AZA], azure B [AZB], and azure C [AZC]), extraction by chloroform of thionin at pH 10, and solid-phase extraction on a disposable carboxylic acid column. LC separation and quantitation are performed with an isocratic acetonitrile-acetate buffer mobile phase on a cyano column. Average recoveries (5-20 ppb levels) of MB, AZA, AZB, and thionin from fortified milk were 83.2,60.0, 84.4, and 22.5%, respectively. Some incurred MB metabolites in milk are bound organically to a fraction of the milk substrate, whereas others are free demethylated forms of MB. Analysis of milk collected 8 h after administration of MB contained the following average levels (n = 6) of free MB and metabolites: MB, 31.0 ppb; AZA, 21.3 ppb; and AZB, 54.1 ppb. Depletion of free MB and metabolites AZA and AZB from milk after administration of MB to lactating cows occurred in less than 40 h. Metabolites of MB that formed complexes with organic compounds were hydrolyzed in part to the free forms. The leuco form of MB cou