Cobalamin as an analytical tool for analysis of oxirane metabolites of 1,3-butadiene: development and validation of the method

The reduced form of vitamin B12 [cob(I)alamin] is known to be a supernucleophile, with the ability to react 10(5) times faster than standard nucleophiles. Procedures have been developed where cob(I)alamin is used as an analytical tool for the trapping of electrophilically reactive compounds. In the present work, a sensitive and accurate method for determination of reactive metabolites produced in vitro has been developed and validated. Diepoxybutane (DEB), a metabolite of 1,3-butadiene, was used as a model compound. The intermediate precursor 1,2-epoxybutene (EB) was incubated in a mouse liver S9 metabolic system and the formation of DEB was studied. Samples were taken at different times from the incubation mixture and added to the cob(I)alamin. The alkyl-cobalamins (alkyl-Cbl) formed were directly analysed by a miniaturized LC-MS/MS method and column switching. The assay was linear over the concentration range of 1.5-500 microM with acceptable precision and accuracy.