A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO), it forms methyl radicals (CH3) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO formation. However, we recently reported that CH3 might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated α-[4-pyridyl 1]-N-tert-butyl nitrone, d0/d9-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO and DMSO. In addition to identifying the two well-known free radicals, CH3 and OCH3, from this interaction, we also characterized two additional free radicals, CH2OH and CH2S(O)CH3. Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/CH3 was not detected. However, with the addition of the LC/MS technique, we found ∼0.75 μM POBN/CH3 hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.