Detection of glutathione conjugates derived from 4-ipomeanol metabolism in bile of rats by liquid chromatography-tandem mass spectrometry

Earlier studies postulated that bioactivation of 4-ipomeanol by cytochrome P450 enzymes may occur through oxidation of its furan ring, following a mechanism similar to the bioactivation of other furan-containing compounds. This would lead to the formation of furan epoxides and alpha,beta-unsaturated di-aldehyde-reactive metabolites that can conjugate with glutathione. These metabolites are thought to be responsible for the cytotoxic and anticancer properties of 4-ipomeanol. We hypothesized that if 4-ipomeanol is metabolized following this pathway, its glutathione conjugates would be isobaric (molecular ion mass=492 Da) and would be excreted in bile. To investigate this hypothesis, we analyzed by liquid chromatography-tandem mass spectrometry the bile of rats administered d(0)/d(6) 4-ipomeanol (1:1 ratio) intravenously. Hexadeuterated 4-ipomeanol had all deuterium atoms incorporated on its aliphatic chain. Multiple reaction monitoring scans of bile for the mass transition: MH+/(MH-129)(+), which is characteristic of glutathione conjugates, detected four glutathione conjugates. The observation of the isotope cluster (M+1)(+)(d(0))/(MH+6)(+)(d(6)) in a 1:1 molar ratio confirmed that these conjugates were derived from 4-ipomeanol. Retention of the six deuterium atoms in the glutathione conjugates detected, (MH+6)(+), indicates that the bioactivation of 4-ipomeanol took place on the furan ring moiety. Rat hepatic microsomal incubations provided additional evidence. From this study, the mass of the reactive metabolites of 4-ipomeanol can be inferred. The inferred mass (186 Da) matches the mass postulated. A pathway of 4-ipomeanol bioactivation is proposed here. This work represents one step forward to understanding the mechanism of bioactivation of 4-ipomeanol.