Synthesis of 13 C 2 15 N 2 ‐labeled anti‐inflammatory and cytoprotective tricyclic bis (cyanoenone) ([ 13 C 2 15 N 2 ]‐TBE‐31) as an internal standard for quantification by stable isotope dilution LC‐MS method

Tricyclic bis (cyanoenone), TBE‐31, one of the most potent activators of the Keap1/Nrf2/antioxidant response element pathway, has been developed as a new anti‐inflammatory and cytoprotective agent. 13 C 2 15 N 2 ‐labeled TBE‐31 ([ 13 C 2 15 N 2 ]‐TBE‐31), which has two 13 C and two 15 N atoms in two cyano groups, was designed to develop a method for quantification of cell, tissue, and plasma levels of TBE‐31 that involves chromatography/mass spectrometry coupled with the use of a stable isotope‐labeled internal standard. [ 13 C 2 15 N 2 ]‐TBE‐31 was successfully synthesized in four steps from a previously reported intermediate, which is prepared in 11 steps from cyclohexanone, by introduction of two 13 C atoms with ethyl [ 13 C]formate and two 15 N atoms with hydroxyl[ 15 N]amine. The stable isotope dilution liquid chromatography–mass spectrometry method for quantification of TBE‐31 was successfully developed using [ 13 C 2 15 N 2 ]‐TBE‐31 to compensate for any variables encountered during sample processing and analysis.