Fusaric acid as a novel proton-affinitive derivatizing reagent for highly sensitive quantification of hydroxysteroids by LC-ESI-MS/MS

A highly sensitive derivatization method for liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry of dehydroepiandrosterone (DHEA), testosterone (T), pregnenolone (P5), and 17α-OH-pregnenolone (17-OHP5) was developed based on the use of fusaric acid as a reagent. DHEA, P5, and 17-OHP5 were rapidly and quantitatively converted to the 3-fusarate esters by treatment with fusaric acid and 2-methyl-6-nitrobenzoic anhydride. The positive ESI-mass spectra of the fusarate esters of each steroid were dominated by the appearance of [M + H]+ as base peaks. The fusarate derivatization of these steroids showed 17.6-fold (DHEA), 11.9-fold (P5), 3.3-fold (17-OHP5), and 1.8-fold (T) higher sensitivity to those of the corresponding picolinate derivatives in LC-selected reaction monitoring.