Specific derivatization of internal alkynes for improved electrospray analysis

Many plant and fungal species, including foods, produce specialized metabolites that contain internal alkynes. Specific detection and analysis of naturally produced internal alkynes is often difficult, because of poor ionization, thermal instability, and a lack of internal-alkyne-specific chemical tools to facilitate analysis. We developed a derivatization protocol for improved LC-MS analysis of internal alkynes. The method exploits the ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) reaction to form trisubstituted triazoles, species with vastly improved electrospray ionization efficiencies compared to many naturally occurring internal alkynes. The method can be used in crude biological extracts and other complex samples, and increases the ionization efficiency by up to 32,000-fold. Additionally, using a fast (1 minute), direct LC-MS analysis of RuAAC reactions during derivatization, we observed the kinetics of formation and depletion of variety of catalyst-derived ruthenium complexes, some of which may provide mechanistic insight into the limitations of the RuAAC derivatization. We use this derivatization in the course of isolating a dietary internal alkyne from a hedgehog mushroom. This molecule proved to be a previously unreported analog of repandiol.