Detection of ergosterol by liquid chromatography/electrospray mass spectrometry - investigation of unusual in source reactions.

RATIONALE: In the field of natural products, dereplication of complex mixtures hasbecomes usual practice to annotate known compounds and avoid their re-isolation. To this purpose, many groups rely on liquid chromatography coupled to high resolution mass spectrometry to deduce molecular formulae of compounds allowing comparison with public or in-house databases. Electrospray ionisation is usually considered as the method of choice to investigate a large panel of compounds but, in some cases, it may lead to unusual results as described in the following article for ergosterol. METHODS: Ergosterol and other fungal sterols in methanolic solution were analysed using different chromatographic gradients by HPLC/MS on both IT-TOFMS and Orbitrap-MS instruments fitted with an ESI source. Further flow injection analyses were performed to investigate the influence of the solvent composition. MS/MS fragmentation data were acquired to annotate the different ions observed. RESULTS: Contrary to other fungal sterols, ergosterol was found to be highly sensitive to oxidation during electrospray ionisation. Putative structures were proposed based on MS/MS studies and known oxidation mechanisms of ergosterol by reactive oxygen species (ROS) that could be formed in the ESI process. The proportion of acetonitrile in the eluent was found to influence this in-source oxidation, with increased proportion of oxidized sodium adducts with higher proportions of acetonitrile. CONCLUSIONS: While ergosterol is a major sterol found in fungi, this study investigates its ionisation by electrospray for the first time. The results reported here will help further detection and annotation of this compound in fungal extracts after HPLC/ESI-MS analyses.