Detection of ergosterol using liquid chromatography/electrospray ionization mass spectrometry: Investigation of unusual in-source reactions

Rationale In the field of natural products, de-replication of complex mixtures has become a usual practice to annotate known compounds and avoid their re-isolation. For this purpose, many groups rely on liquid chromatography coupled to high-resolution mass spectrometry (HPLC/MS) to deduce molecular formulae of compounds allowing comparison with public or in-house databases. Electrospray ionization (ESI) is usually considered as the method of choice for investigating a large panel of compounds but, in some cases, it may lead to unusual results as described in this article for ergosterol. Methods Ergosterol and other fungal sterols in methanolic solution were analysed using various chromatographic gradients with HPLC/MS using both ion trap time-of-flight MS 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 various ions observed. Results Contrary to other fungal sterols, ergosterol was found to be highly sensitive to oxidation during ESI. Putative structures were proposed based on MS/MS studies and known oxidation mechanisms of ergosterol by reactive oxygen species that could be formed in the ESI process. The proportion of acetonitrile in the eluent was found to influence this in-source oxidation, with an 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 ionization 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.