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

Abstract

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 highresolution 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-offlight 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.

Adapting an Ergosterol Extraction Method with Marine Yeasts for the Quantification of Oceanic Fungal Biomass

“…For the developed LC-APCI-MS/MS method, the observed ionization was in agreement with results obtained from Ory et al [ 29 ], with the [M+H-H 2 O] + and the [M+H-2H 2 ] + ions being the most intense. An LOD of 1.5 nM (S/N = 3/1) and an LOQ of 5.0 nM (2 ng/mL) at S/N = 10/1 could be obtained, thus being about 25 times more sensitive than with the HPLC-UV method.…”

“…To ensure the identity of the analyte applying the HPLC-UV method and to compare quantification, LC-MS/MS analysis was also performed. Ergosterol concentrations were quantified with LC-MS/MS similar to the protocol of Ory et al [ 29 ]. Briefly, an Agilent (Waldbronn, Germany) 1290 UHPLC System was used together with a Sciex (Framingham, MA, USA) QTrap 6500+ mass spectrometer and a Phenomenex (Aschaffenburg, Germany) Gemini column (150 × 4.6 mm, 5 µm particle size).…”

Metabolites Profiling and In Vitro Biological Characterization of Different Fractions of Cliona sp. Marine Sponge from the Red Sea Egypt

“…In addition, ion fragments at m / z 229 [M + H-H 2 O-C 12 H 24 ] + corresponded to the loss of the side chain beside the C 2 H 3 moiety ( Figure 4 D). With these data, and with the aid of the available literature, compound 10 was tentatively identified as β-sitosterol [ 43 ].…”

Metabolic Profiling and In Vitro Assessment of the Biological Activities of the Ethyl Acetate Extract of Penicillium chrysogenum “Endozoic of Cliona sp. Marine Sponge” from the Red Sea (Egypt)

“…Moreover, remaining fragments at m/z 173, 69, and 55 were also detected. Hence, compound 35 was identified as Ergosta-4,6,8(14),22-tetraen-3-one by comparison with the available literature [ 64 ]. Ergosta-4,6,8(14),22-tetraen-3-one was previously isolated from the terrestrial Penicillium ssp .…”