Aggressive dereplication using UHPLC–DAD–QTOF: screening extracts for up to 3000 fungal secondary metabolites

Abstract

In natural-product drug discovery, finding new compounds is the main task, and thus fast dereplication of known compounds is essential. This is usually performed by manual liquid chromatography-ultraviolet (LC-UV) or visible light-mass spectroscopy (Vis-MS) interpretation of detected peaks, often assisted by automated identification of previously identified compounds. We used a 15 min high-performance liquid chromatography–diode array detection (UHPLC–DAD)–high-resolution MS method (electrospray ionization (ESI)+ or ESI−), followed by 10–60 s of automated data analysis for up to 3000 relevant elemental compositions. By overlaying automatically generated extracted-ion chromatograms from detected compounds on the base peak chromatogram, all major potentially novel peaks could be visualized. Peaks corresponding to compounds available as reference standards, previously identified compounds, and major contaminants from solvents, media, filters etc. were labeled to differentiate these from compounds only identified by elemental composition. This enabled fast manual evaluation of both known peaks and potential novel-compound peaks, by manual verification of: the adduct pattern, UV–Vis, retention time compared with log D, co-identified biosynthetic related compounds, and elution order. System performance, including adduct patterns, in-source fragmentation, and ion-cooler bias, was investigated on reference standards, and the overall method was used on extracts of Aspergillus carbonarius and Penicillium melanoconidium, revealing new nitrogen-containing biomarkers for both species.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-013-7582-x) contains supplementary material, which is available to authorized users.

<i>Aspergillus flavus</i> Secondary Metabolites: More than Just Aflatoxins

“…Coupling HPLC-DAD to a mass spectrometer (MS) provides more information for identification 127,128) . High resolution mass spectrometry (HRMS) allows molecular formula estimation and simplifies dereplication 129) . Databases of characterized natural products searchable by monoisotopic mass, such as Antibase, can assist in dereplication 130) .…”

Metabolomics and Secondary Metabolite Profiling of Filamentous Fungi

“…Complementary to such a targeted, low-resolution tandem MS approach, LC-ESI-HRMS has successfully been used for untargeted profiling of fungal metabolites in the full scan mode. LC-ESI-HRMS-based methods are less suited for accurate quantification, however, they show the great advantage of allowing to inspect the presence of fungal metabolites by retrospective data analysis [23,55,56]. LC-ESI-HRMS-based untargeted metabolite profiling currently enables the most comprehensive, unbiased coverage of the secondary metabolome.…”

“…LC-MS and GC-MS combine high sensitivity and detector linearity over 3-4 orders of magnitude with excellent selectivity (physical metabolite separation by chromatography and separation of co-eluting analytes according to m/z ratio of intact ionized molecule-or metabolitespecific fragment ions) and thus allow to determine hundreds of metabolites in a single analytical run. Other techniques such as liquid chromatography-ultraviolet (LC-UV) are used complementary for metabolite profiling (e.g., [23,24]). …”

Four novel Talaromyces species isolated from leaf litter from Colombian Amazon rain forests

“…Three agar plugs from the Talaromyces isolates grown on CYA, MEA, YES and OA at 25°C for 7 days were extracted as described by Smedsgaard (1997). The detected metabolites were identified by comparison with standards according to Kildgaard et al (2014) and Klitgaard et al (2014). Colony texture and ascomata on OA after 2-week incubation.…”