A DOE-optimized supercritical fluid chromatography-mass spectrometry workflow complementing liquid chromatography for comprehensive profiling of Trichoderma reesei specialized metabolism.

Fungal secretomes are prolific sources of structurally diverse bioactive natural products, including peptaibols, lipopeptides, and polar metabolites. Their chemical complexity makes comprehensive untargeted profiling a persistent analytical challenge, particularly for highly polar compounds that are poorly retained in conventional reversed-phase liquid chromatography (RPLC). This study presents the development and optimization of a supercritical fluid chromatography (SFC) method coupled to high-resolution tandem mass spectrometry (HRMS/MS) for the untargeted metabolite profiling of a Trichoderma reesei secretome extract. The SFC method was systematically compared to an established RPLCHRMS/MS workflow to evaluate their respective analytical performances and chemical space coverage. RPLC demonstrated superior overall metabolite detectability, with 3726 features detected compared to 2301 in SFC, and excelled at resolving structurally related isomers, particularly within the peptaibol class where up to six isomers were baseline-separated. SFC, by contrast, provided complementary class-specific retention behavior and uniquely revealed highly polar amino sugar-like compounds alongside a series of 14-residue harzianine-like peptaibols that remained undetected under RPLC conditions. Molecular networking analysis confirmed that 43% of SFC-detected features were absent from RPLC datasets, demonstrating substantial orthogonality between the two separation modes. Neither approach alone was sufficient to capture the full chemical diversity of the secretome. These findings establish that integrating SFC and RPLC workflows substantially expands the accessible chemical space in fungal metabolomics and provides a robust framework for the discovery of bioactive natural products.