2.3. Extraction of Siderophores and Calibration

For extraction of siderophores, two-step liquid-liquid extraction was performed. Briefly, mycelia were sonicated and centrifuged to release intracellular content. FeCl₃ solution (100 μM) was added to the supernatant to saturate iron-free siderophores. Supernatants (50 or 100 µL for extracellular or intracellular metabolite screening, respectively) were spiked with ferrioxamine E (FoxE) as an internal standard at a final concentration of 100 or 50 ng/mL for extracellular or intracellular metabolites, respectively. The supernatants were extracted twice with ethyl acetate (triple volume) and dried under reduced pressure. The remaining aqueous phase was mixed with four volumes of methanol and frozen at −80 °C, 1 h. Precipitated proteins were removed by centrifugation (14,000× g, 4 °C, 10 min), and the supernatant layer was transferred to a vial with the evaporated ethyl acetate fraction and concentrated under reduced pressure.

Standard triacetylfusarinine C (TafC), FoxE, and ferricrocin (FC) ferriforms were obtained from EMC Microcollections GmbH (Tübingen, Germany) and used for calibration curve construction. The hydroxyferricrocin (HFC) was quantified using the FC calibration curve, assuming the same ionization efficiency. The calibration sequence consisted of 0.5, 1, 5, 10, 50, 100, 500, and 1000 ng/mL of TafC and FC final siderophore concentrations in an HPLC vial. The limits of detection (LOD) and quantitation (LOQ) were defined as the lowest concentrations for which the standard deviation of the intercept equaled 3.3 and 10, respectively. All samples were analyzed in triplicate, and results expressed as means ± standard deviation. The instrument performance was checked by a system suitability test using the HPLC peptide standard mixture (Sigma-Aldrich, Prague, Czech Republic).