*3.6. Extraction and LC-MS*/*MS Analysis*

For LC-MS/MS analyses of Ncps, the lyophilized (10 mg DW) biomass of *N*. *edaphicum* CCNP1411 was homogenized by grinding with mortar and pestle, and extracted in 1 mL of milliQ water, 20% methanol (pH 3.5, 6.0 and 8.0) and 50% methanol in water. The pH was adjusted with 0.5 M HCl and 1.0 M NaOH. In addition, the fresh material (500 mg FW) was extracted in 20% methanol in water. The samples were vortexed for 15 min and centrifuged at 14,000 rpm for 10 min, at 4 ◦C. The collected supernatants were directly analyzed by LC-MS/MS system.

The LC-MS/MS was carried out on an Agilent 1200 HPLC (Agilent Technologies, Waldbronn, Germany) coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer QTRAP5500 (Applied Biosystems MDS Sciex, Concord, ON, Canada). The separation was achieved on a Zorbax Eclipse XDB-C18 column (4.6 mm ID × 150 mm, 5 μm; Agilent Technologies, Santa Clara, CA, USA). The extract components were separated by gradient elution from 10% to 100% B (acetonitrile with 0.1% formic acid) over 25 min, at a flow rate of 0.6 mL/min. As solvent A, 5% acetonitrile in MilliQ water with 0.1% formic acid was used. The mass spectrometer was operated in positive mode, with turbo ion source (5.5 kV; 550 ◦C). An information-dependent acquisition method at the following settings was used: for ions within the *m*/*z* range 500–1100 and signal intensity above the threshold of 500,000 cps

the MS/MS spectra were acquired within the *m*/*z* range 50–1000, at a collision energy of 60 eV and declustering potential of 80 eV. Data were acquired with the Analyst ® Sofware (version 1.7 Applied Biosystems, Concord, ON, Canada).
