Agilent 1290 infinity 2 accurate mass q tof lc ms spectrometer

A 5000 mL scale-up of P. igniarius was cultured according to the above-mentioned fermentation conditions. After the 0.1 mg/mL TL was added, the co-culture was grown for another 3 days. The filtrate of the fermentation broth was concentrated to 500 mL and partitioned with EtOAc (3 × 500 mL). The EtOAc extract was then evaporated under reduced pressure to yield 290 mg of residue, which was subjected to Sephadex LH-20 column chromatography, eluted with petrol-CHCl₃-MeOH (5:4:1) to produce five fractions (A-E). Fraction B was purified through reverse-phase preparative HPLC using a mobile phase of MeOH-H₂O (45:55) to afford compound A (25.5 mg). Fraction D was separated by preparative RP-HPLC using MeOH-H₂O (62:38) to afford compound B (7.0 mg). The structures of the target compounds were identified by 1D and 2D NMR as well as HRESIMS analysis. 1D- and 2D-NMR spectra were obtained at 400 MHz for ¹H and 100 MHz for ¹³C, respectively, on Bluker 400 MHz spectrometers in methanol-d₄ with solvent peaks used as references. HRESIMS data were measured using an Agilent 1290 Infinity II Accurate Mass Q-ToF LC/MS spectrometer.

Nuclear Magnetic Resonance (NMR) spectra were detected at 400 MHz for ¹H and 100 MHz for ¹³C on Bruker AVIII 400 MHz spectrometers (Bruker Daltonic Inc., Billerica, MA, United States) in methanol-d₄ (CD₃OD) and chloroform-d (CDCl₃) with solvent peaks used as references. (–)-HRESIMS data were measured using an Agilent 1290 Infinity II Accurate Mass Q-TOF-LC/MS spectrometer (Agilent Technologies, Santa Clara, CA, United States). Column chromatography (CC) was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex LH-20 (Amersham Biosciences Inc., Shanghai, China). Preparative high-performance liquid chromatography (HPLC) was performed by using a Harbor NP7005C pump system (Harbor Sci. & Tech., China) equipped with Mgres/headra C18 (250 × 20 mm, 10 μm, Harbor Sci. &Tech., China). Thin layer chromatography (TLC) was carried out on precoated silica gel GF₂₅₄ glass plates. Spots were visualized under ultraviolet (UV) light or by spraying with 10% H₂SO₄ in 95% ethanol (EtOH) followed by heating.