Q exactive obitrap mass spectrometer

Optical rotations were measured with an Autopol IV polarimeter (Rudolph, Hackettstown, NJ, USA). UV spectra were measured on a UV-2450 spectrometer (Hitachi High-Technologies, Tokyo, Japan). CD spectra were recorded with an Applied Photophysics spectrometer (Chirascan, New Haven, CT, USA). One-dimensional and 2D spectra were recorded on a Bruker AV-600 spectrometer (Bruker, Karlsruhe, Germany) with TMS as an internal standard. HRESIMS spectra were recorded on Q Exactive Obitrap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA). Medium pressure liquid chromatography (MPLC) was performed on a Biotage SP1 System and column packed with RP-18 gel (Biotage, Uppsala, Sweden). Silica gel (Qingdao Marine Chemical Factory, Qingdao, China), RP-18 gel (Fuji Silysia Chemical Factory, Kasugai, Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Factory, Uppsala, Sweden) were used for column chromatography (CC). Semi-preparative HPLC experiments were carried on Agilent 1260 HPLC with Zorbax SB-C₁₈ column (Agilent, Palo Alto, CA, USA, 5 μm, 9.4 mm × 150 mm). Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Factory, Qingdao, China), and spots were visualized by heating silica gel plates sprayed with vanillin and 10% H₂SO₄ in EtOH.

UV spectra were afforded with a UH5300 UV-VIS Double Beam Spectrophotometer. ECD spectra were obtained on a Chirascan CD spectrometer (Applied Photophysics, London, UK). HRESIMS spectra were recorded on a Q Exactive Obitrap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) and UPLC-ESI-Q-TOF-MS (1290 UPLC-6540, Agilent Technologies Inc., Palo Alto, CA, USA) HRMS spectrometer. IR spectra were obtained with a Shimadzu Fourier Transform Infrared Spectrometer using KBr pellets. NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer with TMS as an internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. Column chromatography (CC) was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China) and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden). Medium Pressure Liquid Chromatography (MPLC) was performed on a Biotage SP1 System and columns packed with RP-18 gel. Preparative high-performance liquid chromatography (prep-HPLC) was performed on an Agilent 1260 liquid chromatography system equipped with Zorbax SB-C18 columns (Agilent, 5 μm, 9.4 mm × 150 mm) and a DAD detector. Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China).

Each prepared metabolite sample was injected onto a Thermo Fisher Scientific Vanquish UHPLC with a Waters Acquity UPLC BEH C18 column (1.7μm, 2.1×100mm; Waters Corp., Milford, MA, USA) and analyzed using a Thermo Fisher Q Exactive obitrap mass spectrometer in negative ionization mode. LC separation was performed over a 25 minute method with a 14.5 minute linear gradient of mobile phase (buffer A, 97% water with 3% methanol, 10mM tributylamine, and acetic acid-adjusted pH of 8.3) and organic phase (buffer B, 100% methanol) (0 minute, 5% B; 2.5 minute, 5% B; 17 minute, 95% B; 19.5 minute, 5% B; 20 minute, 5% B; 25 minute, 5% B, flow rate 0.2mL/min). A quantity of 10μL of each sample was injected into the system for analysis. The ESI settings were 30/10/1 for sheath/aux/sweep gas flow rates, 2.50kV for spray voltage, 50 for S-lens RF level, 350C for capillary temperature, and 300C for auxiliary gas heater temperature. MS1 scans were operated at resolution = 70,000, scan range = 85–1250m/z, automatic gain control target = 1 × 10⁶, and 100ms maximum IT. Metabolites were identified and quantified using El-MAVEN (v0.12.1-beta) with metabolite retention times empirically determined in-house. Metabolite levels were compared using the peak AreaTop metric.