Drx 600 spectrometer

The HR-ESI-MS data were obtained on an Agilent 6210 time of flight LC-MS instrument (Agilent Technologies Inc., Palo Alto, CA, USA). NMR experiments were conducted on a Bruker DPX-400 NMR spectrometer (400 MHz for ¹H NMR and 100 MHz for ¹³C NMR) or Bruker DRX-600 spectrometer (600 MHz for ¹H NMR and 150 MHz for ¹³C NMR) (Bruker Corporation, Karlsruhe, Germany). The chemical shifts were given in δ (ppm) and referenced to the solvent signal (DMSO-d₆, δ_H 2.50, δ_C 39.5; acetone-d₆, δ_H 2.05, δ_C 29.8). Column chromatography (CC) was accomplished on silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), ODS (40–70 µm, Merck Company, Darmstadt, Germany) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Semi-preparative reverse-phase (RP) HPLC was performed on a Hitachi HPLC system with a L-7110 pump, a L-7420 UV/vis detector and an Hypersil RP-C₁₈ column (5 µm, 250 × 10.0 mm, Thermo Fisher Scientific, Waltham, MA, USA). Thin-layer chromatography (TLC) was conducted on silica gel GF254 (10–20 µm, Qingdao Marine Chemical Inc., Qingdao, China). All chemicals used were of HPLC grade or analytical grade.

To analyze the transformation of DON, C20 was added to broth containing 10 μg ml⁻¹ DON and incubated at 30°C on a rotary shaker (180 rpm) for 72 h. Culture broth containing C20 alone was used as a control. Samples were prepared as described above and detected by HPLC. The mobile phase was methanol/water (50:50, v/v) with the flow rate of 0.8 ml min⁻¹.
To investigate the structure of DON degradation products, C20 was inoculated into MM medium with 100 μg ml⁻¹ DON and cultured for 72 h at 30°C with shaking at 180 rpm. The DON degradation products were extracted three times with ethyl acetate. The organic phase was pooled, concentrated and applied to a Waters 1525 Prep-HPLC system (Milford, MA, USA) equipped with a UV detector. Each 20 μl extract was injected onto an XBridge™ Prep C₁₈ column (19 mm × 100 mm, 5 μm, Waters) with methanol/water (50:50, v/v) at a flow rate of 2 ml min⁻¹. DON and its degradation products were detected at a wavelength of 218 nm at 35°C. The resulting degradation products were dried under an N₂ stream and re-dissolved in CDCl₃ for nuclear magnetic resonance (NMR) analysis.
The ¹H NMR spectra of the biodegradation products were measured by a Bruker DRX-600 spectrometer operated at 600 MHz. The chemical shift (δ) was recorded in parts per million (ppm) relative to the solvent signals [δ(H) 7.26], and the coupling constant (J) was measured in Hz.

The High-performance liquid chromatography (HPLC) system includes two LC-20AD pumps (Shimadzu, Japan), an SPD-20A UV-Vis detector, a thermostat column, and an Agilent ZORBAX SB-C18 column (5 µm, 4.6 × 250 mm). The eluation system consists of water with 0.1% formic acid (mobile phase A) and methanol (mobile phase B), and the flow rate was 0.8 mL/min during the following gradient: 0.00–35.00 min, 30–100% mobile phase B; 35.00–40.00 min 100 mobile phase B. The 1D NMR spectra were recorded in CD₃OD using a Bruker DRX-600 spectrometer (Bruker, Rheinstetten, Germany) with tetramethylsilane (TMS) as the internal standard. Cells were cultured in a constant temperature incubator shaker (Zhicheng Analytical Instrument, Shanghai, China). The ligand fishing process was completed by a high-speed centrifuge (DLABsci Instrument, Beijing, China) and a vortex oscillator (Crystal Instrument, HYQ-3110, USA). HPLC-MS/MS analysis was performed on a Waters ACQUITY system coupled with a XEVO TQ MS triple-quadrupole mass spectrometer (Waters, Milford, PA, USA). A microplate reader (ThermoFisher, Multiskan GO, USA) was used for enzymatic activity assay. A Shimadzu 8030 LC-MS (Shimadzu, Japan) was used for compound identification.

Spectroscopic analysis of urine (n = 47 for discovery cohort, n = 322 for replication cohort) and plasma samples (n = 48 for discovery cohort, n = 328 for replication cohort) was performed on a Bruker DRX600 spectrometer equipped with either a 5-mm TXI probe operating at 600.13 MHz or a 5-mm BBI probe operating at 600.44 MHz. The 90° pulse length was determined prior to each run and field frequency was locked using D₂O as solvent. Detailed procedures are included in supplementary materials.

NMR spectra were recorded on a Bruker DRX-600 spectrometer. Aβ(1–42) peptide (500 µM) was dissolved in HFIP/water-D₂O 50/50 v/v. The 1D ¹H homonuclear spectrum was recorded in the Fourier mode, with quadrature detection. The 2D ¹H homonuclear TOCSY and NOESY experiments were acquired in the phase-sensitive mode using quadrature detection in ω1 by time-proportional phase incrementation of the initial pulse [48 (link),49 (link),50 (link)]. The water signal was suppressed by excitation sculpting experiments [51 (link)]. Before Fourier transformation, the time domain data matrices were multiplied by shifted sin² functions in both dimensions. A mixing time of 80 ms was used for the TOCSY experiments. NOESY experiments were run at 298 K with mixing times of 200 ms.

UV spectra were recorded on a PerkinElmer Lambda spectrophotometer. The NMR experiments were performed on a Bruker DRX-600 spectrometer at 300 K. The mass spectrometry analyses were performed with a Q-TOF premier spectrometer (Waters, Milford, MA, USA), coupled with an Alliance HPLC module (Waters, Milford, MA, USA). TLC was performed on precoated Kieselgel 60 F₂₅₄ plates (Merck, Darmstadt, Germany). Column chromatography was performed over Sephadex LH-20 (Pharmacia); reversed-phase (RP) HPLC separations were conducted on a Shimadzu LC-8A series pumping system equipped with a Shimadzu RID10 A refractive index detector and a Shimadzu injector, using a Bondapak C₁₈ column (30 cm × 7.8 mm, 10 µm, Waters, Milford, MA, USA). A Metrohm 827 pH meter, a two-channel laboratory pH measuring instrument for measuring pH/mV and temperature, was used for the pH measurement. The UE was carried out using a 320 W Ultrasonic bath (Branson 2510E-MTH, Bransonic®). The sample was placed in an Erlenmayer flask with the corresponding amount of solvent and was treated with ultrasound at 25°C for a given duration (Table 1). MAE was performed using a multimodal household microwave oven Silvercrest SMW 700 A1 at 700 W and a 100 mL flask exposed to microwave irradiation (irradiation cycle: 10 s power on, followed by 10 s power off) for a given duration (Table 1) [13 (link)–15 ].

Measurements of the T₁ and T₂ relaxations and the [¹H]-¹⁵N heteronuclear nuclear Overhauser effect (NOE) were performed at 25°C on a Bruker DRX600 spectrometer. T₁ delays of 10, 50, 100, 200, 400, 600, 800, 1000, 1200 and 1500 ms were used with repeated 50, 400 and 1200 ms. T₂ delays of 0, 17, 34, 51, 68, 102, 136, 170, 204 and 238 ms were used with repeated 34, 102 and 204 ms. For the [¹H]-¹⁵N NOE measurement, independent saturated and unsaturated spectra were recorded in an interleaved manner. The spectral data were processed using NMRpipe [53 (link)]. The ¹⁵N T₁ (= 1/R₁) and T₂ (= 1/R₂) relaxation values were analyzed by fitting the series of peak intensities to an exponential decay curve in Sparky [54 ]. The NOE data were obtained by calculating the peak intensity ratios between the saturated and unsaturated NOE spectra. The relaxation parameters containing R₁, R₂ and NOE with error values were fitted to model-free equations using the Tensor2 program [55 (link)]. The rotational diffusion tensors were estimated using the X-ray monomer structure (PDB code 1DCF) as the template.