6530 accurate mass q tof lc ms system

Metabolite separation was performed using an Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS system (Santa Clara, CA, USA). The mobile phase consisted of water and 0.1% formic acid (eluent A) and acetonitrile (eluent B). Serum analyses were archived on an Agilent Zorbax extend C18 column (150 × 4.6 mm i.d., 5 μm). The flow rate was 1.0 mL/min with solvent A (water with 0.1% formic acid) and solvent B (acetonitrile). The chromatographic gradient was started at 5% phase B for the first minute, followed by an increase of phase B to 95% (from 0 to 50 min), and was then kept at 95% for 10 min (from 50 to 60 min). The gradient returned to initial conditions (5% phase B) in 0.5 min, which were maintained for 10 min. The injection volume of all samples was 10 μL. A calibrating solution containing reference masses at m/z 121.0509 (protonated purine) and m/z 922.0098 (protonated hexakis [1H,1H,3H-tetrafluoropropoxy]) in positive ion mode was continuously introduced. Mass spectrometry was performed with an electrospray ionization ion source in the positive (ESI+) ion mode. The MS parameters were set as follows: fragmental voltage at 120 V, nebulizer gas at 35 psig, capillary voltage at 4000 V, drying gas flow rate at 9 L/min, and temperature at 325 °C. The data were collected in centroid and profile mode with a mass range of 50–1500 m/z using the high-resolution mode (4 GHz).

An Agilent 1260 HPLC with a refrigerated autosampler (4 °C) equipped with a Synergi Hydro-RP column (50 × 2.0 mm, 2.5 µm, 100 Å, Phenomenex, Torrance, CA USA; Mobile phase in Table S1) was used for chromatography and samples were kept at 4 °C during the time of measurement with column temperature at 35 °C. An Agilent 6530 Accurate Mass Q-TOF LC–MS system (Santa Clara, CA USA) was used for detection with the settings shown in Table S1. Two scans per second were acquired between m/z 100 and 3200 with constant infusion of reference ions to maintain mass accuracy.

Optical rotations were determined on a JASCO P-2000 polarimeter (Hachioji, Tokyo, Japan). The CD spectrum was recorded with a Chirascan spectropolarimeter (Applied Photophysics, U.K.). The high-resolution electrospray ionisation mass spectra (ESI-MS) were obtained from an Agilent 6530 Accurate-Mass Q-TOF LC/MS system (CA, U.S.A.). The NMR spectra were recorded on Bruker AM600 FT-NMR and Bruker BioSpin 400 NMR spectrometers (MA, USA). Column chromatography (CC) was performed on silica gel (Kiesel gel 60, 70–230 mesh and 230–400 mesh, Merck, Darmstadt, Germany), YMC*GEL (ODS-A, 12 nm S-150 µm, YMC Co., Ltd., Japan) and Sephadex LH-20 gel (Pharmacia Biotech, Sweden). TLC used pre-coated silica gel 60 F₂₅₄ (1.05554.0001, Merck) and RP-18 F_254S plates (1.15685.0001, Merck, Germany). Preparative high-performance liquid chromatography (HPLC) was carried out using a Shimadzu LC-6AD (Shimadzu, Japan) instrument with a YMC-Pack ODS-A column (20 mm I.D.×250 mm, S-5 µm, 12 nm) and a SPD-20A wavelength detector at 210 nm. Soluble epoxide hydrolases (10011669) and PHOME (10009134) were purchased from Cayman (Cayman, MI, USA).

Optical rotations were determined using a DIP-370 automatic polarimeter (Jasco, Easton, MD, USA). The FT-IR spectra were measured using a Jasco Report-100 infrared spectrometer; The NMR spectra were recorded using an ECA 600 spectrometer (¹H, 600 MHz; ¹³C, 150 MHz, JEOL, Tokyo, Japan). Mass spectra were recorded on an LCQ advantage trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA) equipped with an electrospray ionization (ESI) source. High-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained using a 6530 Accurate-Mass Q-TOF LC/MS system (Agilent, Santa Clara, CA, USA). Column chromatography was performed using silica gel (Kieselgel 60, 70–230, and 230–400 mesh, Merck, Darmstadt, Germany) and YMC RP-18 resins (Merck), and thin layer chromatography (TLC) was performed using pre-coated silica-gel 60 F₂₅₄ and RP-18 F₂₅₄S plates (both 0.25 mm, Merck).

Serum metabolite separation was performed on a SynergiTM Fusion-RP C18 column (50 mm × 2 mm internal diameter (i.d.), 2.5 μm) using an Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS system (Santa Clara, CA, USA). The mobile phase consisted of solvent A (0.1% aqueous formic acid solution) and solvent B (0.1% acetonitrile formic acid). The chromatographic conditions were as follows: 0–2 min, 0–5% B; 2–10 min, 5–60% B; 10–15 min, 60–70% B; 15–20 min, 70–80% B; 20–22 min, 80–95% B; 22–28 min, 95–60% B; 28–30 min, 60–30% B, and back to initial conditions (with 2 min for equilibration). The flow rate was 0.4 mL/min. Each sample injection volume was 5 μL. The MS was performed with an electrospray ionization (ESI) ion source in the positive (ESI+) and negative (ESI−) ion modes. The data were collected in both positive and negative ion modes with a mass range of 50–2000 Da. The MS parameters were set as follows: fragmental voltage, 120 V; nebulizer gas, 35 psig; capillary voltage, 4000 V; drying gas flow rate, 9 L/min; temperature, 325 °C.