Agilent 6530 accurate mass q tof mass spectrometer

The metabolomics analysis was performed with an Agilent 1290 Infinity LC system coupled to an Agilent 6530 Accurate-mass Q-TOF mass spectrometer (Agilent Technologies, Palo Alto, CA, United States). Chromatographic separation of serum samples was performed on an Agilent ZORBAX SB-C18 threaded column (2.1 × 150 mm, 1.8 µm, Agilent Technologies, Palo Alto, CA, United States) maintained at 35°C. The mobile phase consisted of solvent A (0.1% formic acid in water, v/v) and B (0.1% formic acid in acetonitrile, v/v). The optimized gradient program was established. The post time was set to 3 min for equilibration. Mass spectrometry was performed in both positive (ESI+) and negative (ESI-) electrospray ionization modes. The fragment voltage was set to 135 V and the skimmer voltage was set to 65 V. The capillary voltages were set to 4.0 KV in positive mode and 3.5 KV in negative mode. The drying gas (nitrogen) was used at a flow rate of 10 L/min at 350°C and the nebulizer pressure was set to 45 psig. Data were collected in centroid mode from 50 to 1000 m/z using an extended dynamic model.
Serum samples were analyzed in random order during the analysis. In addition, QC samples were detected once every 6 subject samples for conditioning of the analytical system, signal correction, and quality assurance.

Samples
were reconstituted in 100 μL of Nanopure water prior to analytical
separation, which was carried out using an Agilent 1260 Infinity II
HPLC (Agilent Technologies, Santa Clara, CA, USA). Chromatographic
separation was performed on a 150 mm × 1 mm Hypercarb column
from Thermo Scientific (5 μm particle size). The column compartment
was set at 40 °C. A binary gradient was employed and consisted
of solvent A: (3% (v/v) ACN, 0.1% FA in water) and solvent B: (90%
ACN, 0.1% FA in water). A 45 min gradient with a flow rate of 0.132
mL/min was used: 3–25% B, 0–15 min; 25–25% B,
15–18 min; 25–99% B, 18–30 min; 99–99%B,
30–32 min; 99–3% B, 32–34 min; 3–3% B,
34–45 min.
HPLC was coupled to Agilent 6530 Accurate-Mass
Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).
The MS detector was run in the positive mode with the following electrospray
source parameters: drying gas temperature = 150 °C. drying gas
flow rate = 11 L/min, fragmentor = 175 V, skimmer = 60 V, octupole
1 RF = 750 V. Acquisition mode was set to data-dependent mode, where
top 5 most abundant precursor ions were selected for fragmentation.
Dynamic exclusion was enabled for 30 s. The acquisition rate was set
to 0.63 spectra/s. For tandem MS fragmentation, a linear function
for collision energy (CE), where CE = 1.45*(m/z)-3.5, was employed.

Optical rotations were measured on a JASCO P-2000 polarimeter (JASCO Corporation, Tokyo, Japan), UV/Vis data were obtained using a Beckman DU800 spectrophotometer (Beckman Coulter, Brea, CA, USA), and IR spectra were recorded on a Nicolet 100 FT-IR spectrometer (Nicolet, Madison, WI, USA). NMR data were obtained on a Bruker AVANCE III 600 MHz NMR with a 1.7 mm dual tune TCI cryoprobe (Bruker, Billerica, MA, USA) and the 1D TOCSY experiments were performed on a JEOL ECZ 500 NMR spectrometer equipped with a 3 mm inverse probe (H3X) (JEOL Ltd., Tokyo, Japan). Data were recorded in either pyridine-d5 or methanol-d4 and adjusted to the residual solvent peak (pyridine-d5 δ_H 7.22, δ_C 123.87; methanol-d4 δ_H 3.31, δ_C 49.00). Deuterated NMR solvents were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). For high resolution electrospray mass spectrometric analysis (HR–ESI–MS), an Agilent 6530 Accurate Mass QTOF mass spectrometer was used in the positive ion mode (Agilent, Santa Clara, CA, USA). Semi-preparative HPLC was performed on a Thermo Fisher Scientific HPLC system with a Thermo Dionex UltiMate 3000 pump, RS autosampler, RS diode array detector, and automated fraction collector (Thermo Fisher Scientific, Waltham, MA, USA). All solvents were HPLC grade except for water, which was purified by a Millipore Milli-Q system before use.