Agilent 1290 series uplc system

The samples were ground into fine powder using A100 with liquid nitrogen. A total of 0.8 g of the sample was extracted with 40 mL methanol by ultrasonication (30 min, 100 Hz) on ice. After centrifugation, the supernatant was filtered and stored at −20 °C before LC–MS analysis.
The analysis was performed on an Agilent 1290 series UPLC system (Agilent, Santa Clara, CA, USA) coupled with an Agilent G4212 UV detector. Samples were separated on an Agilent ZORBAX SB-C18 column (4.6 × 50 mm, 1.8 μm) at 25 °C. Liquid phase A, water (0.1% formic acid); Liquid phase B, acetonitrile (0.1% formic acid); the gradient program: 80:20 v/v at 0 min, 70:30 v/v at 15 min, 50:50 v/v at 25 min, 25:75 v/v at 35 min, 0:100 v/v at 40 min, 0:100 v/v at 50 min, and 80:20 v/v at 60 min; flow rate, 0.75 mL/min; temperature, 30 °C; and injection volume, 10 μL. The DAD was set to 283 nm for naringin, 310 nm for isoimperatorin, 320 nm for bergapten, and 330 nm for rhoifolin. The external standard method was used for quantitative identification. Naringin (Lot. No. 1226D021), rhoifolin (Lot. No. 727B021), isoimperatorin (Lot. No. 1229A025) and bergapten (Lot. No. 619B021) were purchased from Aladdin Reagent Co., Ltd., China. These standard samples were serially diluted with methanol to obtain the standard working solutions for quantification.

An Agilent 1290 series UPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump, an autosampler, and a thermostatically controlled column compartment was used for the chromatographic analysis. A Waters ACQUITY™ BEH C18 column (100 × 2.1 mm, 1.7 μm; Milford, MA, USA) was used for sample separation at 40°C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) using a gradient program of 0–2 min, 20% B; 2–12 min, 20–28% B; 12–20 min, 28–45% B; 20–35 min, 45–48% B; and 35–46 min, 48–75% B. The sample volume injected was 2 μL, and the solvent flow rate was set at 0.4 mL/min. For mass spectrometric determination, the UPLC system was hyphenated with an ultrahigh definition accurate mass quadrupole time-of-flight mass spectrometry (MS) system (Agilent Technologies G6540A) by a multimode ionization source (G1978-65339) interface. The conditions of MS analysis were optimized as follows: drying gas (N₂) flow rate, 8.0 L/min; drying gas temperature, 300°C; nebulizer, 45 psi; capillary, 2500 V; and fragmentor voltage, 150 V. Mass spectra were recorded across the range m/z 100–1700 in both positive and negative modes. All operations and data analysis were controlled by Agilent MassHunter Workstation software version B.04.00.

Chromatographic analysis was performed on an Agilent1290 series UPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump, an online degasser, an autoplay sampler, and a monitoring column thermostat. Chromatographic separation was carried out at 30°C on a Waters Acquity UPLC C₁₈ column (2.1×100 mm, 1.7 μm) (Waters, Milford, MA, USA). A mobile phase was consisting of 0.1% acetic acid (A) and acetonitrile (B) in a gradient elution manner as following: 10-14% B at 0-3 min, 14-16% B at 3-9 min, 16-25% B at 9-13 min, 25-80% B at 13-14 min, 80% B at 14-16 min. The re-equilibration time of gradient elution was 2 min between each run. The sample injection volume was 2 μL, and the flow rate was 0.35 mL/min. MS detection was performed on an Agilent 6460 Triple Quadrupole Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ionization (ESI) source. The mass spectrum was chosen in positive mode, and the MS spectra were acquired in MRM mode. The drying gas (N₂) flow rate was 11.0 L/min; the drying gas temperature was 300°C; the nebulizer was 15 psig, and the capillary voltage was 4000 V. The precursor ion, production, fragment, and collision energy (CE) were adjusted to obtain the highest abundance of each analyte (Table 1).

Freshly-thawed plasma samples were analyzed at Baylor College of Medicine Metabolomics Core facility. Metabolites were extracted from plasma using previously described standard procedures for targeted metabolomic profiling using ultrahigh-performance liquid chromatography (UPLC)-tandem mass spectrometry [4–8 (link)]. Pooled plasma samples were used as quality controls. For extraction of the metabolome, 100 μl of plasma was mixed with a methanol mixture containing equimolar amounts of eight internal standard compounds, and metabolic extraction was performed using consecutive application of ice-cold organic and aqueous solvents (water: methanol: chloroform: water, with a ratio of 1:4:3:1) followed by deproteinization and drying of the extract. The dried extract was resuspended in injection solvent and analyzed using UPLC-tandem mass spectrometry (Agilent 1290 series UPLC system equipped with a degasser, binary pump, thermostatted autosampler and column oven, Agilent Technologies, CA, USA). The multiple reaction monitoring-based measurement of relative metabolite levels was performed using normal phase chromatographic separation. All samples were kept at 4°C, and analysis was performed on aliquots of 5 and 10 μl.

Reaction stoichiometry was determined by correlating the amounts of O₂ consumed and vanillin produced using an oxygraph and HPLC-based assays. The sample was prepared by reacting 50 μM lignostilbene or DCA-S with ∼0.1 μΜ LSD4 in air-saturated 40 mM HEPES (I = 0.1 M, pH 7.5) at 25 °C. The reaction was quenched upon the consumption of ∼50 μM O₂ with 1% (v/v) glacial acetic acid. The protein was removed by centrifugation and passage through 0.22 μm filter. Vanillin produced from the LSD4-catalyzed reaction was quantified using an Agilent 1290 series UPLC system equipped with a Luna 2.5 um C18(2)-HST 100 Å column (Phenomenex) and a gradient of acetonitrile in 0.16% formic acid. Vanillin was detected at 265 nm using an Agilent 1290 diode array detector. Peak areas were converted to concentrations using a calibration curve established with authentic vanillin (R² > 0.995).

The γ-[Glu]_{(n=1, 2, 3, 4)}-Trp peptides in the samples were analyzed by the UPLC-Q-TOF-MS/MS system [21 (link)]. Agilent 1290 series UPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with an Agilent ZORBAX RRHD SB-C18 column (2.1 mm × 50 mm, 1.8 μm; maintained at 30 °C) was used to separate the peptides. A maXis Impact Q-TOF MS/MS system (Bruker Daltonics, Beijing, China) equipped with an electrospray ionization (ESI) probe was used for detection. Mobile phase A was 0.1% formic acid-methanol water solution, and mobile phase B was 0.1% formic acid-water solution. The flow rate was maintained at 0.5 mL/min throughout the analysis, and the elution conditions were 0–5 min, 90–85% (A); 5–10 min, 85–20% (A); 10–15 min, 20–90% (A); 15–25 min, 90% (A). A 10-μL aliquot of each sample was injected for analysis, and the mass range was from 50 to 1000 m/z.