Agilent 1290 series uhplc system

The serum samples were measured on an Agilent 1290 series UHPLC system (Agilent Technologies, Santa Clara, USA) coupled with a ZORBAX RRHD 300 SB-C18 column (100 × 2.1 mm, 1.8-μm, Agilent Technologies, Santa Clara, USA) for chromatography and separation. During the analysis, the setting conditions were set as follows: sample maintaining temperature, 4 °C; injection volume: 4 μL; column temperature: 30 °C; flow rate, 0.30 mL/min. The mobile phases were composed as solvent A (0.1% formic acid in acetonitrile), and solvent B (0.1% formic acid in water). The gradient elution was setting as Table 1.Table 1

Mobile phases for serum metabolomics analysis

T (min)	A (v/v)%	B (v/v)%
0–1.0	95	5
1.0–9.0	95–60	5–40
9.0–19.0	60–10	40–90
19.0–21.0	10–0	90–100
21.0–25.0	0	100

A, 0.1% formic acid in acetonitrile; B, 0.1% formic acid in water

In this study, an Agilent 1290 series UHPLC system (Agilent Technologies, United States) was used to analyse the intracellular metabolic profile of H9c2 cardiomyocytes. A ZORBAX RRHD 300 SB‐C18 column (100 mm × 2.1 mm, 1.8 μm, Agilent Technologies) was applied for chromatographic separation. Mobile phase A was 0.1% formic acid in acetonitrile, and phase B was 0.1% formic acid in water. The column temperature was kept at 30°C, and the flow rate was 0.30 mL/min. The gradient elution of mobile phase A was set as follows: 0‐1.0 minutes, 95%; 1.0‐9.0 minutes, 95%‐60%; 9.0‐19.0 minutes, 60%‐10%; 19.0‐21.0 minutes, 10%‐0%; 21.0‐25.0 minutes, 0%. All samples were preserved at 4°C with a 4 μL injection volume during the analysis.
Then, mass spectrometry was carried out on an Agilent 6550A Q‐TOF/MS (Agilent Technologies, United States) with electrospray ionization (ESI) mode. The mass range was set from 80 to 1000 m/z, and the gas flow was set as 11 L/min. The conditions in positive ionization mode were set as follows: electrospray capillary voltage, 4.0 kV; gas temperature, 225°C; nebulizer, 45 pisg. And conditions in negative ionization mode were set as follows: electrospray capillary voltage, 3.0 kV; gas temperature, 200°C; nebulizer, 35 pisg. The other parameters were as follows: sheath gas temperature, 350°C; sheath gas flow rate, 12 L/min; nozzle voltage, 500 V.

HPLC experimental conditions: the atomized gas and dry gas are nitrogen, and the collision gas is helium. The capillary voltage is 3500 eV, the atomization temperature is 350 °C, the dry gas is 10.0 L / min, and the atomized gas is 206.85 kPa. The MS scanning range is m/z 80 ~ 1000, and the data storage mode is the centroid. The ion source was double ESI spray, and the data were collected by mass spectrometry through 2 known standards [Hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazine And 7H-purine, corresponding to m/z 922.00980 and 121.0509] for real-time correction. The reference solution was injected into the mass spectrum at a rate of 0.01 ml/min by an Agilentisonic pump. The AutoMS/MS experiment uses CID collision. Liquid chromatography is an Agilent 1290 series UHPLC system. The liquid phase is separated by a reversed-phase column ( Atlantis, T3, 150 m m × 2.1 mm, 3 μm,Waters,IRELAND), Mobi le phase A phase is 0.1% formic acid–water, and phase B is 0.1 percent formic acid- (methanol: acetonitrile = 1: 1), Gradient elution conditions under positive ion conditions: 0–40 min, 92% B; 17–32 min, flow rate 0.3 ml/min, column temperature 35 °C, injection volume 10 μl; Negative ion gradient elution conditions: 0–5 min, 33–35% B; 5–40 min, 35% -65% B, The column temperature was 35 °C and the injection volume was 10 μL.

Chromatography was performed using an Agilent 1290 series UHPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with quaternary pump, online degasser, autosampler, and thermostatted column compartment. The volume of injection samples was fixed at 4 μL. All samples were maintained at 4°C during the analysis. The separation was performed on a ZORBAX RRHD 300 SB-C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent Technologies, Santa Clara, CA, USA). The column temperature was set to 30°C. The mobile phases consisted of 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in water (solvent B). The flow rate was set to 0.30 mL/min. The following gradient was used: a linear gradient of 95% A for the first minute, 95–60% A from 1.0 to 9.0 min, 60–10% A from 9.0 to 19.0 min, 10–0% A from 19.0 to 21.0 min, and 0% A from 21.0 to 25.0 min. The eluent was directly introduced to the mass spectrometer. After the injection of 10 samples, a pooled sample, the QC sample, followed by a blank was injected in order to ensure the stability and repeatability of the LC–MS systems.

For cannabinoid analysis, 150 µl of ice-cold methanol and 10 µl of isotope-labeled internal standard mixture were added to 50 µl of each individual sample (PBS as blank sample, calibration standards, QC standards, and serum samples) for a 1-h protein precipitation. All the samples were then centrifuged at 13,000 × g for 20 min. For each sample, 180 µl of the supernatant was loaded into the center of corresponding wells of a 96-well deep well plate, followed by the addition of 90 µl of water to each well. The plate was then shaken at 600 rpm for 15 min before LC–MS/MS analysis. Mass spectrometric analysis was performed on an AB Sciex 5500 QTRAP^® tandem mass spectrometry instrument (Applied Biosystems/MDS Analytical Technologies, Foster City, CA) equipped with an Agilent 1290 series UHPLC system (Agilent Technologies, Palo Alto, CA). Data analysis was done using Analyst 1.6.3.

An Agilent 1290 series UHPLC system coupled to a 6460 Triple Quadrupole (QqQ) mass spectrometer (both Agilent Technologies, Waldbronn, Germany) was used to analyze the samples. Precursor and product ion selection as well as the optimization of collision energies were performed with flow injection of single-analyte solutions. UHPLC separations were performed in a reversed-phase C18 analytical column of 50 mm × 2.1 mm and 1.8 μm particle size (ZORBAX RRHD Eclipse Plus C18) by Agilent Technologies (Waldbronn, Germany).
The chromatographic solvents were water 0.1% formic acid solution (eluent A) and acetonitrile 0.1% formic acid (eluent B). The gradient program was as follows: 0.0 min, 10% B; 2.4 min, 42% B; 6.0 min, 51% B; 6.2 min, 95% B; 7.0 min, 10% B. A subsequent re-equilibration time (5 min) should be performed before next injection. The constant flow rate was 0.3 mL/min while the injection volume was 2 μL. Moreover, the column temperature was maintained at 30 °C.
MS/MS analyses of mycotoxins were performed on an 6460 QqQ mass spectrometer with Agilent Jet Stream Technology under the dynamic multiple reaction monitoring (DMRM) conditions in ESI⁺. The following settings were used: nebulizer, 40 psi; drying gas temperature, 350 °C; drying gas flow, 10 L/min; capillary voltage, 4000 V.