Agilent mass hunter software

All of the animals were deeply anesthetized with chloral hydrate and decapitated after the last behavioural test. Their brains were rapidly removed and put on ice. Adhered blood was rinsed by ice‐cold normal saline, and then, the hippocampi were dissected, frozen in liquid nitrogen and stored at −80°C. 5‐HT, NE and DA content in the hippocampus were determined by high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS), using an Agilent 1200 series HPLC system (Agilent, USA). The chromatographic separation was carried out on an Agilent XDB C18 column (50 × 4.6 mm, 5 m; Waters) at 30°C. The samples were separated using a gradient mobile phase consisting of 5% methanol and 95% water at a flow rate of 0.2 mL/min. The injection volume was 10 μL. The mass spectrometer (Agilent, 6410B, USA) was operated in the positive ion electrospray mode with MRM. Data were acquired and processed using Agilent Mass Hunter software.

Samples from the colorimetric enzyme assay, as described above, were analyzed by RP-HPLC with an Agilent 1200 series chromatograph on a Vydac C₁₈ column (218TP5205, Hesperia, CA). Samples were injected (25 to 45 μl), and pNA substrate, pNA product, and native and modified forms of M^pro were eluted with a 2%/min acetonitrile gradient beginning with 95% solvent A (0.1% FA)/0.02% TFA) in RP-HPLC/MS grade water and 5% solvent B (0.1% FA/0.02% TFA in acetonitrile). The 2% gradient continued for 30 min and then was ramped to 95% acetonitrile for 2 min followed by a 5-min reequilibration to the starting conditions. Elution of samples was monitored at 320 nm (for pNA substrate) and 390 nm (for pNA product) with an Agilent diode array detector followed by MS analysis with an Agilent 6230 time of flight MS configured with Jetstream. M^pro and its glutathionylated forms eluted between 24 and 26 min (approximately 57% acetonitrile). The mass of the protein was determined by protein deconvolution using Agilent’s Mass Hunter software. The TOF settings were the following: gas temperature, 350°C; drying gas, 13 liters/min; nebulizer, 55 lb/in²; sheath gas temperature, 350°C; fragmentor, 145 V; skimmer, 65 V. The mass determination for peptides was done by deconvolution (resolved isotope) using Agilent Mass Hunter software (Agilent).

Using similar methods to Zeng et al. (2015 (link)), gas chromatography mass spectroscopy (GC-MS) analysis was performed using the Agilent 6890 N Gas Chromatograph system (Agilent Technologies, USA) to identify the component profiles in the tested oils. Data acquisition and analysis was performed using the Agilent MassHunter software (Agilent Technologies, USA) based on the retention times and mass spectra.

All CE-TOF-MS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany), Agilent G1969A and G6220A Accurate-Mass TOF LC-MS system (Agilent Technologies, Palo Alto, CA, USA), Agilent 1100 and 1200 series isocratic high-performance LC pumps, G1603A Agilent CE-MS adapter, and Agilent CE electrospray ionization (ESI)-MS sprayer kit (G1600AX and G7100A). An Agilent G1607-60001 platinum ESI needle was used for anion analysis. The Agilent ChemStation software (ver. A.10.02, B.02.01.SR1, and B.03.02, C.01.07.SE1, Agilent Technologies, Waldbronn, Germany) for CE and the Agilent MassHunter software (ver. B.02.00, Agilent Technologies, Palo Alto, CA, USA) was used for the system control and data acquisition.

An Agilent 1200 HPLC (Agilent Technologies, Palo Alto, CA, USA) was used for chromatographic separation of analytes, and an Agilent 6410 QqQ triple quadrupole (Agilent Technologies, Palo Alto, CA, USA) equipped with ESI was used for the generation and detection of the eluted analyte ions. Agilent Mass Hunter software (Agilent Technologies, Palo Alto, CA, USA) was used for instrument data analysis and control. LC–MS/MS analytical parameters were optimized to achieve optimum separation of ENF, BNB, and AVB; AVB was used as an internal standard (IS) (Table 6). We used Agilent triple quadrupole mass analyzer operated in the positive ion mode with electrospray ionization (ESI) for mass analysis. Nitrogen (12 L/min) was used to dry the spray in the ESI source and the collision cell (60 psi) for dissociation studies. Direct injection was used to optimize all mass spectrometric analytical parameters to achieve the highest ion intensity. ESI source temperature (T) was set at 350 °C, while capillary voltage (V) was adjusted to 4000 V. Data acquisition was managed with the Mass Hunter software (Agilent Technologies, Palo Alto, CA, USA). The multiple reaction monitoring (MRM) mode of the QqQ was used to increase the selectivity and avoid interference of the HLM matrix in estimating ENF, BNB, and the IS, thereby elevating the LC–MS/MS method’s sensitivity [38 (link),39 (link),40 (link)].

Cells were extracted with 800 μl of methanol:chloroform:water, 2:1:1 (v:v) containing an internal standard (20 nM 2-isopropylmalic acid and D2-oleic acid). Samples were sonicated and centrifuged, and the upper aqueous phases were transferred to a glass test tube, and the metabolites such as those from glycolysis, TCA cycle, and pentose phosphate pathway (PPP) (group 1) were analyzed. The lower organic phase was transferred to new tubes for the analysis of fatty acids (group 2). All samples were evaporated and derivatized by using 20 mg/ml methoxyamine hydrochloride in pyridine coupled with MSTFA (group 1, Sigma, 69479) and BCl₃-methanol (12% w/w, group 2, Sigma, 33033) according to the manufacturer’s instructions. All samples were injected into the GC–MS system. Metabolites were analyzed using an Agilent 7000B gas chromatography system coupled with a 7000 C tandem mass spectrometric detector (Agilent GC-QQQ-MS/MS, Agilent Technologies) and equipped with an ultra HP-5 ms capillary column (30 m × 0.25 μm, i.d., 0.25 μm film thickness, Agilent J&W Scientific). The data processing for both qualitative and quantitative analyses was performed using Agilent MassHunter software (Agilent Technologies).