Agilent 6540 q tof mass spectrometer

Manufactured by Agilent Technologies

Sourced in United States

The Agilent 6540 Q-TOF mass spectrometer is a high-resolution, accurate-mass, time-of-flight (TOF) instrument designed for advanced analytical applications. It utilizes quadrupole technology to isolate and select specific ions, which are then analyzed by the TOF mass analyzer to provide high-resolution, accurate mass measurements.

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11 protocols using agilent 6540 q tof mass spectrometer

LC-TOF-MS Analytical Protocol for Metabolomics

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LC-TOF-MS analyses were performed by using an Agilent-1200 LC system coupled with an electrospray ionization (ESI) source (Agilent Technologies, Palo Alto, CA, USA) and an Agilent-6540 Q-TOF mass spectrometer. Separation of all samples were performed on an ACQUITY UPLC T₃ C₁₈ column (2.1 mm × 100 mm I.D., 1.8 μm) with a column temperature set at 35 °C. The flow rate was 0.45 mL/min, and the mobile phase was composed of 0.1% FA in acetonitrile and 0.1% FA in water. The following gradient program was used: 1% acetonitrile for 0–1 min; 1–70% acetonitrile for 1–10 min; 70–99% acetonitrile for 10–11 min; 99% acetonitrile for 11–13 min; re-equilibration step for 4 min. The sample injection volume was 2 μL.
Mass detection was operated in both positive and negative ion modes with the following setting: drying gas (N₂) flow rate, 8 L/min; gas temperature, 350 °C; pressure of nebulizer gas, 35 psig; capillary voltage, 4500 V; fragmentor, 145 V; skimmer voltage, 65 V; scan range, m/z 50–1400. All analyses were acquired using the instrument mass spray to ensure accuracy and reproducibility. Leucine enkephalin was used as the instrument reference mass (m/z 556.2771) at a concentration of 50 fmol/μL with the flow rate 40 μL/min. Data profile was recorded at a speed of 0.15 s/scan and the scanning delay of 0.01s during analysis.

Su T., Tan Y., Tsui M.S., Yi H., Fu X.Q., Li T., Chan C.L., Guo H., Li Y.X., Zhu P.L., Tse A.K., Cao H., Lu A.P, & Yu Z.L. (2016). Metabolomics reveals the mechanisms for the cardiotoxicity of Pinelliae Rhizoma and the toxicity-reducing effect of processing. Scientific Reports, 6, 34692.

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Optimized QTOF Mass Spectrometry Protocol

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MS data were recorded using an Agilent 6540 QTOF mass spectrometer (Agilent Technologies) equipped with a QTOF mass spectrometer and a jet stream electrospray ion source. Data acquisition were controlled by Mass Hunter Qualitative Analysis B.06 and Quantitative Analysis B.04 (Agilent Technologies). The optimized operating parameters in the negative ion modes were as follows: nebulizing gas (N₂) flow rate, 8.0 L/min; nebulizing gas temperature, 300°C; jet stream gas flow, 8 L/min; sheath gas temperature, 350°C; nebulizer pressure, 45 psi; capillary voltage, 3000 V; skimmer, 65 V; Octopole RFV, 600 V; and collision energy, 15 eV. Mass spectra were recorded across the range m/z 100–1300 with accurate mass measurement of all mass peaks. Deprotonated molecular ions of the 10 analytes were selected as their respective extraction ions in quantitative analysis using the extracting ion mode.

Zhong W.F., Tong W.S., Zhou S.S., Yip K.M., Li S.L., Zhao Z.Z., Xu J, & Chen H.B. (2017). Qualitative and quantitative characterization of secondary metabolites and carbohydrates in Bai-Hu-Tang using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and ultraperformance liquid chromatography coupled with photodiode array detector. Journal of Food and Drug Analysis, 25(4), 946-959.

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Affinity-based Proteomic Profiling of CSG Interactions

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Matrigel extract at 0.5 mg/ml in buffer containing 50 mM Tris, 5 mM EDTA‐Na and protease cocktail inhibitor (Thermo Scientific) was incubated with CSG‐coated Sulfolink beads (Pierce Biotechnology) at 4°C. After washing, bound proteins were eluted with the same buffer containing 2 mmol/l free CSG or CREKA peptide and separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Gel bands excised from silver‐stained gels were digested, and peptides were extracted as described previously (Bringans et al, 2008). Samples were analysed an Agilent 6540 Q‐TOF mass spectrometer (Agilent Technologies) with an HPLC Chip Cube source. The analyses were performed in the WA Proteomics Facility, supported by Lotterywest and Bioplatforms Australia, at the Harry Perkins Institute for Medical Research.

Yeow Y.L., Kotamraju V.R., Wang X., Chopra M., Azme N., Wu J., Schoep T.D., Delaney D.S., Feindel K., Li J., Kennedy K.M., Allen W.M., Kennedy B.F., Larma I., Sampson D.D., Mahakian L.M., Fite B.Z., Zhang H., Friman T., Mann A.P., Aziz F.A., Kumarasinghe M.P., Johansson M., Ee H.C., Yeoh G., Mou L., Ferrara K.W., Billiran H., Ganss R., Ruoslahti E, & Hamzah J. (2019). Immune‐mediated ECM depletion improves tumour perfusion and payload delivery. EMBO Molecular Medicine, 11(12), e10923.

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Comprehensive Chromatographic Separation Techniques

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Column chromatography was done by using silica gel (60–120 and 100–200 mesh size), HP-20, HP-20SS, Sephedex, and silver nitrate-incorporated silica gel. Merck Kieselgel (Aufoilen) 60 F254 plates were used for thin-layer chromatography (TLC). High resolution mass spectra were obtained on Agilent 6540 (Q-TOF) mass spectrometer, in the electrospray (ESMS) mode. All solvents used for high performance liquid chromatography (HPLC) analysis were obtained from Merck Chemicals (Mumbai India). Water used for extraction and isolation purposes was obtained at Central Drug House (P) Ltd., Delhi. Spectroscopic data (NMR) of isolated compounds was gathered on a ¹H NMR at 400 and 500 MHz and on a¹³C NMR at 100 and 125 MHz (Bruker Advance spectrometer). The reference point was TMS (δ_H and δ_C: 0.00 ppm). Chemical shifts (δ) were referenced internally to the residual solvent peak (CD₃OD: ¹H- 3.30, ¹³C- 49.0 ppm). In 2D NMR, all heteronuclear ¹H and ¹³C correlations were established based on gradient-enhanced inverse-detected Heteronuclear Multiple Bond Correlation (HMBC) and Heteronuclear single quantum coherence (HSQC) experiments. HP-20, HP-20SS, Sephedex, silver nitrate, dimethylsulphoxide (DMSO), and all other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, United States).

Lone B.A., Tabassum M., Bhushan A., Rani D., Dhiman U., Ahmad A., Mir H.A., Gupta P.N., Mondhe D.M., Gairola S, & Gupta P. (2023). Trilliumosides K and L, two novel steroidal saponins from rhizomes of Trillium govanianum, as potent anti-cancer agents targeting apoptosis in the A-549 cancer cell line. Frontiers in Chemistry, 11, 1306271.

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Optimizing Cory-B Encapsulation in Exosomes

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Loading of Cory-B in Fe65-EXO was carried out using the sonication method and loading efficiency calculated using UV-Visible spectroscopy and liquid chromatography-mass spectroscopy (LC–MS). Briefly, for drug loading into exosomes by sonication, an equal amount of Cory-B and exosome was mixed and sonicated (20% amplitude and 6 cycles of 30 s on/150 s off). After sonication, the Cory-B and exosome solution was incubated at 37 °C for 60 min. Excess free drug was separated from EXO-Cory-B by size exclusion chromatography using a Sephadex G25 column. For further validation, the samples were prepared in high performance liquid chromatography (HPLC) grade methanol, and an internal control used for analysis using Agilent 1290 (Agilent Technologies) UHPLC system. Briefly, 2 ml of sample was injected into C18 column (1.7 µm 2.1 × 100 mm) (Acquity UPLC BEH) for its separation at 40 °C. The mobile phase constituted 0.1% formic acid (A) in water and 0.1% formic acid in ACN (B), with a constant flow rate of 0.1 ml/min. The MS and MS/MS data were obtained by an Agilent 6540 Q-TOF mass spectrometer (Agilent Technologies) equipped with a jet stream ESI source in positive ion mode. The data was analyzed by Mass Hunter (Agilent Technologies) B.03 software and for all mass peaks, the mass spectral data were analyzed at a range of 100e1700 m/z.

Iyaswamy A., Thakur A., Guan X.J., Krishnamoorthi S., Fung T.Y., Lu K., Gaurav I., Yang Z., Su C.F., Lau K.F., Zhang K., Ng R.C., Lian Q., Cheung K.H., Ye K., Chen H.J, & Li M. (2023). Fe65-engineered neuronal exosomes encapsulating corynoxine-B ameliorate cognition and pathology of Alzheimer’s disease. Signal Transduction and Targeted Therapy, 8, 404.

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High-Resolution Mass Spectrometry Protocol

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MS data were
collected using an Agilent 6540 QTOF mass spectrometer (Agilent Technologies)
equipped with a JetStream electrospray ion (ESI) source. Data acquisition
software was MassHunter Qualitative Analysis B.06.00 (Agilent Technologies).
The optimized operating parameters in negative ion mode were as follows:
nebulizing gas (N₂) flow rate at 7 L/min, nebulizing gas
temperature at 300 °C, JetStream gas flow at 7 L/min, sheath
gas temperature at 350 °C, nebulizer pressure at 40 psi, capillary
voltage at 3000 V, skimmer at 65 V, Octopole RFV at 600 V, and fragmentor
voltage at 130 V. An MS/MS technique was applied to provide parallel
alternating scans for acquisition at low collision energy to obtain
precursor ion information or at a ramping of high collision energy
to acquire a full-scan accurate mass data of fragments and precursor
ions and to obtain neutral loss information. The collision energies
for auto MS/MS analysis were 20 and 35 V.

Chan Y.M., Lu B.W., Zhang W.H., Chan K.C., Fang J., Luo H.Y., Du J., Zhao Z.Z., Chen H.B., Dong C, & Xu J. (2023). Impact of Sulfur Fumigation on the Chemistry of Dioscoreae Rhizoma (Chinese Yam). ACS Omega, 8(23), 21293-21304.

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Stable Isotope Labeling for Metabolomic Analysis

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Stable isotope labeling was accomplished by culturing cells in media containing 400 μM uniformly labeled ¹³C OA (Cambridge Isotope Laboratories, Tewksbury, MA). Briefly, SiHa cells were labeled for 24 hours, irradiated with 6Gy, incubated for additional 24 hours with label and then washed with PBS and LC/MS-grade water before quenching, with cold methanol and transferring, to microfuge tubes with a cell scraper. Methanol was evaporated by using a SpeedVac. Pellets were extracted with 2:2:1 methanol: acetonitrile: water. Extracts were reconstituted in 1:2 water: acetonitrile, with volume normalized to the dry mass of the cell pellet. The mixture was then sonicated, centrifuged, and the supernatant was taken for LC/MS analysis. Ultra-high performance LC (UHPLC)/MS was performed with an Agilent 1290 Infinity II LC system interfaced with an Agilent 6540 QTOF mass spectrometer (Agilent Technologies, Santa Clara, CA). For reversed-phase liquid chromatography (RPLC) separations, a Waters CORTECS C8 column (50 mm × 2.1 mm, 1.6 μm) was coupled to the MS detector. For hydrophilic interaction liquid chromatography (HILIC) separations, a ZIC-pHILIC column (100 mm × 2.1 mm, 5 μm) was coupled to the MS detector. LC/MS data were processed and analyzed with the Agilent Profinder software and a natural-abundance correction of ¹³C was applied.

Muhammad N., Ruiz F., Stanley J., Rashmi R., Cho K., Jayachandran K., Zahner M.C., Huang Y., Zhang J., Markovina S., Patti G.J, & Schwarz J.K. (2022). Mono- and diunsaturated fatty acids sensitize cervical cancer to radiation therapy. Cancer research, 82(24), 4515-4527.

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Analytical Methods for Natural Products

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UV spectra were taken on a Shimadzu UV-2401PC (Shimadzu, Kyoto, Japan) spectrometer with KBr pellets. 1D- and 2D-NMR spectra were acquired on Bruker AVANCE III-600 (Bruker BioSpin GmbH, Rheinstetten, Germany) instruments, using tetramethylsilane (TMS) as an internal standard: chemical shifts (δ) are given in ppm, coupling constants (J) in Hz, the solvent signals were used as references (CD₃OD: δ_C = 49.0 ppm; residual CH₃OH in CD₃OD: δ_H = 4.78 ppm). Electrospray ionization (ESI) and High-resolution electrospray ionization (HRESI) mass spectra were carried out using an Agilent 6540 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). MCI gel CHP 20P (75–150 μm, Mitsubishi Chemical Corp., Tokyo, Japan), Sephadex LH-20 (25–100 μm, Pharmacia Fine Chemical Co. Ltd.), and LiChroprep RP-18 gel (40–63 μm, Merck, Darmstadt, Germany), and silica gel (200–300 mesh, Qingdao Haiyang Chemical Co.) were used for normal pressure column chromatography (CC). Thin-layer chromatography (TLC) was carried on silica gel G precoated plates (Qingdao Haiyang Chemical Co.) and spots were visualized by ultraviolet light (254 nm) or 9% sulfuric acid/EtOH.

Yang L., Jiang R., Li H.H., Pan Y.P., Lu J.J., Zhang H., Liu S.J., Shen J.L, & Hu J.M. (2020). Three new compounds from the flower branch of Gastrodia elata Blume and anti-microbial activity. RSC Advances, 10(25), 14644-14649.

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UPLC-QTOF Mass Spectrometry Protocol

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Agilent 1290 UPLC system (Agilent Technologies), equipped with an auto sampler, a binary pump and a thermostatted column compartment, was used for chromatographic analysis. The samples were separated on ACQUITY UPLC BEH C18 column (2.1 mm × 100 nm, 1.7 μm) at 40 °C. The mobile phase was composed of 0.1% formic acid in water (A) and 0.1% formic acid in ACN (B). Programmed gradient elution was performed as follow: 0–3 min, 2% B; 3–9 min, 2–12% B; 9–24 min, 12–32% B; 24–29 min, 32–75% B; 29–29.1 min, 75–100% B; 29.1–32 min, 100% B; 32.1–35 min, 100-2%B. The flow rate was 0.4 mL/min. Agilent 6540 Q-TOF mass spectrometer (Agilent Technologies) equipped with a jet stream electrospray (ESI) ion source was utilized to acquire MS and MS/MS data in positive ion mode. Data acquisition was managed by MassHunter B.03 software (Agilent Technologies). The working parameters were as follow: nebulizing gas (N₂) flow rate, 8.0 L/min; nebulizing gas temperature, 300 °C; jet stream gas flow, 9 L/min; sheath gas temperature, 350 °C; nebulizer, 45 psi; capillary, 3000 V; skimmer, 65 V; Oct RFV, 600 V; fragment voltage, 150 V. Mass spectrum was documented with mass range at 100–1700 m/z of all mass peaks.

Liu L.F., Song J.X., Lu J.H., Huang Y.Y., Zeng Y., Chen L.L., Durairajan S.S., Han Q.B, & Li M. (2015). Tianma Gouteng Yin, a Traditional Chinese Medicine decoction, exerts neuroprotective effects in animal and cellular models of Parkinson’s disease. Scientific Reports, 5, 16862.

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Synthesis and Characterization of THIQ Derivatives

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Unless otherwise noted, all commercially
available reagents were used without further purification. Solvents
and reagents were purchased from commercial sources and used without
further purification. Flash column chromatography was performed using
silica gel (200–300 mesh). Analytical thin-layer chromatography
was performed using glass plates precoated with 200–300 mesh
silica gel impregnated with a fluorescent indicator (254 nm). NMR
spectra were recorded in CDCl₃ or DMSO-d₆ on Bruker NMR-300 (300 MHz) and NMR-400 (400 MHz) spectrometers
with TMS as an internal reference. HRMS was performed on an Agilent
6540 Q-TOF mass spectrometer (ESI). IR spectra were recorded on a
Thermo Fisher IS50 FT-IR spectrometer. X-ray crystallographic data
were collected using a SMART APEX II X-ray diffractometer. THIQ derivatives
were prepared according to the previously reported literature procedures.^{16 (link)}

Hou G.Q., Zhao W., Deng C., Dong C., Wang C., Liu L, & Li J. (2022). Acid-Promoted Redox-Annulation toward 1,2-Disubstituted-5,6-dihydropyrrolo[2,1-α]isoquinolines: Synthesis of the Lamellarin Core. ACS Omega, 7(42), 37050-37060.

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