The endogenous Se in plant tissues was extracted by the method as reported earlier [11 (link)]. The measurement of endogenous plant MT was carried out with some modifications [47 (link)]. Fresh samples (0.5 g) of leaf and root were grounded and homogenized in 5 mL methanol containing 50 ng mL− 1 [2H6]-MT (Toronto Research Chemicals Ltd., Toronto, Ontario, Canada) which was used as internal standard. After shaking the homogenate overnight in the dark at 4 °C and centrifuged at 15,000 g for 10 min. Later after transferring the supernatant into a new tube, the segments were again extracted with 2 mL of methanol and mixed with the fraction of supernatant. For the purification of MT, the supernatant was transferred to the C18 solid-phase extraction (SPE) cartridge (Waters, Milford, MA, USA). Then extracted material was rigorously dehydrated under nitrogen. The obtained residue was dissolved in 0.5 mL of methanol (70%) and subjected to HPLC electrospray ionization/MS-MS analysis on an Agilent 6460 triple quad LC/MS with an Agilent-XDB18 column (2.1 mm × 150 mm, an Agilent Technologies, Frankfurt, Germany). The recovery rate was estimated by the quantification of [2H6]-MT as an internal standard [48 (link)].
Agilent 6460 triple quad lc ms
Manufactured by Agilent Technologies
Sourced in United States, Germany
The Agilent 6460 Triple Quad LC/MS is a high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) system. It is designed to provide accurate and sensitive quantitative analysis of a wide range of analytes in complex matrices.
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Lab products found in correlation
Ce tofms system, by Agilent Technologies (2 mentions)
C18 solid phase extraction cartridge, by Waters Corporation (1 mentions)
Sep pak c18 cartridge, by Waters Corporation (1 mentions)
Agilent xdb c18 column, by Agilent Technologies (1 mentions)
Agilent 6550 ifunnel q tof lc ms, by Agilent Technologies (1 mentions)
500 mhz nmr instrument, by Bruker (1 mentions)
Agilent 1200 series chromatograph, by Agilent Technologies (1 mentions)
Masshunter software, by Agilent Technologies (1 mentions)
Agilent 1290 infinity hplc, by Agilent Technologies (1 mentions)
Zorbax eclipse xdb c18, by Agilent Technologies (1 mentions)
Agilent lc msd tof, by Agilent Technologies (1 mentions)
1200 series rrlc system, by Agilent Technologies (1 mentions)
Ce system, by Agilent Technologies (1 mentions)
10 protocols using agilent 6460 triple quad lc ms
1
Quantification of Endogenous Plant Metallothioneins
2
Microwave-Assisted Synthesis and Characterization
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Solvents and reagents of analytical grade were purchased from Merck and used without any further purification. All reactions were carried out in a commercially available LG microwave oven (MB-3947C) having a maximum output of 800 W operating at 2450 MHz. The 1H NMR spectra were taken by a Bruker Advance DPX 500 MHz spectrometer in CDCl3 from “Wazed Miah Science Research Centre (WMSRC), Jahangirnagar University, Dhaka, Bangladesh using tetramethylsilane (TMS) as a standard. FTIR spectrophotometer (Model-8900, Shimadzu, Japan) was used to record spectra on KBr pellets. Samples were dissolved in CHCl3 to take mass spectra using Agilent 6460 Triple Quad LC/MS. Autoclave Machine (ALP Co. Ltd. Japan), Laminar Airflow (BIOQUELL Medical Ltd.UK) and Incubator were adjusted at 35 ± 2 °C.
3
Metabolic Response to Nigericin Stimulation in BMDMs
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Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 90 min with medium or nigericin. Cells were washed with 5% mannitol and the metabolites were extracted with methanol containing internal standard. The metabolites were analysed by CE-TOFMS (Agilent CE-TOFMS system) and CE-QqQMS (Agilent CE system and Agilent 6460 TripleQuad LC/MS). Absolute value of metabolite was determined, and the relative ratios of nigericin-treated group to medium group, and of febuxostat/nigericin-treated group to nigericin-treated group were calculated to be expressed as the heat map.
4
Quantifying Melatonin Levels in Plant Samples
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MT contents were determined according to Li et al.18 (link). In brief, 0.3 g fresh samples were crushed in liquid nitrogen and homogenized in 3 mL of methanol containing 50 ng mL−1 [2H6]-melatonin (M215002, Toronto Research Chemicals Ltd., Toronto, Ontario, Canada) as an internal standard. The homogenate was shaken overnight in the dark at 4 °C and then centrifuged at 15,000 g for 10 min. The supernatant fraction was removed to a new tube, and the pellet was then re-extracted and mixed with the supernatant fraction. The samples was then eluted through a Sep-Pak C18 cartridge (WAT020805, Waters, Milford, MA, USA) to remove the polar compounds and dried under nitrogen. Dried samples were resuspended in 0.5 mL methanol (70%), and subjected to analysis by HPLC electrospray ionization/MS-MS at 40 °C, coupled to an Agilent 6460 triple Quad LC/MS and Agilent-XDB C18 column (3.5-µm, 150-mm × 2.1-mm, Agilent Technologies, Frankfurt, Germany). The [2H6]-MT was also quantified as an internal standard to estimate the recovery rate18 (link).
5
Analytical Techniques for Chemical Characterization
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Commercially available reagents and solvents were used without further processing. Thin-layer chromatography analysis was used to monitor reactions using thin-layer plates on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany). Flash column chromatography was performed using silica gel 60 (230–400 mesh). 1H NMR and 13C NMR spectra were recorded in δ units relative to the deuterated solvent as an internal reference using a 500 MHz NMR instrument (Bruker, Billerica, MA, USA). Liquid chromatography-tandem mass spectrometry (Agilent 6460 Triple Quad LC/MS, Santa Clara, CA, USA) analysis was performed using electrospray ionization (ESI) mass spectrometer with photodiode array detector (PDA). High-resolution mass spectrometry spectra were obtained using a TOF LC/MS system (Agilent 6550 iFunnel Q-TOF LC/MS).
6
Capillary HPLC-ESI-MS/MS Analysis Protocol
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Capillary HPLC–ESI–MS/MS analyses were performed with an Agilent 1200 series chromatograph (Agilent Technology, Waldbronn, Germany) equipped with a binary capillary pump, degasser, autosampler, thermostatically controlled column compartment and capillary Zorbax SB C18 column coupled to an electrospray ionization triple quadrupole mass spectrometer (Agilent 6460 Triple Quad LC/MS, Agilent Technologies, Santa Clara, CA, USA). All the operations, data acquisition and analysis were processed by MassHunter Software (Agilent Technology, USA). Operational parameters are summarized in Table S1 (see Electronic Supplementary Material).
A Bandelin Sonorex model 1210 ultrasonic bath (Germany) and MPW model 350R centrifuge (MPW Warsaw, Poland) were used for extraction procedures. Sample mineralization was performed with a Speedwave®4 microwave digestion system (Berghof, Germany).
A Bandelin Sonorex model 1210 ultrasonic bath (Germany) and MPW model 350R centrifuge (MPW Warsaw, Poland) were used for extraction procedures. Sample mineralization was performed with a Speedwave®4 microwave digestion system (Berghof, Germany).
7
Microbial Ink Biofilm Binds BPA
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The 3D printed structures of microbial ink (CsgA-αγ) embedded with PQN4-BPA biofilm were immersed in water with or without 1 mM BPA and incubated for 24 h. After 12 and 24 h the water spiked with BPA was collected to test BPA binding by the microbial ink embedded with PQN4-BPA biofilm. The presence of BPA was detected using Agilent 6460 Triple Quad LC/MS and Agilent 1290 Infinity HPLC by Small Molecule Mass Spectrometry Facility at Harvard University, MA, USA. Data obtained from at least three independent samples were reported.
8
HPLC-MS/MS Analysis of Flazasulfuron and Dicamba
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All the samples were analyzed by HPLC- MS/ MS on an Agilent 6460 Triple Quad LC/MS (Agilent, Palo Alto, USA). The analytical column was an Agilent Zorbax Eclipse XDB-C18 (150 mm × 2.1 mm, 3.5 μm). The MS system was electrospray ionization (ESI) source. The mobile phases were 0.1% (v/v) formic acid (A) and 100% acetonitrile (B). The column oven was maintained at a temperature of 35 ℃. The flow rate was 0.3 mL min−1 and the injection volume was 5 μL.
The flazasulfuron samples were separated with the following gradient program: 0–5 min, followed by a linear gradient from 10% B to 60% B; 5–8 min, followed by a linear gradient from 60% B to 75% B; 8–10 min, followed by a linear gradient from 75% B to 95% B. The instrument was operated in positive ionization mode in the multiple reaction monitoring (MRM) mode at m/z 408 and 182 [38 ]. The dicamba samples were separated with the following gradient program: 0–8 min, followed by a linear gradient from 0% B to 35% B; 8–10 min, maintaining 95% B. The instrument was operated in negative ionization mode under the multiple reaction monitoring (MRM) mode at m/z 219 and 175 [39 ].
The flazasulfuron samples were separated with the following gradient program: 0–5 min, followed by a linear gradient from 10% B to 60% B; 5–8 min, followed by a linear gradient from 60% B to 75% B; 8–10 min, followed by a linear gradient from 75% B to 95% B. The instrument was operated in positive ionization mode in the multiple reaction monitoring (MRM) mode at m/z 408 and 182 [38 ]. The dicamba samples were separated with the following gradient program: 0–8 min, followed by a linear gradient from 0% B to 35% B; 8–10 min, maintaining 95% B. The instrument was operated in negative ionization mode under the multiple reaction monitoring (MRM) mode at m/z 219 and 175 [39 ].
9
Quantification of Phytohormones in Tomato Leaves
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Endogenous ABA and JA were extracted from tomato leaves and determined by LC/MS‐MS on an Agilent 1290 Infinity HPLC system coupled to an Agilent 6460 Triple Quad LC‐MS device (Agilent Technologies, Amstelveen, the Netherlands), as described previously (Wang et al., 2016 ). GA was extracted from 1‐g samples of tomato leaves and quantified by a derivation approach coupled with nano‐LC‐ESI‐Q‐TOF‐MS analysis as described previously (Chen et al., 2012 ; Wang et al., 2019 ). For the determination of GA levels, D2‐GA1, D2‐GA3, D2‐GA4, D2‐GA9, D2‐GA19 and D2‐GA20 were added to the extraction solution as internal standards.
10
Targeted Metabolite Analysis by CE-MS and LC-MS
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Targeted quantitative analysis of metabolites was performed by HMT using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS), capillary electrophoresis triple quadrupole mass spectrometry (CE-QqQMS), and liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS). Analytic methods were described previously (6) . CE-TOFMS measurement was performed on an Agilent CE-TOFMS system, CE-MS/MS measurement was performed on an Agilent CE system with Agilent 6460 TripleQuad LC/MS, and LC-TOFMS measurement was performed using an Agilent 1200 series RRLC system . CC-BY-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which this version posted July 17, 2020. ; https://doi.org/10.1101/2020.07. 16 .207712 doi: bioRxiv preprint SL with Agilent LC/MSD TOF (all machines: Agilent Technologies). Peaks detected by CE-TOF/MS and LC-TOF/MS were extracted using MasterHands ver2.17 were performed by HMT using in-house analysis software developed by the company.
The copyright holder for this preprint (which this version posted July 17, 2020. ; https://doi.org/10.1101/2020.07. 16 .207712 doi: bioRxiv preprint SL with Agilent LC/MSD TOF (all machines: Agilent Technologies). Peaks detected by CE-TOF/MS and LC-TOF/MS were extracted using MasterHands ver2.17 were performed by HMT using in-house analysis software developed by the company.
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