All raw materials required for chemical synthesis were purchased from Shipek Chemical Co., Ltd. (Zhengzhou, China). The 1H NMR spectra were recorded at 600 MHz and recorded the 13C NMR spectra at 150 MHz on a Varian NMR spectrometer. DMSO‑d6 was used as a solvent to measure all chemical shifts, recorded ESI-MS data on a Finnigan MAT LC-MS. Analytical TLC had performed on silica gel 60 F254 plates (Qingdao Haiyang Chemical Co., Ltd., China) and visualized by UV and potassium permanganate staining. Flash column chromatography was performed on Gel No. 60 (40–63 mm) (Qingdao Haiyang Chemical Co., Ltd., China). The melting point was determined by an electrothermal melting point instrument and was not corrected. High-resolution mass spectrometry (HRMS) was performed using an Agilent Accurate-Mass Q-TOF 6530 (Agilent, Santa Clara, California, USA) in ESI mode.
Agilent 6530 accurate mass q tof
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 6530 Accurate-Mass Q-TOF is a high-resolution quadrupole time-of-flight mass spectrometer. It provides accurate mass measurements for the identification and characterization of chemical compounds.
Automatically generated - may contain errors
Lab products found in correlation
Av 400 spectrometer, by Bruker (2 mentions)
Masshunter software, by Agilent Technologies (2 mentions)
Nmr spectrometer, by Agilent Technologies (1 mentions)
Av 600 spectrometer, by Bruker (1 mentions)
Agilent mass hunter qualitative analysis software, by Agilent Technologies (1 mentions)
Deuterated chloroform, by Bruker (1 mentions)
6130 quadrupole lc ms, by Agilent Technologies (1 mentions)
Uv 1700 pharmaspec, by Shimadzu (1 mentions)
Alpha ftir, by Bruker (1 mentions)
Plc 2050, by Gilson (1 mentions)
Avance 3 spectrometer, by Bruker (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Buchi rotary evaporator, by Büchi (1 mentions)
Sephadex lh 20, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Dichloromethane, by Merck Group (1 mentions)
Nano chip column, by Agilent Technologies (1 mentions)
Nano hplc system, by Agilent Technologies (1 mentions)
Mascot, by Matrix Science (1 mentions)
13 protocols using agilent 6530 accurate mass q tof
1
Synthesis and Characterization of Novel Compounds
2
Spectroscopic Characterization of Compounds
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Unless otherwise noted, all solvents and reagents were obtained from commercial suppliers and used without further purification. 1H-NMR and 13C-NMR spectra were measured with an AV-600 spectrometer (Bruker Bioscience, Billerica, MA, USA), with tetramethyl silane as an internal standard. The 200–300 mesh silica gel (Qingdao Haiyang Chemical, Qingdao, China) was used for Column chromatography. TLC analysis was performed on silica gel plates GF254 (Qingdao Haiyang Chemical, Qingdao, China). ESI-MS were obtained on Agilent ESI-QTOF instrument (Agilent, Santa Clara, CA, USA). High resolution mass spectra (HRMS) were recorded on an Agilent Accurate-Mass Q-TOF 6530 (Agilent, Santa Clara, CA, USA) in ESI mode.
3
Comprehensive Analytical Techniques for Compound Characterization
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Column chromatography was carried out on the 200–300 mesh silica gel (Qingdao Haiyang Chemical Co. Ltd., Qingdao, Shandong, China). Analytical thin-layer chromatography (TLC) was performed on silica gel precoated GF254 plates (Qingdao Haiyang Chemical Co. Ltd.). 1H-NMR and 13C-NMR spectra were recorded on an AV-400 spectrometer (Bruker Bioscience, Billerica, MA, USA), with tetramethylsilane as an internal standard. 1H and 13C-NMR spectra of these compounds are available in the Supplementary Materials . ESI-MS spectra were obtained on an Agilent ESI-QTOF instrument. High resolution mass spectra (HRMS) were measured with an Agilent Accurate-Mass Q-TOF 6530 (Agilent, Santa Clara, CA, USA) in ESI mode and are available in the Supplementary Materials . Melting points were determined using a X-4 microscope melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) without calibration.
4
Melting Point and Spectroscopic Analysis
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Melting points were recorded on an X-4 microscope melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) without calibration. The 1H-NMR and 13C-NMR spectra were measured by a Bruker AV-400 spectrometer (Bruker Bioscience, Billerica, MA, USA), with tetramethylsilane as an internal standard. High-resolution mass spectra (HRMS) were measured with an Agilent Accurate-Mass Q-TOF 6530 (Agilent, Santa Clara, CA, USA) in ESI mode. Reaction progress was monitored by TLC on silica gel precoated GF254 plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, Shandong, China). Preparative flash column chromatography was performed on the 200–300 mesh silica gel (Qingdao Haiyang Chemical Co. Ltd., Qingdao, Shandong, China). Unless otherwise noted, all solvents and reagents were commercially available and used without further purification.
5
Rapid High-Throughput Mass Spectrometry
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Assays were performed on Agilent 6530 RapidFire QTOF mass spectrometer in 384-well polypropylene plates (Greiner, code 781280) with ∼0.1 mg/ml protein samples in 0.1% formic acid. Samples were aspirated under vacuum and adsorbed onto a C4 solid-phase extraction (SPE) cartridge (Agilent Technologies), equilibrated and washed with LC-MS grade water containing 0.1% formic acid to remove buffer components. Following the aqueous wash, analytes were eluted from the C4 SPE onto an Agilent 6530 accurate mass Q-TOF in an organic elution step containing 85% acetonitrile and 0.1% formic acid. Masses of fragments were determined using the Agilent Masshunter Qualitative Analysis software (Agilent).
6
Analytical Techniques for Natural Product Characterization
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LC-MS analysis was performed with Agilent Technologies® 6130 Quadrupole LC/MS (Santa Clara, CA, USA). UV–vis spectra were obtained using Shimadzu® UV-1700 PharmaSpec (Kyoto, Japan). IR spectra were determined using Bruker® Alpha FT-IR (Billerica, MA, USA). NMR spectra of the compounds were acquired on a Bruker® Avance III spectrometer operating at 600 MHz for 1H and 150 MHz for 13C in deuterated chloroform (Billerica, MA, USA). HRESIMS analysis of compounds 1 –6 were performed using Agilent® 6530 Accurate Mass Q-TOF (Santa Clara, CA, USA), while the HRESIMS data of turcicasulphide (7 ) were acquired on a Thermo Scientific-Q Exactive® (Waltham, MA, USA). Optical rotation data were acquired using a Rudolph Analytical Autopol V Plus® in dichloromethane (Hackettstown, NJ, USA). A Buchi rotary evaporator was used to evaporate the solvent of the extract (Buchi, Flawil, Switzerland). A Sephadex LH-20 (Sigma Chem. Co. 25–100 µm) (GE Healthcare, Chicago, IL, USA) column (5 × 100 cm) was used for the initial fractionation. A Gilson® PLC 2050 was used for the further purification of the compounds (Saint-Avé, France). Hexane, dichloromethane, methanol, and acetonitrile (Merck, Darmstadt, Germany) were used during the chromatographic analyses.
7
Nano LC-MS/MS Analysis of Peptides
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Nano LC-MS/MS analysis was performed with a nano HPLC system (Agilent), and the nano chip column (150 mm × 0.075 mm, Agilent) was used for peptide separation. The mobile phase A for LC separation was 0.1% formic acid in deionized water and the mobile phase B was 0.1% formic acid in acetonitrile. The chromatography gradient was designed for a linear increase from 5% B to 30% B in 25 min, 40% B to 60% B in 5 min, 90% B in 10 min and 5% B in 15 min. The flow rate was maintained at 300 nL/min. Product ion spectra were collected in the information-dependent acquisition (IDA) mode and were analyzed by Agilent 6530 Accurate-Mass Q-TOF (Agilent) using continuous cycles of one full scan TOF MS from 200 to 1500 m/z (1.0 s) plus three product ion scans from 50 to 1800 m/z (1.5 s each). Precursor m/z values were selected starting with the most intense ion, using a selection quadrupole resolution of 3 Da. The rolling collision energy feature was used, which determines collision energy based on the precursor value and charge state. The dynamic exclusion time for precursor ion m/z values was 60 s.
8
Identification of LPS-induced Protein Interactors
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After Co-IP with NCLΔAS-GFP-expressing RAW 264.7 cells stimulated with LPS for 12 h, eluted samples were separated by 8% SDS-PAGE and visualized by silver staining. The bands of interest were excised from the gel and polypeptides were digested by trypsin proteases in 50 mM ammonium bicarbonate on ice for 45 min followed by incubation at 37 °C for 12 h. Tryptic peptides were analyzed by Agilent 6530 Accurate-Mass Q-TOF with a nanochip column (Agilent, 150 mm × 0.075 mm) as previously described95 (link). MASCOT (Matrix Science, London, U.K.; v.2.2.04) was used to identify peptide sequences in the protein sequence database (nr_mouse). Database search parameters for LC-MS/MS were as follows: fixed modification, carboxyamidomethylated at cysteine residues; variable modification, oxidized at methionine residues; maximum allowed missed cleavage, 1; peptide MS tolerance, 100 ppm; and fragment MS tolerance (LC-MS/MS), 0.1 Da. Individual ion scores showing greater than 42 for LC-MS/MS were regarded as significant (P < 0.05).
9
HPLC-QTOF-MS Analysis of Compounds
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Chromatography was performed on Agilent 1100 series HPLC system (Agilent Technologies, Inc., USA).[18 (link)] The separation was performed on a 4.6 mm × 100 mm ZORBAX SB-C18 column (Agilent Technologies, Inc., USA). The column temperature was maintained at 45°C. The gradient mobile phase consisted of a solvent A (0.1% formic acid in water), while mobile phase B (0.1% formic acid in acetonitrile), the flow rate was 1 mL/min. The gradient condition of the mobile phase was as follows: 0–5 min, B 65%–80%; 5–7 min, B 80%–94%; 7–10 min, B 94-100%. Mass spectrometry was performed on an Agilent 6220 Accurate-Mass Time-of-Flight mass spectrometer (Agilent Technologies, Inc., USA) equipped with an ESI source (Agilent Technologies, Inc., USA), the optimal conditions were as follows: Drying gas temperature at 350°C, drying gas flow rate of 9 L/min, fragmentor voltage of 175 V, nitrogen was used as the dry gas and nebulizer. MS data were collected in the full scan mode from m/z 50–1700. HPLC-QTOF-MS system (Agilent 6530 Accurate-Mass Q-TOF, Agilent Technologies, Inc., USA) was used to make further identification, the ESI ion source was set in the positive ion polarity mode for acquiring all mass spectrometry data. All data collected in centroid mode were acquired with MassHunter software (Agilent Technologies, Inc., USA).
10
Quantification and Identification of Bisphenol Transformation Products
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Kinetic experiment samples were analysed using a 7890B series GC coupled to an Agilent 7000 series triple quad MS/MS, (Agilent Technologies, USA) 24 (link) . The analysis is described in detail in SI-3.4.1, Chapter 3. Information on quantification, retention times (RT) and MS/MS optimised conditions are summarised in Table SI-3, while matrix-matched validation using isotopically labelled internal standards was performed as described in SI-3.5. Limits of quantification (LOQ) for BPF and BPS were 0.02 or 0.1 mg L -1 , respectively, and the average concentrations of analytes in the blanks were < LOQ.
Non-target screening for the identification of the formed BTPs was conducted using an Agilent 1290 Infinity UPLC coupled to an Agilent 6530 Accurate-Mass QTOF (Agilent Technologies, USA). An Agilent Jet-Stream electrospray source was operated in negative ionisation mode only since no transformation products of bisphenols were identified in our previous studies. The instrument was run in datadependent acquisition mode with a mass range of 50 to 1000 m/z and a scan rate of 2 scans s -1 for MS and 5 scans s -1 for MS/MS (SI-3.4.2, Chapter 3). MassHunter software (Version B.07.00, Agilent Technologies) was used for data analysis.
Non-target screening for the identification of the formed BTPs was conducted using an Agilent 1290 Infinity UPLC coupled to an Agilent 6530 Accurate-Mass QTOF (Agilent Technologies, USA). An Agilent Jet-Stream electrospray source was operated in negative ionisation mode only since no transformation products of bisphenols were identified in our previous studies. The instrument was run in datadependent acquisition mode with a mass range of 50 to 1000 m/z and a scan rate of 2 scans s -1 for MS and 5 scans s -1 for MS/MS (SI-3.4.2, Chapter 3). MassHunter software (Version B.07.00, Agilent Technologies) was used for data analysis.
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