High-resolution LC–MS (LC-HRMS) was carried out as described by Santiago et al.12 (link). Briefly, a concentration of 1 mg/ml of each crude extract (OPEM + mGb, OPEM + dGb and OPEM) was prepared. The samples were analyzed using the Agilent 1290 Infinity LC system coupled to the Agilent 6520 Accurate-Mass Q-TOF mass spectrometer (Agilent, California, USA) with electrospray ionization (ESI) interface in negative ion mode equipped with an Agilent Eclipse XDB-C18 column (150 mm × 2.1 mm column, Agilent, California, USA). The column conditions were as described by Santiago et al.12 (link). The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The run time for the analysis was 25 min followed by a 5-min post-run time to minimize carry-over between injections. The collected data were processed using the Agilent MassHunter Qualitative Analysis B.07.00 (Agilent, California, USA). The secondary metabolites were identified by comparing with those recorded in the METLIN database. Three technical replicates were used in this study.
Agilent 6520 accurate mass q tof mass spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 6520 Accurate-Mass Q-TOF mass spectrometer is a high-performance instrument designed for precise and reliable mass analysis. It utilizes quadrupole-time-of-flight (Q-TOF) technology to provide accurate mass measurements for a wide range of applications.
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Lab products found in correlation
Agilent 1290 infinity lc system, by Agilent Technologies (3 mentions)
1290 infinity lc system, by Agilent Technologies (3 mentions)
Agilent masshunter qualitative analysis b 07, by Agilent Technologies (2 mentions)
Zorbax eclipse xdb c18, by Agilent Technologies (2 mentions)
Agilent eclipse xdb c18 column, by Agilent Technologies (1 mentions)
Agilent 1290 infinity system, by Agilent Technologies (1 mentions)
Spectrum 100, by PerkinElmer (1 mentions)
P 2000 polarimeter, by Jasco (1 mentions)
High performance liquid chromatography (hplc), by Waters Corporation (1 mentions)
Agilent zorbax eclipse xdb c18, by Agilent Technologies (1 mentions)
Masshunter software, by Agilent Technologies (1 mentions)
10 protocols using agilent 6520 accurate mass q tof mass spectrometer
1
Comprehensive LC-HRMS Analysis of Crude Extracts
2
Quantitative Identification of Compounds
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A total of 2.5 mg/mL sample of each QI semipurified fractions was prepared in ultrapure water. The compound detection utilizes Agilent-1290 Infinity system coupled to Agilent-6520 Accurate-Mass Q-TOF mass-spectrometer with dual ESI. Compounds then searched with Metlin_AM_PCDL-N-130328.cdb database.
3
LC-MS Analysis of Intact Proteins
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The protein was loaded onto an Agilent Zorbax Eclipse XDB-C18 chip column mounted on the Agilent 1290 Infinity LC system coupled to the Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with a dual ESI source (Agilent Technologies, Santa Clara, CA, USA). The chip column was run at 0.5 mL/min using 0.1% formic acid in water (solution A) and 0.1% formic acid in acetonitrile (solution B). The chip was equilibrated with 5% solution B, and the gradient used during the run was 5–100% solution B for 5 to 20 min and maintained with 100% buffer B for another 5 min. The polarity of the Q-TOF was set at positive, the capillary voltage at 4000 V, the fragmentor voltage at 125 V, the drying gas flow at 10 L/min, and a gas temperature of 300 °C. The intact protein spectrum was analyzed in MS-only mode with a range of 100–3200 m/z. The spectrum was deconvoluted using Agilent MassHunter Qualitative Analysis B.07.00 (Agilent Technologies, Santa Clara, CA, USA).
4
Peptide Characterization Using Analytical Techniques
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Melting point of peptides and copper(ii )-peptides were measured using digital melting point apparatus. Plastic plates of thin layer chromatography (TLC) were pre-coated with silica gel 60 F254 (0.2 mm thickness), Merck brand (fluorescent). High performance liquid chromatography-ultraviolet detector (HPLC-UV) analysis were carried out using Waters HPLC (Binary HPLC pump 1525 and UV-Waters 2489) employing gradient system (deionized water with 0.1% trifluoroacetic acid (TFA) and acetonitrile with 0.1% TFA) with 0.5 mL min−1 flowing through reversed phase C18 column XBridge (4.6 × 250 mm). Liquid chromatography-mass spectroscopy (LC-MS) analysis were conducted using Agilent 1290 Infinity LC system coupled to Agilent 6520 accurate-mass Q-TOF mass spectrometer with dual ESI source with the same gradient system and flow rate as HPLC. The column used was Agilent Zorbax 300SB-C18 Narrow-Bore RR (2.1 × 100 mm × 3.5 μm). Peptides and their copper complexes were analysed using Perkin Elmer spectrum 100 with wavenumbers ranging from 4500–450 cm−1. Secondary structure and optical rotation of peptides and Cu(ii )-peptides in phosphate buffer (KH2PO4) were studied using the JASCO P1050 spectrometer and JASCO P-2000 polarimeter, respectively.
5
Metabolome Analysis by LC-QTOF-MS
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A 10 mg/mL of SF1 was injected using Agilent-1290-Infinity liquid chromatography (LC) system and separated using a C18 column (Agilent ZORBAX Eclipse XDB-C18, Narrow Bore 2.1mm × 150mm, 3.5 micron). The LC was coupled to an Agilent-6520 Accurate-Mass Q-TOF mass spectrometer with a dual Electrospray Ionisation source. The total run time was 30 min. The analysis was set to mass spectrometry (MS) mode with negative and positive polarity.
6
Crocodile Gut Metabolite Identification
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To determine identity of the molecules in the CM from the selected gut bacteria of crocodile, LC-MS analysis was employed as discussed earlier [31 (link)]. The molecules were extracted from the CM using chloroform as an extraction solvent in a ratio of 1:3 of chloroform to conditioned media. Next, the metabolite suspension was dried under reduced pressure utilizing a rotatory evaporator. The metabolites were then re-suspended 1:1 ratio of methanol to water. Analysis of samples was completed with the Agilent 1290 Infinity liquid chromatography (LC) system equipped with a Zorbax SB-C18 column, 100 × 2.1 mm i.d., 3.5 μm particle size, coupled to an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with dual electron-spray ionizing (ESI source) as described previously (Righi et al., 2016). Chromatographic runs were carried out with a gradient of acetonitrile with the solvent flow rate of 0.3 mL per min at 25 °C, and the injector volume of 2 μL. The chromatograms generated from mass spectrometry were used to establish the identity of molecules from the Metlin_AM_PCDL-N-170502.cdb, Metabolite and Chemical Entity Database. Scifinder software was used to identify biological activity of the molecules and their novelty.
7
LC-MS/ESI-MS Identification of Bioactive Compounds
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LC-MS/ESI-MS analysis was performed with an Agilent 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent 6520 accurate-mass Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with dual ESI sources operated in positive-ion mode. The MS was operated with the electrospray voltage set to 4000 V, a sheath gas flow of 10 L/min, fragmented voltage of 125 V, gas temperature of 300 °C and nebulizer gas at 45 psig. Chromatographic separation of metabolites was achieved using an Agilent Zorbax Eclipse XDB-C18 (Agilent Technologies, Santa Clara, CA, USA) narrow-bore 2.1 × 150 mm, 3.5 micron (particle size) operated at 25 °C. The column was eluted at a flow rate of 0.5 mL/min with aqueous solvent A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile. The chemical structure of all the identified bioactive compounds were visualized using ChemDraw JS Sample Page (version 19.0.0-CDJS-19.0.x+da9bec968, PerkinElmer, Waltham, MA, USA).
8
Peptide Characterization by ESI-MS/MS
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The molecular mass and amino acid sequence of the purified peptide were determined by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) on an Agilent 6520 accurate-mass Q-TOF mass spectrometer (Agilent, USA) equipped with an electrospray ion source in positive ion mode. The freeze-dried sample obtained by RP-HPLC was dissolved in distilled water containing 5% DMSO. The solution was injected continuously, and the setting parameters were 40 psi ESI nebulizer gas (N2) pressure, 8.00 L/min ESI drying gas flow rate operating on positive mode with a capillary voltage of 4000 V and capillary temperature of 350 °C, and a fragmentor voltage of 120 V. The MS/MS spectra were acquired in the data-dependent mode, in which collision energy was 70 V. Positive ion intensities were recorded over a mass range (m/z) of 100–1700. A de novo peptide sequencing method was performed by a manual calculation.
9
UHPLC-MS Analysis of Secondary Metabolites
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The AA was examined for secondary metabolites through ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis. For this study, Agilent 1290 Infinity LC system along with Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with dual ESI source (Agilent technology, USA) was used. The column used for UHPLC analysis was Agilent Zorbax Eclipse XDB-C18, narrow-bore 2.1 × 150 mm, 3.5 µm. The temperature was regulated at 25 °C for the column while at 4 °C for auto-sampler. The 1.0 µL of the sample was injected and its flow was maintained at 0.5 ml/minute. The sample run time was 25 min and the post-run time was 5 min. The mobile phases used in the analysis were formic acid (0.1 %) (A) and in acetonitrile (B). Full scan MS analysis was done using negative electrospray ionization mode, over a range of m/z 100–1000. Nitrogen was supplied at flow rates of 25 and 600 L/hour as nebulizing and drying gas, respectively and the drying gas temperature was kept at 350 °C. The capillary voltage was set to 3500 V while the fragmentation voltage was optimized to 125 V. Obtained data was examined through Agilent Mass Hunter software (B.05.00) for qualitative analysis (Method: Metabolomics-2017–00004.m) and the detected compounds were identified through Search Database (METLIN_AM_PCDL-N- 170502.cdb).
10
Metabolomic Profiling of C. hirsuta
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Reversed-phase ultrahigh-performance
liquid chromatography (RP-UHPLC) coupled to diode array detection
(DAD) and mass spectrometry (MS) was used for secondary metabolites
profiling of defatted hydro-methanolic extract of C.
hirsuta by an Agilent 1290 Infinity LC system coupled
to an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with a dual
ESI source.14 (link)
liquid chromatography (RP-UHPLC) coupled to diode array detection
(DAD) and mass spectrometry (MS) was used for secondary metabolites
profiling of defatted hydro-methanolic extract of C.
hirsuta by an Agilent 1290 Infinity LC system coupled
to an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with a dual
ESI source.14 (link)
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