Analyses were performed using a reversed-phase high-performance liquid chromatography (HPLC) system (Agilent model 1260 series, Santa Clara, CA, USA) with a Capcell pak C18 column (5 μm × 4.6 mm × 250 mm; Shiseido, Japan) and Agilent 6120 (Santa Clara, CA, USA) in the single-quadrupole positive ion mode. Chromatography was performed at room temperature at a flow rate of 1 mL/min, and 10 μL was analyzed for 50 min. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) in a ratio specified by the following binary gradient with linear interpolation: 0 min 5% B, 40 min 80% B and 50 min 5% B.
Agilent 6120
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 6120 is a single-quadrupole liquid chromatography-mass spectrometry (LC-MS) system. It is designed for routine analytical applications, providing reliable and reproducible results. The Agilent 6120 features a compact footprint and is capable of performing qualitative and quantitative analyses.
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Lab products found in correlation
Capcell pak c18 column, by Shiseido (1 mentions)
Methanol, by Agilent Technologies (1 mentions)
Avance 3, by Bruker (1 mentions)
Avance 3 400 mhz spectrometer, by Bruker (1 mentions)
Lcms it tof mass spectrometer, by Shimadzu (1 mentions)
Agilent 1290 infinity system, by Agilent Technologies (1 mentions)
Agilent 1260, by Agilent Technologies (1 mentions)
Toc vcph, by Shimadzu (1 mentions)
Agilent 1290, by Agilent Technologies (1 mentions)
Agilent 1100 series, by Agilent Technologies (1 mentions)
Agilent 1200, by Agilent Technologies (1 mentions)
Lc 20a, by Shimadzu (1 mentions)
Tripletof 5600, by AB Sciex (1 mentions)
Melting point b 540 apparatus, by Büchi (1 mentions)
Arx 600 mhz spectrometer, by Bruker (1 mentions)
Avance 3 400 mhz, by Bruker (1 mentions)
14 protocols using agilent 6120
1
HPLC Analysis of Chemical Compounds
2
Analysis of Fatty Acid Methyl Esters by GC-MS
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Chromatographic separation was performed as follows: Methanol extracts were brought to dryness in a Rotavapor Fischer rotary evaporator (USA) and later in the Telstar lyophilizer (Barcelona, Spain). The amount of the total sample obtained was weighed. The samples were then subjected to derivatization with a 0.2 N methanolic solution of m-trifluoromethylphenyl trimethylammonium hydroxide Meth Prep II (Fisher, Loughborough, UK). This one-step reagent simplifies the transesterification of triglycerides to methyl esters. In total, 5 µL was injected into GC/MS Agilent 6120 (Santa Clara, CA, USA). All standards were from Sigma Aldrich (Sant Louis, MI, USA) at ≥95.0% (HPLC).
The chromatography-mass spectrometry was carried out with the Interdepartmental Research Service at the Universidad Autónoma de Madrid (UAM).
The chromatography-mass spectrometry was carried out with the Interdepartmental Research Service at the Universidad Autónoma de Madrid (UAM).
3
Comprehensive mTOR Inhibitor Evaluation
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All compounds tested for mTOR inhibitions were acquired from the SPECS and GSMTL. Compound purity was assessed by HPLC equipped with a XDB-C18 column (250 mm × 4.6 mm, 5 μm particle size) and a UV/VIS detector setting of λ = 254 nm. Compounds were eluted with the two solvent systems (CH3OH as the organic phase in method I and CH3CN as the organic phase in method II). HPLC analysis of the compounds assayed confirmed the purity to be ≥95% (Table S1 ). 1H-NMR and MS spectra data were recorded on a Bruker AvanceIII spectrometer at 400 MHz using TMS as reference (Bruker Company, USA) and Agilent 6120 using methanol as solvent(Agilent, German), respectively. Detailed results can be found in Figure S7 .
4
Chromatographic Separation of Triglycerides
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Chromatographic separation was performed as follows: all of the supplied volume was first dried in a rotary evaporator (Rotavapor Fischer) and later in a lyophilizer Telstar. The total amount of sample obtained was weighed. They were derivatized with Meth-Prep Fisher (EEUU). Meth-Prep II is a 0.2 N methanolic solution of m-trifluoromethylphenyl trimethylammonium hydroxide. This one-step reagent simplifies the transesterification of triglycerides to methyl esters. They were injected into GC/MS.
The chromatographic separation was done in a HPLC-MS Agilent 6120 (Santa Clara, CA, USA) using a C20 column maintained at 35 °C. The chromatography mass spectrometry was carried out at the Servicio Interdepartamental de Investigación from the Universidad Autónoma de Madrid (UAM).
The chromatographic separation was done in a HPLC-MS Agilent 6120 (Santa Clara, CA, USA) using a C20 column maintained at 35 °C. The chromatography mass spectrometry was carried out at the Servicio Interdepartamental de Investigación from the Universidad Autónoma de Madrid (UAM).
5
Synthesis and Characterization of Quinoline-5,8-dione Derivatives
6
TMAO Quantification by LC/MS
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The serum TMAO concentrations were quantified using an LC/MS method that employed an Agilent 6120 LC/MS model mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and Phenomenex Kinetex Biphenyl Column, 100 × 4.6 mm, 5 µm (Phenomenex, Torrance, CA, USA). The isocratic mobile phase was composed of 50% water and 50% acetonitrile containing 0.1% formic acid at a flow rate of 1 mL/min. The Single Ion Monitoring (SIM) was at m/z 76.1+ [M + H]+ for TMAO serum samples and [M + H]+ at m/z 85.1+ for the internal standard TMAO-d9.
7
HPLC-MS Analysis of Organic Compounds
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HPLC were performed using Agilent 1290 infinity system (Agilent Technologies Inc, Waldbronn, Gremany) coupled with quadrupole LC/MS Agilent 6120 (Agilent Technologies Inc). The samples were injected on to a C-18 column (4.6 25 cm, 5l m; phenomenex, Torrance, CA, USA). The solvent used were A—90% acetic acid–water and B—10% MeOH, establishing following elution gradient; isocratic 10% B for 5 min, 10–100% B over 10 min, 100% B for 6 min, and re-equilibration of the column using flow rate of 0.1 mL/min. The spectra were recorded in negative and positive ionization mode between m/z 50 and 1200.
8
Quantitative Analysis of Phenol-Soluble Modulins
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PSM secretion was assayed as before (19 (link)) with modifications. Starter cultures were diluted into TSB without glucose supplemented with chloramphenicol and tetracycline to an OD600 = 0.200. Cells were incubated, shaking at 37 °C, 180 rpm for 4 h. D-xylose was added to 0.5% (w/v), and cells were cultured for one additional hour. Cells were pelleted by centrifugation at 13,200 rpm for 2 min in a microcentrifuge, and the supernatants (extracellular fraction) were collected and analyzed for PSMs by injecting 100 µL into a HPLC (Agilent 1260) connected inline to a quadrupole mass spectrometer (MS, Agilent 6120) performed exactly as before (44 (link)) using a 2.1 × 5 mm C8 column with increasing gradients of acetonitrile (ACN) + 0.1% trifluoroacetic acid (TFA) to wash the column and elute PSMs. Peptides were detected based on elution time and ion m/z. PSM secretion was quantified by summing the extracted ion chromatogram (EIC) peak area from two ionized (m/z) species per PSM.
9
Analytical Techniques for TOC, HBCD, and Debrominated Products
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Total organic carbon (TOC) was determined using a Total Organic Carbon Analyser (Shimadzu TOC-VCPH, Kyoto, Japan). Because of low microbial content in 2 mL samples, the biomass content in the solution could not be measured directly by the VSS content. In this study, protein content was converted to VSS, which was analyzed according to the reported method (Zubkov et al., 1999 (link)). HBCD was analyzed by gas chromatography-mass spectrometry (GC-MS) with a fused silica column DB5-MS (30 m × 0.25 mm id, 0.25 μm dj) using He as the mobile phase. Three diastereomers of HBCD were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the model of Agilent 6120. The analytical conditions were 10 mM ammonium acetate in water as phase A and 2% mobile phase B (methanol) at flow rate of 0.3 mL/min. The debrominated products were analyzed by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS, Agilent 1290, Palo Alto, CA, United States; Bruker, Germany) using electrospray ionization (ESI) positive mode.
10
Phlorotannin Analysis by HPLC-MS
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The purified phlorotannin sample was analyzed by High-Performance Liquid Chromatography (HPLC) (Agilent 1100 series; Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an autosampler/injector, a column compartment, a binary pump, and a diode array detector. The column was a Zorbax Analytical Rx-C18 (4.6 × 250 mm, 5 μm, Agilent Technologies Inc.). The flow rate was 1 mL/min. A mobile phase was 0.1% formic acid in aqueous solution, and B was 0.1% formic acid in acetonitrile. The linear gradient was as follows: 0–8 min (5–15% B), 8–10 min (15–30% B), 10–18 min (30–35% B), 18–23 min (35–100% B), 23–35 min (100% B), and 35–40 min (100–5% B). A wavelength at 280 nm was recorded. Mass spectrometric analysis (Agilent 6120; Agilent Technologies Inc., Santa Clara, CA, USA) was performed. Qualitative analysis was done by LC/MSC Chem Station B.04.03 software (Agilent Technologies Inc. address, Santa Clara, CA, USA).
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