Analyses of previously prepared urine samples, with the use of capillary electrophoresis coupled with time-of-flight mass spectrometry, were performed with a 7100 CE coupled with 6224 TOF/MS system (Agilent Technologies, Germany) equipped with an ESI ion source with a sheath liquid delivery system. Separation of the compounds was performed with a fused silica capillary (50 μm × 100 cm; Agilent Technologies, USA). Before each analysis, the capillary was flushed with BGE (0.8 ml/L, formic acid in 10% methanol in water) for 5 min at 950 mbar. Prepared urine samples were injected into the capillary for 50 s under 50 mbar pressure. Next, BGE was injected for 10 s under 100 mbar pressure. Compound separation was performed under a pressure of 25 mbar, voltage of 30 kV and amperage of 22 μA. The sheath liquid was delivered to the ion source with a flow rate of 0.6 ml/min. The drying gas temperature and flow rate were 200 °C and 10 L/min, respectively, with the nebulizer pressure set to 10 psi. The capillary, fragmentor and skimmer voltages were 3500 V, 125 V and 65 V, respectively. The mass spectrometer was operated in scanning mode with a m/z range from 50 to 1000.
Fused silica capillary
Manufactured by Agilent Technologies
Sourced in United States
Fused silica capillary is a type of analytical laboratory equipment used to facilitate the separation and analysis of chemical compounds. It is a narrow, flexible tube made of pure fused silica glass, which is an inert material that can withstand high temperatures and pressures. The capillary is used in various analytical techniques, such as gas chromatography and capillary electrophoresis, to transport and separate the components of a sample mixture.
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Lab products found in correlation
6224 tof ms system, by Agilent Technologies (1 mentions)
Chemstation software, by Agilent Technologies (1 mentions)
7100 ce instrument, by Agilent Technologies (1 mentions)
Aqua c18 resin, by Phenomenex (1 mentions)
Cosmosil 5c18 paq, by Nacalai Tesque (1 mentions)
Agilent 3d ce instrument, by Agilent Technologies (1 mentions)
9 protocols using fused silica capillary
1
Capillary Electrophoresis-Mass Spectrometry for Urine Analysis
2
CE-MS Analysis of Small Molecules
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An Agilent 7100 (CE) system, coupled to a TOF Mass Spectrometer (6224 Agilent), was used for sample analysis. In brief, a fused-silica capillary (Agilent Technologies; total length, 96 cm; i.d., 50 μm) was pre-conditioned with 1 M NaOH for 30 min, followed by MilliQ® water and background electrolyte-BGE (0.8 M formic acid in 10% methanol) for 30 min. Before each analysis, the capillary was flushed for 5 min (950 mbar pressure) with BGE. The MS was operated in positive polarity, with a full scan from 80 to 1000 m/z at a rate of 1.4 scan/s. Drying gas was set to 10 L/min, nebulizer to 10 psi, voltage to 3.5 kV, fragmentor to 125 V, gas temperature to 200 °C and skimmer to 65 V. The sheath liquid composition was methanol/water (1/1, v/v), containing 1.0 mmol/L formic acid with two reference masses (121.050873-purine (C5H4N4) and 922.009798-HP-921 (C18H18O6N3P3F24)), which allows for correction and provides more accurate mass determination. Flow rate was 0.6 mL/min and split was set to 1/100. Samples were injected at 50 mbar for 50 s. After each injection, along with the samples, BGE was co-injected for 10 s at 100 mbar pressure to improve repeatability. Separations were performed at a pressure of 25 mbar and a voltage of +30 kV; current under these conditions was 100 μA.
3
Capillary Electrophoresis Protocol for Separation
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Using an Agilent 7100 CE instrument supplied with an autosampler, and a diode array detector, electrophoretic separations were performed. The management of the CE system, data collection, and inspection were done using the ChemStation software (Agilent Technologies, Germany). Fused silica capillary, purchased from Agilent Technologies (USA), of total length 48.5 cm, 40 cm effective length and internal diameter of 50 µm was utilized. Separation was achieved using optimized background electrolyte (BGE) of 40.0 mM borate buffer at pH = 9.0, applied voltage of + 15 kV and detection wavelength of 200.0 nm. Each run pass through three main steps; first step is pre-conditioning where 0.1 mol/L NaOH was flushed for 1 min followed by deionized water and background electrolyte (BEG), in order, for 3 min each. Second step is sample injection where sample is injected hydro-dynamically at 50 mbar for 5 s. Finally, the capillary is flushed with deionized water for 5 min as post-run step. During working day, the capillary was flushed with running buffer for 120 s between each two sequential injections. To preserve the repeatability of the injections from run to run, buffer vials were replenished after every five subsequent runs.
4
CE-TOF/MS Metabolome Analysis Protocol
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The equipment consisted on a CE (7100 Agilent) coupled to a TOF/MS analyzer (6224 Agilent). Separation was carried out using a fused-silica capillary (total length of 100 cm; i.d. of 50 μm; Agilent technologies). The capillary was pre-conditioned with 1 M NaOH for 30 min, followed by MilliQ® water and background electrolyte-BGE (0.8 M of FA in 10% methanol) for 30 min, at 20 °C, using normal polarity. Before each analysis, the capillary was flushed for 5 min (950 mbar pressure) with BGE. The MS was operated in positive polarity, with a full scan from 80 to 1000 m/z at a rate of 1.4 scans/s. Drying gas was set to 10 L/min, nebulizer to 10 psi, voltage to 3.5 kV, fragmentor to 125 V, gas temperature to 200 °C and skimmer to 65 V. Sheath liquid composition was methanol/water (1:1, v/v) containing 1.0 mM of FA and two reference masses which allowed mass correction and higher mass accuracy m/z 121.0509 (purine) and m/z 922.0098 (HP-921). Samples were injected at 50 mbar for 50 s and stacked by applying background electrolyte at 100 mbar for 10 s. The separation voltage was 30 kV with 25 mbar of internal pressure and a total running time of 30 min24 (link),25 (link).
5
Preparation of Monophasic C18 Columns
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Monophasic C18 loading columns were prepared by polymerizing a Kasil 1624 (next advance) frit into a 150-μm-inner-diameter fused silica capillary (Agilent) and then packing with 2.5-cm-long reversed-phase 5 μm Aqua C18 resin (125 Å, Phenomenex). C18 loading columns were washed with MeOH and Buffer A prior to analysis. Analytical columns were prepared by pulling 100 μm diameter fused silica columns (Agilent) with a P-2000 laser tip puller (Sutter Instrument Co., Novato, CA), followed by packing with at least 15-cm reversed-phase 3 μm Aqua C18 resin (Phenomenex).
6
N-Glycoamidase F Purification
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N-Glycoamidase
F (PNGaseF) was obtained from Roche Diagnostics (Minato-ku, Tokyo,
Japan). The ultrafiltration membrane (MWCO: 3000 Da) was purchased
from Sartorius Mechatronics (Shinagawa-ku, Tokyo, Japan). The fused
silica capillary (100 μm i.d.) was obtained from Agilent Technologies
(Santa Clara, CA, USA). COSMOSIL 5C18-PAQ (4.6 mm I.D. × 150
mm) was obtained from Nacalai Tesque (Nakagyo-ku, Kyoto, Japan). Other
reagents and solvents were of the highest grade and were commercially
available.
F (PNGaseF) was obtained from Roche Diagnostics (Minato-ku, Tokyo,
Japan). The ultrafiltration membrane (MWCO: 3000 Da) was purchased
from Sartorius Mechatronics (Shinagawa-ku, Tokyo, Japan). The fused
silica capillary (100 μm i.d.) was obtained from Agilent Technologies
(Santa Clara, CA, USA). COSMOSIL 5C18-PAQ (4.6 mm I.D. × 150
mm) was obtained from Nacalai Tesque (Nakagyo-ku, Kyoto, Japan). Other
reagents and solvents were of the highest grade and were commercially
available.
7
CZE Analysis of Analytes
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CZE measurements were performed on an Agilent 3DCE instrument (Agilent Technologies, Waldbronn, Germany). For the analyses, a fused silica capillary (Agilent Technologies) with an internal diameter of 50 μm and an outer diameter of 360 μm was used (Ltot/eff = 60.1/51.7 cm). During measurements, the temperature was kept at 22.5 °C and the electric field strength was 33 kV/m. 200 mM boric acid with 10 mM SDS (pH 8.4, adjusted with NaOH) was used as a background electrolyte (BGE). Analytes were detected at 205 nm with an UV/Vis detector. Prior to measurements, the capillary was conditioned with BGE for 5 min, post-conditioning included flushing with 1 M NaOH and ultra-pure water, 5 min each. As internal standards, benzoic acid (400 μg/mL stock in ultra-pure water diluted 20-fold in samples) and DMSO (5.5 mg/mL stock in ultra-pure water — electroosmotic flow (EOF) marker — diluted 20-fold in samples) were used. Samples (tenfold dilution of the obtained SPE flow through) were injected hydrodynamically (40 mbar, 8 s).
8
Capillary Electrophoresis of Analytes
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An Agilent CE instrument (Agilent Technologies Deutschland, Germany) was used for the analysis. The instrument equipped with data processing Agilent Chem Station® software and IBM computer. Analytes were detected at 205 nm by DAD. A fused silica capillary (Agilent Technologies, Fullerton, CA) with the dimensions: 65 cm total length, 55 cm effective length, and 50 µm internal diameter, was used throughout the analysis. The capillary and the sample were maintained at 20 °C. The background electrolyte (BGE) is a microemulsion consisted of 1.66% Sodium dodecyl sulfate (SDS) as surfactant, 0.91% heptane as oil phase, 6.61% 1-butanol as cosurfactant, and 90.72% borate buffer (20 mM, pH 9.5) as aqueous phase. Samples were introduced into the capillary at 20 mbar for 40 s. A 20 kV voltage was applied to the capillary. The capillary was washed with 0.1 M NaOH (5 min), water (5 min), and BGE (5 min) before the first run. To ensure reproducibility of the assay, the capillary was washed between injections with 0.1 M NaOH (2 min), water (2 min), and BGE (2 min).
9
Capillary Electrophoresis Analysis of Caecal SCFAs
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Shortly after the caecum was opened, pH was measured in the distal end of the caecum. Acetate, propionate, and butyrate in caecal contents were analyzed using capillary electrophoresis and indirect UV detection by a method modified from Westergaard et al. 33 Briefly, approximately 0.1 g of caecal contents was diluted 10 times in alkaline buffer (0.1 M Tris, pH 8.7 with 100 μM malonic acid as internal standard), vortexed for 10 s, centrifuged (14000 x g, 10 min, 4 °C) and the supernatant was filtered using a sterile 0.2 μm filter (Minisart). Samples were kept at -80 °C until analysis. Prior to analysis the samples were diluted 30 times by running buffer (0.2 mM 1,2,4-benzenetricarboxylic acid), 8 mM TRIS and 0.3 mM tetradecyltrimethylammonium bromide, pH 7.6). The fused silica capillary (0.75 μm, 80.5 cm and 72 cm to detector window) purchased from Agilent (Waldbronn, Germany) was rinsed with 1 M NaOH before each sequence and pre-treated with water for 0.5 min, 0.1 M NaOH for 1 min and running buffer for 5 min before each run. Samples were injected by pressure (35 mbar, 2 s) and run at -30 kV for 12 min on a G1600A 3DCapillary electrophoresis Instrument (Hewlett-Packard, Waldbronn, Germany). All chemicals were purchased from Sigma Aldrich, Steinheim, Germany.
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