We used an Accela UHPLC system coupled to a TSQ Quantum Ultra tandem mass spectrometer (both Thermo Fisher Scientific, Waltham, MA) in positive atmospheric pressure chemical ionization (APCI) mode for analysis. Isocratic elution (73% MeOH/water at 0.45 mL/min) for 12 min on an Ascentis F5 analytical column (2.1 mm × 150 mm, 2.7 μm, Sigma-Aldrich, St. Louis, MO) held at 27 °C, followed by a short (1–3 min) column flush with 100% methanol and short (1–3 min) equilibration to initial conditions, resulted in a < 20 min instrument analysis time per sample. The autosampler tray temperature was maintained at 7 °C. Injection volume, using partial loop mode, was 30 μL. Mass detection was carried out under selected reaction monitoring (SRM) conditions using the following transitions: m/z 383.3 → 365.1 (quantitation) and 383.3 → 105.0 (confirmation) for 25(OH)D3, m/z 395.3 → 377.3 (quantitation) and 395.3 → 209.1 (confirmation) for 25(OH)D2, m/z 389.3 → 371.1 for d6-25(OH)D3 and m/z 398.3 → 380.3 for d3-25(OH)D2. Scan time was 0.25 sec. Mass resolution in Q1 and Q3 was set at 0.7 full width at half maximum. We used Xcalibur 2.0.6 software (Thermo Fisher Scientific) for instrument control and data collection and processing.
Accela uhplc system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom
The Accela UHPLC system is a high-performance liquid chromatography instrument designed for ultra-high-pressure liquid chromatography (UHPLC) applications. The system features a high-pressure pump, an autosampler, a column compartment, and a variety of detector options, enabling efficient and precise separation and analysis of a wide range of chemical compounds.
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Lab products found in correlation
Kinetex c18 column, by Phenomenex (2 mentions)
Sieve 2, by Thermo Fisher Scientific (2 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Ltq velos pro, by Thermo Fisher Scientific (2 mentions)
Tsq vantage, by Thermo Fisher Scientific (2 mentions)
Beh c18 column, by Waters Corporation (2 mentions)
Ltq orbitrap xl hybrid mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Xcalibur software, by Thermo Fisher Scientific (2 mentions)
Syncronis c18 column, by Thermo Fisher Scientific (2 mentions)
Hypersil gold aq column, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (2 mentions)
Tsq quantum ultra, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap xl discovery, by Thermo Fisher Scientific (1 mentions)
Nicolet is10 infrared spectrometer, by Thermo Fisher Scientific (1 mentions)
Luna c8, by Phenomenex (1 mentions)
D8 advance x ray diffractometer, by Bruker (1 mentions)
Jsm 5600, by JEOL (1 mentions)
Accela autosampler, by Thermo Fisher Scientific (1 mentions)
Xcalibur, by Thermo Fisher Scientific (1 mentions)
40 protocols using accela uhplc system
1
Quantification of 25-Hydroxyvitamin D by UHPLC-MS/MS
2
Comprehensive Analytical Workflow for Lipid Profiling
3
Characterization of Graphene-based Nanomaterials
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The morphological characterization of graphene, graphene oxide nanosheets, and their magnetic analogs was conducted with scanning electron microscope (SEM) (JSM-7001F, Japan). The corresponding SEM images of the C18 magnetic nanoparticles were obtained using a JOEL microscope (JSM-5600, JEOL, Tokyo, Japan) after gold coating. The crystal structure characterization was conducted by X-ray diffraction (XRD) on a Bruker D8-advance X-ray diffractometer at 40 kV and 40 mA using Cu Kα (λ = 1.5406 Å) radiation and the sample was scanned from 5° to 60°, in steps of 0.02° (2θ), at a rate of 2 s per step. The magnetic properties of the respective nanomaterials were examined on a vibrating magnetometer (LakeShore 7300, Westerville, OH, USA) at room temperature.
The chromatographic analysis was conducted on an Accela UHPLC system (Thermo Fisher Scientific, Bremen, Germany) consisting of an Accela autosampler (model 2.1.1) and an Accela quaternary gradient UHPLC pump (model 1.05.0900). The chromatographic system was coupled to a hybrid LTQ Orbitrap XL Fourier transform mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The linear ion trap (LTQ) part of the hybrid MS system was equipped with an Ion Max electrospray ionization probe, operating in the positive and negative ionization mode.
The chromatographic analysis was conducted on an Accela UHPLC system (Thermo Fisher Scientific, Bremen, Germany) consisting of an Accela autosampler (model 2.1.1) and an Accela quaternary gradient UHPLC pump (model 1.05.0900). The chromatographic system was coupled to a hybrid LTQ Orbitrap XL Fourier transform mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The linear ion trap (LTQ) part of the hybrid MS system was equipped with an Ion Max electrospray ionization probe, operating in the positive and negative ionization mode.
4
Quantitative Analysis of Bioconversion Products
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LC–MS/MS quali-quantitative analyses of the reaction mixtures from different bioconversion experiments were performed on a LTQ-Orbitrap ESI mass spectrometer (Thermo-Fisher Scientific) coupled with an Accela UHPLC system (Thermo-Fisher Scientific). Chromatographic separation was carried out on a Kinetex C18 column (150 × 2 mm, 2.6 μm) (Phenomenex) using aqueous 0.1% formic acid (A) and methanol (B) as mobile phases, a linear gradient from 5 to 30% of B in 10 min and a 200 μL/min flow rate. Full mass (MS1) spectra were acquired in high resolution negative ion mode, over the m/z range from 125 to 500. MS/MS spectra were acquired for the most intense ion detected in the MS1 spectra (dependent scan mode), by in-trap fragmentation.
Relative abundance of 4-OHE2 and reaction products was estimated by generating an extracted ion chromatogram for each compound (m/z 287.2 for 4-OHE2, for 323.2 for 4-OHE2 meta cleavage product and 305.2 for 4-OHE2 cyclization product (hypothetical).
For each compound, the highest peak area registered was assumed as 100% area. Area % at the other time points were calculated compared to the highest peak area. Relative abundances obtained at the different time points were plotted in function of time.
Samples were analysed in triplicate and data were reported as the mean of the measured areas.
Relative abundance of 4-OHE2 and reaction products was estimated by generating an extracted ion chromatogram for each compound (m/z 287.2 for 4-OHE2, for 323.2 for 4-OHE2 meta cleavage product and 305.2 for 4-OHE2 cyclization product (hypothetical).
For each compound, the highest peak area registered was assumed as 100% area. Area % at the other time points were calculated compared to the highest peak area. Relative abundances obtained at the different time points were plotted in function of time.
Samples were analysed in triplicate and data were reported as the mean of the measured areas.
5
Comprehensive Metabolomic Analysis Pipeline
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Both polar and lipid analyses were analyzed on a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Sci., Bremen, Germany). Sources for all solvents and reagents, and more detailed experimental procedures are included in Supplementary Information . Chromatographic separation was performed on a Thermo Scientific (Thermo Fisher Sci., San José, CA, USA) Accela UHPLC system equipped with a quaternary pump, vacuum degasser and an open autosampler with a temperature controller. MS analysis was carried out on a QEx active benchtop Orbitrap detector loading an electrospray (ESI) source simultaneously operating in fast negative/positive polarity switching ionization mode. For all sample types (media, and polar and apolar intracellular fractions), quality control samples were run once every five samples.
All raw MS datasets were processed using Sieve 2.2 (Thermo Scientific) and mined against an in-house database of accurate masses and retention times generated in our laboratory using the IROA 300, MS Metabolite Library of Standards (IROA Technologies, Bolton, MA). In addition, databases of accurate masses taken from the KEGG database65 (link) and the Human Metabolome database66 (link) were also mined. MS data were then combined to the MRS data for the subsequent post-processing, followed by univariate and multivariate statistical analyses.
All raw MS datasets were processed using Sieve 2.2 (Thermo Scientific) and mined against an in-house database of accurate masses and retention times generated in our laboratory using the IROA 300, MS Metabolite Library of Standards (IROA Technologies, Bolton, MA). In addition, databases of accurate masses taken from the KEGG database65 (link) and the Human Metabolome database66 (link) were also mined. MS data were then combined to the MRS data for the subsequent post-processing, followed by univariate and multivariate statistical analyses.
6
Profiling Reduced GOS DP3 Isomers
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Reduced (including non-reducing) GOS DP3 isomers were analyzed
on an Accela UHPLC system (Thermo Scientific) coupled to a mass spectrometer
(LTQ Velos Pro ion trap MS, Thermo Scientific) as described elsewhere,
with some minor modifications.27 Samples
(0.5 μL, 0.25 mg/mL) were injected on a Hypercarb PGC column
(3 μm particle size, 2.1 × 150 mm) in combination with
a Hypercarb guard column (3 μm particle size, 2 × 10 mm,
Thermo Scientific). As mobile phase A, ULC–MS water + 0.1%
(v/v) formic acid was used. Mobile phase B consisted of ACN + 0.1%
(v/v) formic acid. The flow rate was 300 μL/min. The solvents
were eluted according to the following profile: 0–2 min, 3%
B; 2–51.7 min, 3–11% B; 51.7–53.2 min, 11–100%
B; 53.2–61 min, 100% B; 61–62.5 min, 100–3% B;
and 62.5–70.3 min, 3% B. The temperatures of the autosampler
and column oven were controlled at 10 and 25 °C, respectively.
ULC–MS water containing 3% ACN was used to wash the autosampler
needle. DP3 standards β-3-galactosyl-lactose, β-4-galactosyl-lactose,
and β-6-galactosyl-lactose were used for the identification
of GOS isomers. Vivinal GOS was analyzed to identify the DP3 isomers
in the Vivinal GOS mixture. Prior to analysis, both the standards
and Vivinal GOS were reduced and purified using a small-scale SPE
method as described elsewhere.27 Data acquisition
and processing were performed using Xcalibur (version 2.2, Thermo
Scientific).
on an Accela UHPLC system (Thermo Scientific) coupled to a mass spectrometer
(LTQ Velos Pro ion trap MS, Thermo Scientific) as described elsewhere,
with some minor modifications.27 Samples
(0.5 μL, 0.25 mg/mL) were injected on a Hypercarb PGC column
(3 μm particle size, 2.1 × 150 mm) in combination with
a Hypercarb guard column (3 μm particle size, 2 × 10 mm,
Thermo Scientific). As mobile phase A, ULC–MS water + 0.1%
(v/v) formic acid was used. Mobile phase B consisted of ACN + 0.1%
(v/v) formic acid. The flow rate was 300 μL/min. The solvents
were eluted according to the following profile: 0–2 min, 3%
B; 2–51.7 min, 3–11% B; 51.7–53.2 min, 11–100%
B; 53.2–61 min, 100% B; 61–62.5 min, 100–3% B;
and 62.5–70.3 min, 3% B. The temperatures of the autosampler
and column oven were controlled at 10 and 25 °C, respectively.
ULC–MS water containing 3% ACN was used to wash the autosampler
needle. DP3 standards β-3-galactosyl-lactose, β-4-galactosyl-lactose,
and β-6-galactosyl-lactose were used for the identification
of GOS isomers. Vivinal GOS was analyzed to identify the DP3 isomers
in the Vivinal GOS mixture. Prior to analysis, both the standards
and Vivinal GOS were reduced and purified using a small-scale SPE
method as described elsewhere.27 Data acquisition
and processing were performed using Xcalibur (version 2.2, Thermo
Scientific).
7
Metabolic Tracing of Tumor Cells and Tissues
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EL4 and Colo205 cells (107) were incubated with 11 mm [U-13C]glucose for 30 s and then quenched in ice-cold methanol. Tumor-bearing mice were injected intravenously with 0.4 ml of 200 mm [U-13C]glucose with or without 28 mm DHA; the animals were then sacrificed, and tumors were excised rapidly at 1 min after injection and then freeze-clamped immediately. Cells were extracted at 5 × 107 ml−1, and tumors were excised at 50 mg ml−1 in ice-cold 75:25 methanol/acetonitrile containing 0.2% formic acid using metal bead-containing tubes on a Precellys24 homogenizer coupled to a Cryolys® cooler (Stretton Scientific, Stretton, UK) at 4 °C. A second extraction was performed with 200 μl of water, and the organic and aqueous extracts were mixed. Solvent was removed by evaporation, and the extracts were dissolved in 0.75% octylamine in HPLC grade water. LC-MS/MS measurements of 13C-labeling of 6PG, 3PG, and PEP were based on a method published previously (26 (link)). Analytes were separated using octylamine/acetonitrile gradients on an ACQUITY UPLCTM BEH130 C18 ID column (Waters, Elstree, UK) at 30 °C and detected using a triple quadrupole TSQ Vantage mass spectrometer with an Accela UHPLC system (Thermo Scientific, Loughborough, UK) fitted with a HESI probe with a source temperature of 320 °C.
8
LC-MS/MS Analysis of Compounds
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LC–MS/MS analysis was performed using an Accela UHPLC system (Thermo Fisher Scientific, CA, USA) coupled with a high-resolution LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Electron, Bremen, Germany) via an ESI interface. Sample separation was performed at 40 °C using a Waters BEH C18 column (2.1 × 150 mm, 1.7 μm). Water (A) and acetonitrile (B) were used as the mobile phase, with 0.1% formic acid being added to both solvents. The flow rate was adjusted to 400 μL/min. The elution gradient was as follows: 0% B (0–1 min), 0–40% B (1–20 min), 40–100% B (20–24 min), and 100% B (24–27 min). The sample (2 μL) was injected, and the analysis was performed using PDA at 200–600 nm and MS at m/z 100–1000 in the positive ion mode. The conditions of the ESI source were similar to those of a previous study. The compounds were identified using the reference literature [25 (link)], high-resolution mass spectra, and MS/MS spectral library search [26 (link)].
9
Comprehensive U-HPLC-DAD/ESI-MS/MS Analysis of PEESG
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In this study, U-HPLC-DAD/ESI-MS/MS analysis method was carried out to demonstrate and characterize the major constituents of PEESG. The analysis was performed on an Accela U-HPLC system (Thermo Fisher Scientific, San Jose, CA) coupled with LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA), fitted with an ESI source. Samples were separated on a reversed-phase Kinetex C18 column (100 mm × 2.10 mm, 1.7 μm, Phenomenex Inc., USA) using a flow rate of 0.3 mL/min at 25°C. The mobile phase consisted of eluent A (methanol) and eluent B (aqueous formic acid solution, 0.2%, v/v). A gradient program was used for elution: 0–30 min, A from 15% to 95%, B from 85% to 5%. Analytes were determined by ESI-MS/MS selected reaction monitoring in the negative ion mode. The triple quadrupole MS and spray chamber conditions were gas temperature, 300°C; drying gas, nitrogen at 10 L/min; nebulizer pressure, 15 psi; sheath gas temperature, 250°C; sheath gas flow, nitrogen at 7 L/min; capillary voltage, 4 kV.
10
Sensitive Fluorescent Derivatization and UHPLC-MS Analysis
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The derivatization reaction took place in Thermo Scientific Reacti-Therm Heating Module (Metrolab, Athens, Greece). All measurements were performed with a Fluostar Galaxy multifunctional microplate reader (BMG LabTechnologies GmbH, Germany). Fluorescence optics were installed, and the excitation and emission wavelengths were set at 540 and 615 nm, respectively, by selecting the relevant filters. The 96-well black polystyrene plates and 1.5-mL Eppendorf tubes were obtained from Nunc (Pnoi, Athens, Greece).
An Accela UHPLC system (Thermo Fisher Scientific, Bremen, Germany) equipped with a binary pump and an autosampler was utilized to separate the excess of derivatization agent from the product on an Ascentis Fused Core C18 column (100 × 2.1 mm, 2.7 μm). Mass spectra were recorded on a hybrid LTQ™ Orbitrap Discovery XL instrument (Thermo Fisher Scientific), enabling the characterization of the reaction product. The whole system was controlled by the Xcalibur 2.1 software.
An Accela UHPLC system (Thermo Fisher Scientific, Bremen, Germany) equipped with a binary pump and an autosampler was utilized to separate the excess of derivatization agent from the product on an Ascentis Fused Core C18 column (100 × 2.1 mm, 2.7 μm). Mass spectra were recorded on a hybrid LTQ™ Orbitrap Discovery XL instrument (Thermo Fisher Scientific), enabling the characterization of the reaction product. The whole system was controlled by the Xcalibur 2.1 software.
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