LCMS with electrospray ionization and selected ion monitoring (LCMS-ESI-SIM; ThermoScientific TSQ Quantiva) was used to analyze the extract of steroid biomarkers from the glass beads. Four 4mm glass beads from each participant were washed with 50 μL of 70/30 LCMS water/ACN (1% formic acid) in glass LCMS vials and then transferred to a second LCMS vial with a clean glass insert. 2 μL of glass bead extract was injected into the instrument for analysis by LCMS-ESI-SIM. Chromatography of the extract was accomplished using a Restek Raptor Biphenyl column (200 mm × 2.1 mm; 2.7 um bead) held at 30C and a water/ACN (0.1% formic acid) binary solvent system. The solvent flow rate was set to 0.5 mL/min with a gradient of ACN from 10% - 100% over 3 minutes followed by a column washing step for 1-minute at 100% ACN and a 3-minute column equilibrium step at 10% ACN before the next sample injection. Mass spectrometer settings used for detection of steroid biomarkers were as follows: 3kV in positive ion mode, 0.4 resolution, 20usec dwell time, 300 °C vaporizer temperature and ion transfer tube temperature, 50abs N2 sheath gas, 25Arb N2 aux gas. Table 1 shows the steroid biomarkers tracked in the extract samples.
Raptor biphenyl column
Manufactured by Restek
Sourced in United States
The Raptor Biphenyl column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of analytes. The column features a biphenyl stationary phase, which provides unique selectivity for a variety of compound classes, including aromatic and polar compounds. The column is suited for use in various HPLC applications, such as pharmaceutical, environmental, and food analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lcms 8060 triple quadrupole mass spectrometer, by Shimadzu (2 mentions)
1100 hplc system, by Agilent Technologies (2 mentions)
Tsq quantiva, by Thermo Fisher Scientific (1 mentions)
Catechin 2 3 4 13c3, by Merck Group (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Labsolutions lcms software, by Shimadzu (1 mentions)
Xevo tq xs triple quadrupole mass spectrometer, by Waters Corporation (1 mentions)
Kinetex f5 column, by Phenomenex (1 mentions)
Nexera2 uhplc system, by Shimadzu (1 mentions)
9 protocols using raptor biphenyl column
1
Steroid Biomarker Analysis by LCMS-ESI-SIM
2
UHPLC-MS/MS Analysis of Phenolic Compounds
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The stationary phase involved a Raptor Biphenyl column (100 × 2.1 mm, 1.8 µm) acquired from Restek (Milan, Italy), and a Raptor Biphenyl UHPLC (5 × 2.1) was used as a guard column. The mobile phase was a mixture of (A) ultrapure water and (B) methanol, both with the addition of 0.02% of acetic acid, eluting under the following gradient conditions: 0.0–0.3 min 10% B, 0.3–5.0 min 100% B, 5.0–8.0 min 100% B, 8.1 min 10%. B. The final run time was 10.0 min. The flow rate was 0.45 mL min−1, the injection volume was 1.0 µL, and the oven temperature was set at 45 °C.
The ESI worked in negative polarity ion mode (NI) and the mass spectrometer was used in selected reaction monitoring (SRM) mode. The optimized instrumental parameters were set as follows: nebulizing gas flow 3.0 L/min; heating gas flow 10 L/min; drying gas flow 10 L/min; interface temperature 300 °C; DL temperature 250 °C; heat block temperature 400 °C. The unit mass resolution was established and maintained in each mass-resolving quadrupole by keeping a full width at half-maximum (FWHM) of approximately 0.7 u. The SRM transitions with the related dwell times and the optimized compound-dependent parameters, such as voltage potential Q1, Q3, and collision energy (CE), are reported inTable S1 in the Supplementary Materials (SM) .
The ESI worked in negative polarity ion mode (NI) and the mass spectrometer was used in selected reaction monitoring (SRM) mode. The optimized instrumental parameters were set as follows: nebulizing gas flow 3.0 L/min; heating gas flow 10 L/min; drying gas flow 10 L/min; interface temperature 300 °C; DL temperature 250 °C; heat block temperature 400 °C. The unit mass resolution was established and maintained in each mass-resolving quadrupole by keeping a full width at half-maximum (FWHM) of approximately 0.7 u. The SRM transitions with the related dwell times and the optimized compound-dependent parameters, such as voltage potential Q1, Q3, and collision energy (CE), are reported in
3
Quantifying Polyphenol Metabolites in Chicken Plasma
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The chicken plasma samples were first hydrolyzed with β-glucuronidase/sulfatase from Helix pomatia (Type 1H, Sigma-Aldrich, Saint Louis, MO, USA). The identification and quantification of (poly)phenol metabolites in plasma after GSE consumption was performed using micro-elution solid phase extraction (μ-SPE), as previously described [33 (link),34 (link)], followed by UPLC-Q-q-Q MS. The detection of target metabolites was performed on a SHIMADZU Triple Quadrupole Mass Spectrometer (LCMS-8060, SHIMADZU, Kyoto, Japan) through an electro-spray ionization (ESI) source in negative mode. Extracted samples (5 μL) were injected through a Raptor Biphenyl column (2.1 × 50 mm, 1.8 µm, Restek, Bellefonte, PA, USA) with a compatible Raptor Biphenyl Guard Cartridge (5 × 2.1 mm, Restek, Bellefonte, PA, USA) in the UPLC system following a previously validated separation method [30 (link)]. Authentic standards were injected for the identification and quantification of individual (poly)phenols. Recoveries of the compounds during μ-SPE were estimated with the isotopically labeled internal standard (±) catechin-2,3,4-13C3 (Sigma-Aldrich, Steinheim, Germany). Analysis of the plasma (poly)phenols were performed on 4 hens from (control) group, 5 hens from (1% LT) group and 5 hens from (2% ST) group.
4
Uptake of Mito-LND into Mitochondria
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For measuring uptake of Mito-LND into isolated mitochondria59 (link),60 (link), we incubated Mito-LND (10 µM) with mouse liver mitochondria (1.5 mg/mL) in nonionic mannitol plus a sucrose-based buffer (220 mM mannitol, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, 0.2% fatty acid-free BSA, pH 7.2) at room temperature for 2 min, followed by additional 2 min incubation in the presence or absence of mitochondrial uncoupler, FCCP (1 µM). Next, the suspension was centrifuged (7000 g, 1 min) and the concentration of Mito-LND in the supernatant was measured by HPLC (Agilent 1100 HPLC system with UV-Vis and fluorescence detectors). The mitochondrial supernatant was injected onto a Raptor Biphenyl column (Restek, 50 mm × 4.6 mm, 2.7 µm) equilibrated with mobile phase containing water:acetonitrile mixture (1:1 by vol.) and trifluoroacetic acid (0.1% by vol.). Mito-LND was eluted by increasing the acetonitrile content in the mobile phase from 50 to 100% (by vol.) over 3 min, at a flow rate of 2 mL/min. For Mito-LND quantification, fluorescence traces (excitation at 300 nm, emission at 420 nm) were used.
5
Receptor Fluid and Extract Analysis
6
Phytochemical Profiling of Latex using UPLC-ESI-MS/MS
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Latex phytochemicals were identified by employing ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESIMS/MS) [21 (link)]. A Waters Acquity UPLC (Waters Corporation, Milford, MA, USA) chromatography system, coupled with ESI Xevo TQD triple quadrupole mass spectrometer, was used for phytochemicals identification. Latex phytochemicals were separated in a positive ionization mode in a Restek Raptor biphenyl column (100 mm length × 2.1 mm diameter × 1.8 micrometer particle size). Chromatographic analyte separations were carried out using a gradient mobile phase consisting of 0.1% formic acid in 10 mM ammonium formate and 0.1% formic acid in acetonitrile under linear gradient conditions (A:B % v/v, 0–0.5 min: 80:20; 0.5–14 min: 30:70; 14–15 min: 80:20) at 0.6 mL/min flow rate. The column temperature was maintained at 50 °C. Cone gas flow of 10 L/h, desolvation gas flow of 1000 L/h, capillary voltage of 0.70 kV, source temperature of 150 °C, and desolvation temperature of 450 °C were maintained for the source-dependent parameters. Ion detection was performed in the multiple-reaction monitoring (MRM) mode by monitoring the transition pairs. The compounds were identified based on standards prepared in methanol.
7
Plasma Phenolic Metabolite Extraction and Quantification
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The extraction of phenolic metabolites from plasma samples was performed by micro-elution solid phase extraction (μ-SPE) and measured by UPLC-Q-q-Q MS, as per the protocol of a validated method. 62 Briefly, undiluted plasma samples were acidified with 4% phosphoric acid (v : v 1 : 1). This mix (total volume of 600 μL) was then loaded onto Oasis 96-well reversed-phase HLB (hydrophilic-lipophilic balanced) sorbent μ-SPE plates (Waters, Eschborn, Germany), washed with water and 0.2% acetic acid, and eluted using 90 μL of methanol. Identification and quantification of ( poly)phenol metabolites were performed on a SHIMADZU Triple Quadrupole Mass Spectrometer (LCMS8060, SHIMADZU, Kyoto, Japan). Five microliters of the eluded samples were then injected through a Raptor Biphenyl column 2.1 × 50 mm, 1.8 µm (Restek, Bellefonte, USA) with a compatible Raptor Biphenyl Guard Cartridges 5 × 2.1 mm (Restek, Bellefonte, USA) in the UPLC system. A 14-minute gradient followed by a 2-minute equilibration was applied to the run under a flow rate of 0.5 mL min -1 at 30 °C. The identification of metabolites was performed by comparing retention times with authentic standards in corresponding multiple reaction monitoring (MRM) transitions and quantified by calibration curves made from standard mixes using SHIMADZU LabSolutions™ LCMS Software.
8
Quantification of Analytes by UPLC-MS/MS
9
LC-MS/MS Analysis of Lonidamine and Mito-LND
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Cells were grown in 10-cm dishes and incubated with the compounds for 24 h in complete medium. Liquid chromatography–mass spectrometry (LC-MS/MS) analyses were performed on cell extracts using Shimadzu Nexera2 UHPLC system equipped with UV-Vis absorption and triple quadrupole mass spectrometry (LC-MS8030) detectors. Detection of lonidamine was accomplished using Raptor Biphenyl column (Restek, 100 mm × 2.1 mm, 2.7 µm) equilibrated with mobile phase containing water:methanol (MeOH) mixture (4:1). LND was eluted by increasing the content of MeOH from 20 to 100% over 5 min and detected in the multiple reaction monitoring (MRM) mode using the transition of 319.10 > 274.95 (negative mode). Mito-LND was analyzed using a Kinetex F5 column (Phenomenex, 100 mm × 2.1 mm, 1.7 µm) equilibrated with a mobile phase containing water (75%), MeCN (25%) and formic acid (0.1%). Mito-LND was eluted by increasing the content of MeCN in the mobile phase from 25 to 100% over 5 min and detected in the MRM mode using the transition of 720.20 > 262.00 (positive mode). For both LND and Mito-LND analyses, the mobile phase flow rate was set at 0.5 mL/min.
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