In order to facilitate the identification of the bioactive compounds present, CETC was filtered through a 0.22-μm syringe driven filter (Nylon 66) and initially was subjected to LC-MS analysis so as to obtain the Molecular ion mass using Acquity H-Class (Waters) ultra-performance liquid chromatography with BEH C18 column (50 mm × 2.1 mm × 1.7 μm) and a Xevo G2 (Waters) and Mass spectrometer. The mobile phase used was a mixture of acetonitrile and water (10:90) that was delivered at a flow rate of 0.2 ml/min. The electrospray ionization was used in both positive and negative mode with a scan range from m/z 50 to 2000, and the scan time was 9 min. The source temperature and desolvation temperature were 135 and 350 °C, respectively, with the capillary voltage at 4.50 kV. The molecular ion mass which was identified for possible bioactive compound from the LC-MS analysis was further confirmed by detailed LC-MS/MS based analysis using Acquity H-Class (Waters) ultra-performance liquid chromatography with BEH C18 column (50 mm × 2.1 mm × 1.7 μm) and Xevo G2 (Waters) Quadruple Time of Flight (Q-TOF) Mass spectrometer. The compound was further confirmed by the fragmentation pattern analysis.
Acquity h class
Manufactured by Waters Corporation
Sourced in United States, France, United Kingdom, Germany
The Acquity H-Class is a high-performance liquid chromatography (HPLC) system designed for analytical applications. It features an advanced quaternary solvent delivery system, a high-sensitivity UV-Vis detector, and a temperature-controlled autosampler. The Acquity H-Class is capable of achieving precise and reproducible results across a wide range of analytical methods.
Automatically generated - may contain errors
Lab products found in correlation
Beh c18 column, by Waters Corporation (8 mentions)
Synap g2, by Waters Corporation (3 mentions)
Acquity uplc beh c18, by Waters Corporation (3 mentions)
Uplc beh c18 column, by Waters Corporation (3 mentions)
Empower 3, by Waters Corporation (3 mentions)
Qtrap 6500 system, by Waters Corporation (2 mentions)
Xevo tq s triple quadrupole mass spectrometer, by Waters Corporation (2 mentions)
Accq tag ultrac18, by Waters Corporation (2 mentions)
Xevo g2, by Waters Corporation (2 mentions)
Masslynx software v4, by Waters Corporation (2 mentions)
Sqd mass analyzer, by Waters Corporation (2 mentions)
Acquity uplc beh, by Waters Corporation (2 mentions)
Synapt g2 s high definition ms system, by Waters Corporation (2 mentions)
Analyst 1, by AB Sciex (1 mentions)
Ab 6500 qtrap, by AB Sciex (1 mentions)
Turbo ion spray interface, by AB Sciex (1 mentions)
Synapt g2 si high definition mass spectrometer, by Waters Corporation (1 mentions)
Xbridge c18 column, by Waters Corporation (1 mentions)
Avance 400 mhz spectrometer, by Bruker (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
72 protocols using acquity h class
1
Bioactive Compound Identification via LC-MS
2
Quantitative Analysis of Galacturonate Oligosaccharides
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Fluorescently labeled galacturonate-based oligosaccharides were analyzed using a UPLC system (Waters Acquity H-Class, United States) coupled to a fluorescence detector (Waters Acquity H-Class, United States) and an ESI-IT-MSn-detector (Amazon speed ETD, Bruker, Germany). Chromatographic separation was performed on an Acquity UPLC BEH amide column (1.7 m, 2.1 mm × 150 mm) in combination with a Van Guard pre-column (1.7 m, 2.1 mm × 5 mm; Waters Corporation, Milford, MA, United States). HILIC-FLR/ESI-MSn elution procedures and detection methods are described in Supplementary Methods 5 . Quantification of oligogalacturonides was verified using this procedure as illustrated in Supplemental Methods 6 .
3
Purification and Characterization of CaV3.2 Modulator
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P. murinus lyophilized fractions were dissolved in 100 µL of extracellular solution supplemented with 0.1% BSA and tested using manual whole-cell patch clamp on HEK293 cells stably overexpressing CaV3.2 (see protocol above). Fraction 32 was re-purified by RP-HPLC (UltiMate® 3000 Standard LC systems, ThermoFisher Scientific) on a C18 column (Synchronis C18, 254 ⨯ 4.6mm, 5 µm, ThermoScientific) using a gradient of water/ACN in 0.1% formic acid from 20-40% ACN over 20 min. Fractions were collected manually. Sub-fraction 32.6 was evaluated for activity using manual whole-cell patch clamp on HEK293 cells stably overexpressing CaV3.2 (see protocol below) and analyzed by LC-HRMS (Acquity H-Class, Waters, Synapt G2-S, Waters) using a Kinetex C18 100 Å column (100 × 2.1 mm, 2.6 μm particle size) from Phenomenex (France) and a gradient of water/ACN in 0.1% formic acid from 20-40% ACN over 20 min. Peptide mass was determined using electrospray ionization time-of-flight (ESI-TOF) mass spectrometry (Acquity H-Class, Waters, Synapt G2-S, Waters), and the amino acid sequence of the peptide was determined by Edman degradation.
4
Quantifying Metabolites via UPLC-QTOF-MS
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A volume of 10 µL of crude homogenates (HOMG) W and HOMG W/O was used for the mass spectrophotometry analysis that was made with ultra-performance liquid chromatography (UPLC) (Acquity HClass, Waters) coupled to a quadrupole-time-of-flight (QTOF) mass spectrometer (Synap G2, Waters). The chromatographic conditions in [33 (link)] were used. The chromatograms were analyzed with MassLynx V4.1 software to tentatively identify compounds in the crude homogenates based on their mass fragments (MS) and retention times (RT) by the use of the Chemspider database.
5
Lipid Extraction and Characterization from Biomass
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A total of 1 g of biomass was extracted with DMC or EtOAc according to the protocol described by Boutin et al. [30 (link)]. The calibration curve was built up using castor oil and results were expressed as mg of equivalent of castor oil in the extract.
Pigments and total lipid rates were obtained using protocol described in Boutin et al. (2019) [30 (link)]. FFA profiles were obtained using the LC-ESI-MS protocol adapted from Samburova et al. (2013) [50 (link)]. Briefly, LC-ESI-MS analyses were performed on an Acquity H-Class with an SQD detector (Waters, Saint Quentin en Yvelines, France). The system was fitted with a BEH C18 (50 × 2.1 mm; 1.7 µm particle size). The column oven was set at 40 °C. Mobile phases were A Water 0.1% NH3 aq; B acetonitrile 0.1% NH3 aq. Flow rate was 0.25 mL/min and the gradient was set as follows—initial solvent B content was 10%, raised to 40% in 2 min, 90% in 23 min and 100% in 1 min and maintained for 9 min. ESI in negative mode was performed with cone voltage set at 50 V and capillary voltage at 2.8 kV.
Pigments and total lipid rates were obtained using protocol described in Boutin et al. (2019) [30 (link)]. FFA profiles were obtained using the LC-ESI-MS protocol adapted from Samburova et al. (2013) [50 (link)]. Briefly, LC-ESI-MS analyses were performed on an Acquity H-Class with an SQD detector (Waters, Saint Quentin en Yvelines, France). The system was fitted with a BEH C18 (50 × 2.1 mm; 1.7 µm particle size). The column oven was set at 40 °C. Mobile phases were A Water 0.1% NH3 aq; B acetonitrile 0.1% NH3 aq. Flow rate was 0.25 mL/min and the gradient was set as follows—initial solvent B content was 10%, raised to 40% in 2 min, 90% in 23 min and 100% in 1 min and maintained for 9 min. ESI in negative mode was performed with cone voltage set at 50 V and capillary voltage at 2.8 kV.
6
Metabolite Profiling of RAW264.7 Cells
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After 5 days of HA and β‐TCP treatment respectively, RAW264.7 cells were collected and centrifuged at 1200 r min−1 for 5 min at 4 °C, before washing them in cold PBS. Further centrifugation at 1200 r min−1 for 5 min at 4 °C was followed by vigorous vortexing with 1 mL of 80% cold methanol. The supernatant was collected and normalized to the protein concentration. Subsequently, the sample extracts were analyzed using an LC‐ESI‐MS/MS system (Waters ACQUITY H‐Class; MS, QTRAP 6500+ System). The AB 6500+ QTRAP LC‐MS/MS system was equipped with an ESI Turbo Ion‐Spray interface and can operate in both positive and negative ion modes according to Analyst 1.6 software (A B Sciex). Detection and determination of the relative abundances of the targeted energy metabolites were performed using MetWare based on the AB Sciex QTRAP 655 LC‐MS/MS platform.
7
Ion Pairing Chromatography for Metabolite Analysis
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Ion pairing-reversed phase chromatography separations were run on an ultra high performance liquid chromatography system (UHPLC, Acquity H-Class® Waters, Manchester, UK), equipped with a BEH C18 column (100 mm × 2.1 mm, packed with 1.7 μm porosity particles) (Waters, Manchester, UK). The flow rate was of 0.150 mL min−1 and column was heated at 30 °C. A ternary gradient was used (A, pure water; B, pure acetonitrile; and C, 20 mM hexylammonium dissolved in water, and pH value adjusted to 6 by addition of acetic acid), from 16.6% to 35% of solvent B in 10 min, then up to 63.4% at 20 min and maintained at 73.4% for 4.5 min. Percentage of solvent C was kept constant at 25%.
MS measurements were done through a direct coupling with a Synapt G2Si high-definition mass spectrometer (Waters Corp., Manchester, UK) on a mass range of 300–2000 m/z. The instrument was operated in a negative ionization mode in the so-called sensitivity mode, with an ESI capillary voltage of 2.5 kV and a sampling cone voltage of 50 V. Data acquisition was carried out using MassLynx software (V4.1).
MS measurements were done through a direct coupling with a Synapt G2Si high-definition mass spectrometer (Waters Corp., Manchester, UK) on a mass range of 300–2000 m/z. The instrument was operated in a negative ionization mode in the so-called sensitivity mode, with an ESI capillary voltage of 2.5 kV and a sampling cone voltage of 50 V. Data acquisition was carried out using MassLynx software (V4.1).
8
Automated Online SPE-UPLC-MS/MS Analysis
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The online SPE-UPLC-MS/MS system consisted in an isocratic pump (ICS®, Toulouse, France) for online SPE and an UPLC-MS/MS system composed of an UPLC system Acquity® H Class (Waters, Milford, CT, USA) coupled to a Xevo® TQ-S triple quadrupole mass spectrometer (Waters, Milford, CT, USA).
An Xbridge® C18 column (2.1 × 30 mm, 10-µm-particle diameter, Waters, Milford, CT, USA) was used for the fully automated SPE online procedure.
Chromatographic separation was achieved using an Aquity® CSH C18 column (1.7 µm particle size, 2.1 × 100 mm, Waters, Milford, CT, USA) heated at 40 °C and a binary mobile phase (MeOH/water) delivered in the gradient mode at a flow rate of 350 µL.min−1. Quantification was obtained by using MRM mode with two m/z transitions per analyte, one for quantification and one for confirmation, in negative electrospray ionization mode
An Xbridge® C18 column (2.1 × 30 mm, 10-µm-particle diameter, Waters, Milford, CT, USA) was used for the fully automated SPE online procedure.
Chromatographic separation was achieved using an Aquity® CSH C18 column (1.7 µm particle size, 2.1 × 100 mm, Waters, Milford, CT, USA) heated at 40 °C and a binary mobile phase (MeOH/water) delivered in the gradient mode at a flow rate of 350 µL.min−1. Quantification was obtained by using MRM mode with two m/z transitions per analyte, one for quantification and one for confirmation, in negative electrospray ionization mode
9
Quantitative LC-MS Analysis of Compounds
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The drug quantifications were analyzed by LC-MS [43 (link)]. The analysis used UPLC–MS (Acquity H-Class, Waters Corporation, Milford, MA, USA). An integrated vacuum degasser and an ultra-performance binary solvent manager were also used. The instrument was attached to the C18 column (2.1 × 50 mm, 1.7 mm) with a gradient flow pump with autosampler. The study’s mobile phase comprised 0.1% formic acid (A) and acetonitrile (B). The elution was gradient with a constant flow rate of 0.6 mL/min with an injection volume of 5 mL. During the complete analysis, the column was maintained at ambient temperature. The MS conditions consisted of a positive polarity electrospray ionization (ESCi) source; a probe temperature of 450 °C; a sampling cone voltage of 30 V; a source temperature of 150 °C; a source offset voltage of 80 V; a sample infusion flow rate of 5 mL/min, a collision energy ramp of 6 eV (argon, collision gas); and a mass ranging from 50 to 1500 m/z. All of these constitute the acquisition parameters. Furthermore, all acquired data were processed using waters corporation mass lynx software (V4.1, Milford, MA, USA).
10
Purification and Characterization of Synthesized Compounds
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