3D20E22P was purified from MAGs. The purification was performed at analytical scale under the same LC conditions as for HPLC–MS/MS analysis using an ultra-performance liquid chromatography instrument (Acquity, Waters) coupled with a quadrupole mass-based detector (QDa, Acquity, Waters). Fraction collection was triggered when the m/z corresponding to 3D20E22P was detected at the same retention time as that previously identified. The purity of the extracted compounds was then checked by HPLC–MS/MS as described above.
Acquity
Manufactured by Waters Corporation
Sourced in United States, United Kingdom, Germany, Jamaica, France
The Acquity is an analytical instrument designed for high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) applications. It provides precise separation, identification, and quantification of chemical compounds in complex samples.
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Lab products found in correlation
Beh c18 column, by Waters Corporation (8 mentions)
Synapt g2, by Waters Corporation (7 mentions)
Q exactive, by Thermo Fisher Scientific (6 mentions)
Acquity uplc beh c18 column, by Waters Corporation (6 mentions)
Xevo tq s, by Waters Corporation (5 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (4 mentions)
Xevo tqd, by Waters Corporation (4 mentions)
Xcalibur, by Thermo Fisher Scientific (4 mentions)
C18 reversed phase column, by Waters Corporation (4 mentions)
H class system, by Waters Corporation (4 mentions)
Acquity uplc beh, by Waters Corporation (4 mentions)
Acquity beh c18 column, by Waters Corporation (4 mentions)
Amino acid standard, by Merck Group (4 mentions)
Quanlynx software, by Waters Corporation (3 mentions)
Beh amide column, by Waters Corporation (3 mentions)
Kinetex 5 μm c18 column, by Phenomenex (3 mentions)
Exactive mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Microtof 2, by Bruker (3 mentions)
Beh glycan column, by Waters Corporation (3 mentions)
Luna nh2 column, by Phenomenex (2 mentions)
147 protocols using acquity
1
Purification and Validation of 3D20E22P
2
Serum Amino Acid Profiling by HPLC
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The concentrations of Met, Lys, valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp), histidine (His), arginine (Arg), glycine (Gly), alanine (Ala), serine (Ser), proline (Pro), asparagine (Asn), ornithine hydrochloride (Orn), aspartic acid (Asp), glutamine (Gln), glutamic (Glu), tyrosine (Tyr), and threonine (Thr) in the serum were determined using high-performance liquid chromatography (ACQUITY, Waters, Milford, MA, USA) with the column (ACQUITY, Waters, Milford, MA, USA). Metabolite extraction and a computer test were determined following the procedures of previous studies [19 (link),20 (link)].
3
Metabolite and Lipid Profiling of Rice
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Soluble sugars were extracted by shaking vigorously in 80% (v/v) ethanol twice after grinding grain endosperm, and the samples were centrifuged at 12 000 g for 15 min. The supernatant was added chloroform (v/v) for removing the pigment, the solution was centrifuged, and the supernatant was collected. Cell extracts were analysed by ultrahigh‐performance liquid chromatography (Acquity, Waters) coupled to a Q Exactive hybrid quadrupole–orbitrap mass spectrometer (Thermo Fisher Scientific). The injection volume was 10 μL. Metabolites were separated with a Luna NH2 column (100 mm × 2 mm, 3 μm particle size, Phenomenex). The LC‐MS data were analysed as described previously (Zhang et al., 2018b ). Lipids were extracted from dehulled rice seeds and analysed by liquid chromatography–electrospray ionization mass spectrometry (LC‐SI‐MS). Polar lipids were analysed using an Exion UPLC system coupled with a triple quadrupole/ion trap mass spectrometer (6500 Plus Qtrap; SCIEX). MRM transitions were set up for comparative analysis of various polar lipids (Lim et al., 2014 ). Lipid species in each class were quantified by referencing to spiked internal standards corrected by response factor determined by two standards of same class as described previously (Lu et al., 2018 ).
4
UPLC-MS Analysis of Oxidation Products
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Ultra-performance liquid chromatograph
(UPLC) Acquity (Waters Ltd.) equipped with a photodiode array detector
and combined with an LCT Premier XE (Waters Ltd.) mass spectrometer
was used for the chromatographic separation and identification of
AMBB and AAPBA oxidation products. Separation was performed on a reversed-phase
C18 UPLC column (Waters Acquity UPLC BEH C18 1.7 mm, 50 × 2.1 mm) equilibrated with a water/acetonitrile
(CH3CN) (90/10 v/v) mobile phase containing 0.1% (v/v)
trifluoroacetic acid (TFA). The analytes were separated at a flow
rate of 0.3 mL/min. During the first 0.5 min of analysis, the composition
of the mobile phase remained unchanged. The fraction of CH3CN was then gradually increased from 10 to 82% over the next 2 min.
The injection volume was 2 μL, the sample temperature was 20
°C, and the column temperature was 40 °C. The electrospray
source was operated at positive ion mode using the following parameters:
capillary voltage 2.8 kV, sample cone voltage 60 V, desolvation temp.
350 °C, source temp. 100 °C, desolvation gas flow 800 L/h,
and cone gas flow 50 L/h. The MCP detector voltage was 2.5 kV.
(UPLC) Acquity (Waters Ltd.) equipped with a photodiode array detector
and combined with an LCT Premier XE (Waters Ltd.) mass spectrometer
was used for the chromatographic separation and identification of
AMBB and AAPBA oxidation products. Separation was performed on a reversed-phase
C18 UPLC column (Waters Acquity UPLC BEH C18 1.7 mm, 50 × 2.1 mm) equilibrated with a water/acetonitrile
(CH3CN) (90/10 v/v) mobile phase containing 0.1% (v/v)
trifluoroacetic acid (TFA). The analytes were separated at a flow
rate of 0.3 mL/min. During the first 0.5 min of analysis, the composition
of the mobile phase remained unchanged. The fraction of CH3CN was then gradually increased from 10 to 82% over the next 2 min.
The injection volume was 2 μL, the sample temperature was 20
°C, and the column temperature was 40 °C. The electrospray
source was operated at positive ion mode using the following parameters:
capillary voltage 2.8 kV, sample cone voltage 60 V, desolvation temp.
350 °C, source temp. 100 °C, desolvation gas flow 800 L/h,
and cone gas flow 50 L/h. The MCP detector voltage was 2.5 kV.
5
Protein Precipitation for Metabolomics
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For protein precipitation, an ice‐cold methanol/water (3 : 1) solution was added to the serum and centrifuged at 20 817 g for 15 min at 4 °C. The supernatant was diluted with distilled water to half the volume of the supernatant, and it was then injected in the ultra‐performance liquid chromatography (UPLC) (ACQUITY®; Waters, Milford, MA, USA) coupled with a QToF‐MS (SYNAPT™ G2; Waters).
6
Quantifying Glutathione Species in Liver Extracts
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Liver extracts were analyzed with a UPLC system (Acquity, Waters, Manchester) coupled to a Time of Flight mass spectrometer (ToF MS, SYNAPT G2, Waters). A 2.1 × 100 mm, 1.7 mm BEH amide column (Waters), stabilized at 40 °C, was used to separate the analytes before entering the MS. Solvent A (aqueous phase) consisted of 99.5% water, 0.5% formic acid, and 20 mM ammonium formate while solvent B (organic phase) consisted of 29.5% water, 70% MeCN, 0.5% formic acid, and 1 mM ammonium formate. The extracted ion trace was obtained for GSH (m/z = 308.0916) and GSSG (m/z = 613.1598) and in the case of NAPQI–GSH (m/z = 457.139) in a 20 mDa window and subsequently smoothed (2 points, 2 iterations) and integrated with QuanLynx software (Waters, Manchester).
7
Purification and Characterization of YjiC
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Genistein, daidzein, biochanin A and formononetin were purchased from Tokyo Chemical Industry (Japan). UDP-α-D-glucose was purchased from Sigma-Aldrich (USA). Genistein 7-O-β-D-glucoside (genistin) was available in our laboratory. All other chemicals and reagents were of the highest chemical grade available. The high resolution mass spectrometry spectra were obtained in positive ion mode on ACQUITY (UPLC, Waters Corp., USA) coupled with SYNAPT G2-S (Waters Corp.). The details of the methodology for the cloning, expression, and purification of YjiC are described in our previous report (Pandey et al., 2013a (link)).
8
Synthetic Polyurethane Characterization
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The synthetic structure of PUs were measured using nuclear magnetic resonance and Fourier transform infrared. 1H-NMR spectra of PUs were measured using nuclear magnetic resonance (500 MHz, Bruker, Karlsruhe, Germany), with 5% (w/v) polymer solution in Chloroform-d. FT-IR spectra of PUs were measured using a Fourier transform infrared (Varian 640-IR, Varian, Sydney, Australia), and the spectrum ranged from 4000 to 700 cm−1. The number average molecular weights (Mn), weight average molecular weights (Mw), and polydispersity index (PDI) of the PUs were measured by gel permeation chromatography (ACQUITY, Waters, Milford, CT, USA). The measurement conditions were measured using the standard sample polystyrene (Mw = 47,200, 129,000 and 264,000) and using the Tetrahydrofuran solution at a rate of 0.5 mL/min−1. The thermal analysis was measured using a differential scanning calorimetry (Exstar 7020, SEIKO, Tokyo, Japan). The PU sample was sealed in an Al pan, cooled to −70 °C under a nitrogen atmosphere, and then heated to 300 °C at a rate of 10 °C min−1 and measured. Contact angle equipment (Phoenix 300, Surface Electro Optics, Suwon, Republic of Korea) was used to measure the wettability of the polymer surface.
9
Extraction and Analysis of Bacterial Metabolites
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The bacteria were cultured in a TSB medium until the optical depth value measured at a wavelength of 600 nm (OD600) reached 3.5. The secondary metabolites were extracted using ethyl acetate, which has a moderate polarity and low boiling point. Then, 5 mL of the bacterial culture solution was extracted three times using the same volume of acidified ethyl acetate (containing 0.01% glacial acetic acid), and the extract was dried using a centrifugal concentrator. The extract was analyzed via ultra-performance liquid chromatography (UPLC) (Waters ACQUITY, Milford, MA, USA) using a coupled MicroTOF-MS system (Bruker Daltonics GmbH, Bremen, Germany). A 2.1 mm × 150 mm reversed phase chromatographic column (Waters, BEH C18, 1.7 μm, Milford, MA, USA) with a flow rate of 250 μL min−1 was used. The sample was dissolved in acetonitrile and eluted for 30 min using a gradient mobile phase (5% murine, 95% acetonitrile solution containing 1% formic acid). The LC-MS results were processed using DataAnalysis version 3.3 software provided by Bruker Company, Billerica, MA, USA.
10
Metabolite Profiling by Liquid Chromatography-Mass Spectrometry
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