The liquid chromatography (LC) system comprised a Waters (Waters Corporation, Milford, CT, USA) Ultra Performance Liquid Chromatography (UPLC) equipped with an ACQUITY UPLC binary solvent manager, an ACQUITY UPLC sample manager, an ACQUTIY UPLC sample organizer, and an ACQUITY UPLC column heater HT. The mass spectrometric (MS) analysis was performed using a Triple Quad 6500+ (Applied Biosystems/MDS Sciex, Concord, ON, Canada) with an ESI ion source. The data acquisition and control system were created using Analyst 1.6.3 and 1.7.1 Software from Applied Biosystems/MDS Sciex. The LC/MS conditions for JV-DE1 are described in Supplementary Table S1a–c .
Ultra performance liquid chromatography (uplc)
Manufactured by Waters Corporation
Sourced in United States, United Kingdom, Japan
UPLC (Ultra Performance Liquid Chromatography) is a highly efficient liquid chromatography technique developed by Waters Corporation. It utilizes small particle size columns and high pressure to achieve rapid, high-resolution separations of complex samples. The core function of UPLC is to provide enhanced chromatographic performance and increased analytical productivity compared to traditional HPLC (High-Performance Liquid Chromatography) methods.
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Lab products found in correlation
Acquity uplc beh c18 column, by Waters Corporation (6 mentions)
Beh c18 column, by Waters Corporation (6 mentions)
Qtrap 5500, by AB Sciex (5 mentions)
Beh c18, by Waters Corporation (4 mentions)
C18 column, by Waters Corporation (3 mentions)
Beh c8 column, by Waters Corporation (3 mentions)
Qtrap 6500, by AB Sciex (3 mentions)
Synapt g2 quadrupole time of flight mass spectrometer, by Waters Corporation (3 mentions)
Uplc ltq orbitrap xl, by Thermo Fisher Scientific (2 mentions)
Ms frontier software, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (2 mentions)
Xcalibur 2, by Thermo Fisher Scientific (2 mentions)
Multiquant software, by AB Sciex (2 mentions)
Tripletof 6600, by AB Sciex (2 mentions)
Nbd sphingosine, by Avanti Polar Lipids (2 mentions)
Nbd s1p, by Avanti Polar Lipids (2 mentions)
Tripletof 5600 plus, by Thermo Fisher Scientific (2 mentions)
Acquity uplc beh c18, by Waters Corporation (2 mentions)
Xevo tq s, by Waters Corporation (2 mentions)
Tq detector, by Waters Corporation (2 mentions)
167 protocols using ultra performance liquid chromatography (uplc)
1
UPLC-MS/MS Analysis of JV-DE1
2
UPLC Assay for Cembranoid Identification
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For UPLC assay, the cembranoid extract was dried in nitrogen ow and then dissolved in 1 mL of 70% acetonitrile. In the UPLC assay, 5 µL sample was injected into ultra-performance liquid chromatography (UPLC; Waters, USA) under following optimized conditions: BEH C18 column (1.7 µm, 2.1 mm × 100 mm) with the column temperature of 40°C, a gradient mobile phase as indicated in Additional le 1: Table S3 at the ow rate of 0.3 mL/min, and a UV detector for detection of CBT-ol and CBT-diol at 208 nm. The labavailable authentic standards CBT-ol and CBT-diol, which were isolated and puri ed from a 95% EtOH extract of tobacco trichomes using a preparative HPLC system (Waters Technologies Ltd., USA) equipped with a ultraviolet-visible light detector and a Prep C18 OBD (19 mm × 250 mm column, 10 μm) as previously described [28] , were used to distinguish the corresponding peaks in the UPLC chromatograms of the samples.
3
Monocyte Isolation and IL-1β Detection
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Cell culture reagents including media, PBS and TRIzol® were obtained from Invitrogen; Thermo Fisher Scientific, Inc. Methanol and acetonitrile were purchased from Merck KGaA and NH4HCO3 from Sigma-Aldrich; Merck KGaA. The Dynabeads® Untouched™ Human Monocytes kit was purchased from Invitrogen; Thermo Fisher Scientific, Inc. The RevertAid First Strand cDNA Synthesis kit, and Applied Biosystems 7500 and qPCR equipment were purchased from Thermo Fisher Scientific, Inc. Antibodies against NLRP3, ASC and Caspase-1 (NLRP3, cat. no. sc-134306; Caspase-1, cat. no. sc-622; ASC, cat. no. sc-514414) were purchased from Santa Cruz Biotechnology, Inc. The BCA kit was purchased from Pierce; Thermo Fisher Scientific, Inc. The human IL-18 (cat. no. 70-EK1182) and IL-1β (cat. no. 70-EK101B2) ELISA kits were purchased from Hangzhou MultiSciences (Lianke) Biotech. UPLC was purchased from Waters Corporation and the Triple TOF™ 5600+ Mass Spectrometry system was obtained from AB SCIEX LLC.
4
NMR Spectroscopy and UPLC-MS Analysis
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Reagents and solvents
were obtained from Fluka, Sigma-Aldrich or Bachem, and were used without
further purification. NMR spectra in either CDCl3, DMSO-d6 or D2O solution were recorded on
a Bruker DPX 300 spectrometer (300 MHz) or on a Bruker Avance 400
spectrometer (400 MHz); chemical shifts δH are reported
in ppm with reference to the solvent resonance (CDCl3:
δH = 7.26 ppm; DMSO: δH = 2.50 ppm;
H2O: δH = 4.79 ppm); coupling constants
J are reported in Hz. UHPLC analyses were carried out on a Thermo
Scientific Dionex UltiMate 3000 Standard system including an autosampler
unit, a thermostated column compartment and a photodiode array detector,
using UV absorbance detection at λ = 273 nm. HPLC/ESI-MS analyses
were carried out on a Waters UPLC Acquity H-Class system including
a photodiode array detector (acquisition in the 200–400 nm
range), coupled to a Waters Synapt G2-S mass spectrometer, with capillary
and cone voltage of 30 kV and 30 V, respectively, source and desolvation
temperature of 140 and 450 °C, respectively. ESI+ and
ESI– refer to electrospray ionization in positive
and negative mode, respectively. HRMS spectra were recorded on the
same spectrometer, using the same source settings as above.
were obtained from Fluka, Sigma-Aldrich or Bachem, and were used without
further purification. NMR spectra in either CDCl3, DMSO-d6 or D2O solution were recorded on
a Bruker DPX 300 spectrometer (300 MHz) or on a Bruker Avance 400
spectrometer (400 MHz); chemical shifts δH are reported
in ppm with reference to the solvent resonance (CDCl3:
δH = 7.26 ppm; DMSO: δH = 2.50 ppm;
H2O: δH = 4.79 ppm); coupling constants
J are reported in Hz. UHPLC analyses were carried out on a Thermo
Scientific Dionex UltiMate 3000 Standard system including an autosampler
unit, a thermostated column compartment and a photodiode array detector,
using UV absorbance detection at λ = 273 nm. HPLC/ESI-MS analyses
were carried out on a Waters UPLC Acquity H-Class system including
a photodiode array detector (acquisition in the 200–400 nm
range), coupled to a Waters Synapt G2-S mass spectrometer, with capillary
and cone voltage of 30 kV and 30 V, respectively, source and desolvation
temperature of 140 and 450 °C, respectively. ESI+ and
ESI– refer to electrospray ionization in positive
and negative mode, respectively. HRMS spectra were recorded on the
same spectrometer, using the same source settings as above.
5
UPLC-MS/MS Analysis of Plasma Metabolites
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Plasma extract samples (7 μL) were injected into an Acquity UPLC-BEH-C18 column (2.1 × 50 mm, 1.7 μm; Waters, Milford, MA) that was coupled in-line with a UPLC-LTQ-Orbitrap XL (Thermo Fisher Scientific, USA). The injected samples were equilibrated with water containing 0.1% formic acid. Samples were eluted with an acetonitrile gradient containing 0.1% formic acid at a flow rate of 0.35 mL/min for 20 min. Metabolites were separated by UPLC (Waters, Milford, MA), analyzed, and assigned by LTQ-Orbitrap-XL (Thermo Fisher Scientific, USA). The mass spectrometer was operated in ESI-positive mode. The spray voltage was 5 kV. The flow rate nitrogen sheath gas and the auxiliary gas were 50 and 5 (arbitrary units). The capillary voltage (V), tube-lens voltage (V), and capillary temperature (°C) were kept constant at 35 V, 80 V, and 370°C, respectively. The Orbitrap data were collected in the range of m/z 50–1,000. For quality control, a mixture of four standard compounds (acetaminophen, sulfadimethoxine, terfenadine, and reserpine) was injected into every 10th sample. The MS/MS spectra of metabolites were obtained by a collision-energy ramp from 55–65 eV, and analyzed with Xcalibur 2.1 and MS Frontier software (Thermo Fisher Scientific, USA).
6
Quantifying 3-Hydroxybutyrate Release from Wound Dressings
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The integrated wound dressing (about 3.83–4.0 g – 10 cm2) was incubated in buffered saline solution (10 ml, PBS) for 12 h at 37°C. After incubation, the solution was adjusted to pH 3 and extracted 3 times with ethyl acetate. The ethyl acetate fraction was dried under a vacuum, re-suspended in 1 ml of methanol and analysed in Ultra Performance Liquid Chromatography (UPLC; Waters Corp., Milford, USA).
For measuring the release of 3-hydroxybutyrate from dressing, a commercially available assay (β-hydroxybutyrate Assay Kit, Sigma-Aldrich) was used. In this assay, the concentration of β-hydroxybutyrate is determined by an enzymatic reaction wherein the product is determined colorimetrically (at 450 nm).
For measuring the release of 3-hydroxybutyrate from dressing, a commercially available assay (β-hydroxybutyrate Assay Kit, Sigma-Aldrich) was used. In this assay, the concentration of β-hydroxybutyrate is determined by an enzymatic reaction wherein the product is determined colorimetrically (at 450 nm).
7
GXNT Quantification by UPLC-MS
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GXNT was analyzed on a UPLC (Waters, Milford, MA, United States) coupled with a Q-Exactive™ Focus mass spectrometer (Thermo Scientific, Bremen, Germany). Chromatographic separation was performed on a Waters BEH C18 (100 mm × 2.1 mm, 1.7 μm) with acidified water with 0.15% formic acid and acetonitrile with 0.15% formic acid as mobile phases A and B at 30°C. The following solvent gradient was adopted: 0 min, 2% B; 8 min, 98% B. The flow rate was set at 0.6 ml/min, and a volume of 6 μl was injected for the analysis. The parameters for mass spectrometer were as follows: curtain gas, 30 psi; Gas 1 (N2), 55 psi; Gas 2 (N2), 55 psi; ion spray voltage, 3.5 kV (positive mode); 2.5 kV (negative mode) temperature, 350°C; scan range, m/z 120–1,500.
8
UPLC-QTOF/MS Analysis of Metabolites
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An Ultra Performance Liquid Chromatography (UPLC) system equipped with a binary solvent delivery system, an auto-sampler, and a UV detector (Waters, Milford, MA, USA) was used. Aliquots of each sample (2.0 μL) were injected into a 100 × 2.1 mm, 1.7 μm BEH C18 column and eluted at a flow rate of 0.4 mL/min using a chromatographic gradient of two mobile phases (A: water containing 0.1% formic acid; B: acetonitrile containing 0.1% formic acid). A linear gradient was optimized as follows: 0 min, 5% B; 0–16 min, 5–45% B; 16–21 min, 45–80% B; 21–22 min, 80–100% B; 22–23.3 min, 100% B; and 23.3–25 min, 5% B. The Q-TOF/MS system (Vion IMS, Waters) was operated in negative-ion mode under the following conditions: a capillary voltage of 2.3 kV, a cone voltage of 50 V, a source temperature set to 110 °C, and a desolvation temperature set to 350 °C. A sprayer with a reference solution of leucine-enkephalin ([M−H]−m/z 554.2615) was used as the lock mass. The full scan data and MS/MS spectra were acquired using MassLynx software.
9
Quantification of UDCA Levels in Plasma
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Bile acid analysis to determine UDCA levels was carried out on baseline and endpoint plasma samples. Whole blood was collected in heparin tubes, and plasma was isolated and stored at −80° C. UDCA was quantified by an ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UPLC/MS‐MS) assay20 with modifications. Bile acids were first isolated from plasma with a Strata‐X C18 solid‐phase extraction column (#8B‐S100‐TAK; Phenomenex). Standard solutions of ursodeoxycholate (C1020, CAS# 128‐13‐2; Steraloids) were used to generate a standard curve (0‐2.5 µM) to determine UDCA concentration. Separation was achieved using UPLC (Waters Corporation) followed by MS‐MS (TQD Tandem Mass Spectrometer; Waters Corporation) with a Waters Cortecs C18 + UPLC column (#186007367; Waters). Analyte separation and elution were achieved using a 10 mM ammonium acetate, 0.15% ammonium hydroxide aqueous mobile phase and a methanol/10 mM ammonium acetate 0.15% ammonium hydroxide elution phase. We used d4‐ursodeoxycholate (#D3819; CDN, Inc.) as an internal control in each sample.
10
Analytical Techniques for Lovastatin, Lactose, and Biomass
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Lovastatin (LOV) and lactose (LAC) concentrations were determined by liquid chromatography (UPLC®, Waters, USA). Organic nitrogen (N) was determined with the use of carbon and nitrogen analyser IL550TOC-TN (HACH, USA) and biomass (X) was assayed as dry weight [11 (link)]. The accuracy of the applied analytical techniques was as follows. For biomass, the error ranged from 5 to 15 %, for lactose, it did not exceed 4 %, for lovastatin, it was lower than 1 % and for organic nitrogen, it was around 3 %.
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