The conditions of UPLC-QTOF-MS/MS are in accordance with the previous study [12 (link), 13 (link)]. Briefly, the analysis was performed on a Waters Acquity UPLC I-Class system (Waters Corp., Milford, United States), coupled with a Waters QTOF-MS/MS Mass System (Manchester, United Kingdom) equipped with electrospray ionization (ESI). Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm). Mass range, 50-1500 Da; source temperature 100 °C; desolvation temperature, 450 °C; desolvation gas flow, 900 L/h; sampling cone, 40 V; ESI− capillary voltage, 2.5 KV; and ESI+ capillary voltage, 0.5 KV. UPLC-QTOF-MS/MS system was controlled by the Masslynx 4.1 platform. The MSE data collected in a continuum mode were processed using the peak detection and alignment algorithms in UNIFI 1.8, which enabled quasi-molecular ion peaks, adduct ions, and fragment ions to be analyzed as a single entity. RSGB (sugar-free) was filtered through a 0.22 μm microporous membrane before aliquots (2 µL) were transferred to autosampler vials for analysis. Fifteen standards were dissolved in methanol, respectively, and mixed in equal. The final concentration of each standard was 100 ng/mL, and 1µL for analysis.
Waters acquity uplc hss t3 column
Manufactured by Waters Corporation
Sourced in United States
The Waters Acquity UPLC HSS T3 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a sub-2-micron particle size and a hydrophobic C18 bonded silica stationary phase, which enables efficient and high-resolution separations.
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Lab products found in correlation
Waters acquity uplc 1 class system, by Waters Corporation (2 mentions)
Waters acquity uplc system, by Waters Corporation (2 mentions)
Waters synapt g2hdms system, by Waters Corporation (2 mentions)
Methanol, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Dl o chlorophenylalanine, by GL Biochem (1 mentions)
Masslynx 4, by Waters Corporation (1 mentions)
Acquity uplc, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 rs, by Thermo Fisher Scientific (1 mentions)
Thermo scientific orbitrap fusion, by Thermo Fisher Scientific (1 mentions)
Xevo g2 xs qtof high resolution mass spectrometer, by Waters Corporation (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Synapt g2 si hdms qtof mass spectrometer, by Waters Corporation (1 mentions)
Xevo g2 xs qt, by Waters Corporation (1 mentions)
I class plus, by Waters Corporation (1 mentions)
Tq s micro triple quadrupole mass spectrometer, by Waters Corporation (1 mentions)
Waters acquity uplc class system, by Waters Corporation (1 mentions)
13 protocols using waters acquity uplc hss t3 column
1
UPLC-QTOF-MS/MS Analysis of Polysaccharides
2
UPLC-QTOF Analysis of Metabolites in EFR
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The Waters Acquity I-Class PLUS ultra-high-performance liquid tandem Waters Xevo G2-XS QTOF high-resolution mass spectrometer (Waters Corporation, Milford, MA, USA) was used for metabolite analysis of EFR. UPLC fitted the Waters Acquity UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm). The parameter was performed as follows: mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic acid in acetonitrile; gradient with 2% mobile phase B for 0~0.25 min, 2~98% mobile phase B for 0.25~10 min, 98% mobile phase B for 10~13 min, 98~2% mobile phase B for 13~13.1 min, 2% mobile phase B for 13.1~15 min. The parameters of the electrospray ionization-mass spectrometry (ESI-MS) analysis were 2.0 kV (positive ion mode) and −1.5 kV (negative ion mode), cone voltage of 30 V, ion source temperature of 150 °C, desolvent gas temperature of 500 °C, backflush gas flow rate of 50 L/h, and desolventizing gas flow rate of 800 L/h. MassLynx V4.2 with MSe mode (Waters) was utilized for collection of primary and secondary mass spectrometry data. The peak extraction, alignment and data processing operations were conducted by Progenesis QI software. The METLIN database and Biomark’s self-built library (Biomarker Technologies Co., Ltd., Beijing, China) were used for metabolite identification.
3
UPLC-MS/MS Quantification of NBP
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NBP was obtained from Shijiazhuang Pharma Group NBP Pharmaceutical Co. Ltd., Shijiazhuang, Hebei Province, China. DL-o-chlorophenylalanine, used as internal standard (IS), was purchased from GL Biochem (Shanghai) Ltd. Acetonitrile, methanol and ultra-pure water were of chromatographic grade and obtained from Merck Company, Darmstadt, Germany. Formic acid was purchased from CNW, Shanghai, China. The Waters AcquityTM UPLC system was coupled with the Waters XevoTM Q-TOF mass analyzer. The column used for this analysis was the Waters Acquity UPLC HSS T3 column (2.1 mm×100 mm, 1.8 μm), Waters Company, America.
4
Phytochemical Profiling of Artemisia argyi
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The chemical constituents of A. argyi were analyzed using UPLC-Q-TOF-MS method. 0.5 g of A. argyi leaves was mixed with 20 ml of ultra-pure water, 50% ethanol or pure ethanol independently and incubated overnight. After thorough oscillation, samples were subjected to ultrasonication for 1 h and centrifuged at 4000 rpm for 10 min. The supernatants were filtered through a 0.22 μm membrane and collected into a new tube. UPLC-Q-TOF-MS analysis was performed using an Waters Acquity I-Class ultra-performance liquid chromatography combined with a Xevo G2-S quadrupole time-of-flight mass spectrometer. Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) was used. The specific chromatographic and mass spectrometric conditions were presented according to the method reported by Luo, D.D. et al11 .
5
Quantitative Metabolomics Analysis by UPLC-MS
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Chromatography was performed on a Waters Acquity UPLC I-Class system (Waters Corp., Milford, USA) with a Waters Acquity UPLC HSS T3 column (2.1 ×100 mm, i.d. 1.8 µm; Waters, USA) maintained at 40 ℃. The mobile phases consist of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B). A gradient elution program was used as follows: 0–4 min, 2–6% B; 4–18 min, 6–20% B; 18–21 min, 20–24% B; 21–30 min, 24–35% B; 30–33 min, 35–98% B. The flow rate was set at 0.5 mL/min, and a 1-μL aliquot was set as the injection volume.
Mass spectrometry analysis was performed on a Waters SYNAPT G2HDMS system (Waters Corp., Milford, USA) equipped with an electrospray ionization (ESI) source in both positive and negative ion modes. The optimal parameters were set as follows: capillary voltage, 2 kV; cone voltage,40 V; resolvation gas (N2) flow, 900 L/h; source temperature, 100 ℃; resolvation temperature, 450 ℃; scanning time and interval, 0.2 s; scan range, m/z 50–1500; trap collision energy, 20–50 eV; lock mass, [M+H]+ 556.2775 and [M−H]−554.2615. The data were collected in the MSE continuum mode using Masslynx 4.1 software (Waters Corp., Milford, USA).
Mass spectrometry analysis was performed on a Waters SYNAPT G2HDMS system (Waters Corp., Milford, USA) equipped with an electrospray ionization (ESI) source in both positive and negative ion modes. The optimal parameters were set as follows: capillary voltage, 2 kV; cone voltage,40 V; resolvation gas (N2) flow, 900 L/h; source temperature, 100 ℃; resolvation temperature, 450 ℃; scanning time and interval, 0.2 s; scan range, m/z 50–1500; trap collision energy, 20–50 eV; lock mass, [M+H]+ 556.2775 and [M−H]−554.2615. The data were collected in the MSE continuum mode using Masslynx 4.1 software (Waters Corp., Milford, USA).
6
UPLC-MS Analysis of Metabolites
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The ARD and standard solutions were centrifuged at 10,033 g for 10 min, and 2 μL aliquots were respectively injected into the Waters ACQUITY UPLC HSS T3 column (1.7 μm × 2.1 mm × 100 mm; Waters, MA, USA) at 15°C in a Waters ACQUITY UPLC system. The samples were eluted with the flow rate at 0.2 mL/min using acetonitrile (solvent A) and 0.1% v/v formic acid in water (solvent B). The linear gradient was as follows: 0–10 min, 0%–1% A; 10–15 min, 1%–3% A; 15–20 min, 3%–10% A; 20–25 min, 10%–15% A; 25–30 min, 15%–20% A; 30–35 min, 20%–30% A; 35–40 min, 30%–60% A; 40–45 min, 60%–80% A; 45–50 min, 80%–90% A; 50–55 min, 90%–100% A; and 55–60 min, 100%–0% A.
The mass spectra of the above eluents were acquired on the Waters SYNAPT G2 HDMS system (Waters Corp., USA) in positive ion mode by scanning over the m/z range of 50–1200. The following conditions were used: desolvation gas flow at 800 L/h and 450°C, cone gas flow at 50 L/h and 100°C, capillary voltage 3 kV, cone voltage 40 V, and scan time 0.5 s. Leucine enkephalin with an [M+H]+ ion at m/z 556.2771 was used as the lock mass. The Mass Lynx V 4.1 software was used for data analysis.
The mass spectra of the above eluents were acquired on the Waters SYNAPT G2 HDMS system (Waters Corp., USA) in positive ion mode by scanning over the m/z range of 50–1200. The following conditions were used: desolvation gas flow at 800 L/h and 450°C, cone gas flow at 50 L/h and 100°C, capillary voltage 3 kV, cone voltage 40 V, and scan time 0.5 s. Leucine enkephalin with an [M+H]+ ion at m/z 556.2771 was used as the lock mass. The Mass Lynx V 4.1 software was used for data analysis.
7
Analyzing Riboflavin Levels in Rice Calluses
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The homogenized rice calluses were extracted with 1 mL of 50 mM of phosphate buffer containing riboflavin-13C,15N2 as internal standard. The extract was vortexed for 10 s and stored for 2 h at 4 ℃. Then, it was purified with Amicon 3KDa centrifugal filter (15,900 rcf at 4 °C for 20 min) before loading to liquid chromatography coupled with tandem mass spectrometry system (LC–MS/MS). The LC–MS/MS was a Waters ACQUITY UPLC and an Applied Biosystems API 4000 MS equipped with an electrospray ionization source. The extracted riboflavin was separated on a Waters ACQUITY UPLC HSS T3 Column (2.1 mm X 150 mm, 100Å, 1.8 µm) equipped with a Waters ACQUITY UPLC HSS T3 VanGuard Pre-column (2.1 mm X 5 mm, 100Å, 1.8 µm) maintained at 45 ℃.
8
Quantification of 5α-reductase Activity by LC-MS
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As described in the previously established method (Deng et al., 2020 (link)), LC-MS was performed using a LC-8050 triple-quadrupole MS (Shimadzu, Kyoto, Japan). The samples were analyzed on Waters-ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm; Waters, United States) with a mobile phase consisting of 0.5 mM ammonium acetate in water and methanol (30:70, v/v). The flow rate was 0.4 ml/min, column temperature was 40°C and the injection volume was 5.0 μL. The MS was operated in the positive ion mode with an ESI source and the m/z 291.40 (DHT) was selected for detection in multiple reaction monitoring mode. The capillary and heater temperature were 300°C and 250°C respectively and the air flow rate was 10 L/min. The ratio of DHT content in the treated groups to that in the control group was calculated to evaluate the effects of DHT on the 5α-reductase activity.
9
Quantitative Analysis of Afatinib Degradation
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Ultra-high performance liquid chromatograph Thermo Scientific Dionex UltiMate 3000 RS coupled to a high-resolution mass spectrometer (LC-HRMS) Thermo Scientific Orbitrap Fusion (Thermo Fisher Scientific, Waltham, MA, USA) was used for the analysis of afatinib degradation products. The chromatographic separation was achieved on a Waters Acquity UPLC HSS T3 column (1.8 µm, 100 × 2.1 mm) (Waters Corporation, Milford, MA, USA) operated at 50 °C. 0.1% formic acid as mobile phase A (MP A) and a mixture of acetonitrile/methanol in a volumetric ratio 65/35 as mobile phase B (MP B) were used for analysis. Constant flow rate of 0.4 mL/min and gradient as follows were employed: 20% MP B was kept constant for 0.5 min, increased to 50% MP B in 9.5 min, increased to 80% MP B in 2.0 min and kept constant for 2.0 min. The injection volume was 5 µL. HRMS detection was performed in positive ionization mode with a full scan in the range 100 -1000 m/z and extraction of ions 459.1230 m/z and 457.1073 m/z with 5 ppm mass tolerance. Orbitrap detector with resolution 120,000 was used for detection. Ionization was achieved with a heated electrospray probe and voltage 3500 V, vaporizer temperature 375 °C, ion transfer tube temperature 250 °C, sheath gas 50 Arb and auxiliary gas 10 Arb. Divert valve prior to the mass spectrometer was set to waste between 0 min and 3.9 min.
10
Untargeted Metabolite Profiling of Root Exudates
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Untargeted metabolites in the root exudates were determined using a liquid chromatography with tandem mass spectrometry (LC–MS/MS) platform (Biomarker Technologies Co., Ltd.). In brief,1 mL extraction liquid (methanol: acetonitrile: water = 2:2:1) was added into 50 mg of the samples and vortexed for 30 s, and steel balls were added and ground in a 45 Hz grinder for 10 min and ultrasonicated in an ice water bath for 10 min (Dunn et al., 2011 (link)). The sample was then sat at 20°C for 1 h to precipitate the proteins and then centrifugated at 4°C, 12000 rpm for 15 min. 500 μL of supernatant were then moved into an EP tube and dried in a vacuum concentrator; then, 160 μL of an extraction liquid (acetonitrile: water = 1:1) was added to redissolve the sample. It was then vortexed for 30 s, ultrasonicated for 10 min (in ice water bath), and centrifugated for 15 min (4°C, 12,000 rpm). Finally, 120 μL of the supernatant were used for Ultra-High Performance Liquid Chromatography-Q Exactive (UHPLC-QE) orbital trap/mass spectrometry analysis. The LC/MS system for metabolomics analysis is composed of Waters Acquity I-Class PLUS ultra-high performance liquid tandem Waters Xevo G2-XS QT of high-resolution mass spectrometer, and the column used was from Waters Acquity UPLC HSS T3 column (Waters Corp., Milford, CT, United States).
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