The Agilent DD2 NMR spectrometer (JEOL, Tokyo, Japan) at 500 MHz and 125 MHz frequency was used for 1H and 13C NMR spectra respectively. The vacuum column chromatography silica gel (200–300 mesh, Qing Dao Hai Yang Chemical Group Co, Qingdao, China), silica gel plates for thin layer chromatography (G60, F-254, and Yan Tai Zi Fu Chemical Group Co, Yan Tai, China), and reverse phase octadecylsilyl silica gel column were used for the separation of compounds. UPLCMS spectra were measured on Waters UPLC® system (Waters Ltd., Milford, MA, USA) using a C18 column (ACQUITY UPLC® BEH C18, 2.1 × 50 mm, 1.7 μm; 0.5 mL/min) and ACQUITY QDA ESIMS scan from 150 to 1000 Da was used for the analysis of fungal extracts and ESI-MS spectra of the compounds. Semipreparative HPLC was performed on a Hitachi L-2000 system (Hi-tachi Ltd., Tokyo, Japan) using a C18 column (Kromasil 250 × 10 mm, 5 μm, 2.0 mL/min).
Waters uplc system
Manufactured by Waters Corporation
Sourced in United States
The Waters UPLC system is a high-performance liquid chromatography (HPLC) instrument designed for efficient and accurate separation and analysis of chemical compounds. It utilizes ultra-high pressure liquid chromatography (UPLC) technology to achieve superior resolution, sensitivity, and speed compared to traditional HPLC systems.
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Lab products found in correlation
Acquity uplc beh c18, by Waters Corporation (3 mentions)
Acquity beh c18 column, by Waters Corporation (3 mentions)
L 2000 system, by Hitachi (2 mentions)
Jem ecp nmr spectrometer, by JEOL (2 mentions)
Xevo g2 xs qtof system, by Waters Corporation (1 mentions)
Waters masslynx, by Waters Corporation (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
Beh c18 column, by Waters Corporation (1 mentions)
Sephadex lh 20, by Cytiva (1 mentions)
Gemini ultra diffractometer, by Agilent Technologies (1 mentions)
2996 photodiode array detector, by Waters Corporation (1 mentions)
G2 xs qtof ms, by Waters Corporation (1 mentions)
Masslynx v4, by Waters Corporation (1 mentions)
Qtrap 6500, by AB Sciex (1 mentions)
Beh c18, by Waters Corporation (1 mentions)
Multiquant 2, by AB Sciex (1 mentions)
Nicoletnexus470 spectrometer, by PerkinElmer (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Octadecylsilyl silica gel, by YMC (1 mentions)
Micromass q tof spectrometer, by Waters Corporation (1 mentions)
14 protocols using waters uplc system
1
Analytical Methods for Fungal Extracts
2
Peptidoglycan Profiling by UPLC-MS
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Peptidoglycan was purified from three biological replicas normalized to OD600 = 1 and analyzed, using 16M as control, as described elsewhere (Desmarais et al., 2013 (link); Alvarez et al., 2016 (link), 2020 (link)) on a Waters UPLC system (Waters Corporation, Milford, MA, United States) equipped with an ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 150 mm (Waters, United States) and a dual wavelength absorbance detector. Muropeptide identity was confirmed by MS/MS analysis, using Xevo G2-XS QT of system (Waters Corporation, Milford, MA, United States). Quantification of muropeptides was based on their relative abundances normalized to the total amount of PG. Unidentified muropeptides and minor peaks with a relative abundance lower than 0.5% were excluded from further analysis.
3
UPLC-Q-TOF-MS Analysis of Samples
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The Waters UPLC system was used to analyze the samples (Waters Co., Milford, Massachusetts, USA). The sample was separated using an Acquity BEH C18 column (2.1 mm × 100 mm, Waters Co., Milford, Massachusetts, USA). The temperature was kept at 30°C, the flow rate was 0.4 mL/min, and the column equilibration lasted 5 min. The optimum mobile phase comprisedthe linear gradient system (A) 0.1% aqueous solution of formic acid and (B) acetonitrile: 0–5.0 min, B 5–20%; 5.0–20.0 min, B 20–30% and 20.0–30.0 min, B 30–100%. The injection volume was 2 μL. After the column separation, the 5% fraction was delivered directly to the quadrupole time-of-flight mass spectrometry (Q-TOF-MS). The detector was a dual electrospray ionization (ESI) probe. HRMS was obtained from Waters (Waters Corporation, Milford, USA), which was set in positive (ESI+) and negative (ESI−) ionization modes, and optimal analytical conditions were set as follows: the source temperature was 120°C, desolvated gas temperature flow was 350°C and 750 L/h, negative mode voltage was 3.0 kV, and positive voltage was 2.5 kV (Lei et al., 2018 (link)). The Q-TOF collection rate was 0.1 s, the delay was 0.02 s, and the first acquisition rate was 0.1 s. The resolution quadrupole (50–1,800 Da) was performed in wide-pass mode. Waters MassLynx™ (Waters Corp., Milford, MA, USA) was used for data collection and subsequent data processing.
4
Quantification of Cefathiamidine by UPLC-MS/MS
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Cefathiamidine concentrations were detected on an ultra-performance liquid chromatography tandem mass spectrometry (UPLC MS/MS) system comprising a API 4000 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source (Framingham, MA, USA). A series Waters UPLC system (Waters, USA) – consisting of a binary pump, an automatic sampler, and an online degasser – were used for LC-MS/MS analysis. Separation was achieved by using an ACQUITY UPLC® BEH C18 column (1.7 µm, 2.1×50 mm) purchased from Waters (USA). The mobile phase consisted of water and methanol at a flow rate of 0.4 mL/min. Determination of cefathiamidine was based on the internal standard method, using ceftiofur as the internal standard (IS). The multiple reaction monitoring transitions of m/z 473.5+→201.3+ and 524.3+→241.4+ were used to quantify cefathiamidine and IS, respectively. The heated nebulizer was set at 300°C, and the needle voltage (IS) was set at 4,000.00 V; the nebulizer gas (GS1), auxiliary nitrogen gas (GS2), curtain gas (CUR), and collision gas (CAD) were set at 20 psi, 20 psi, 10 psi, and medium, respectively. Nitrogen was used as the collision gas. The calibration curve ranged from 30 to 10,000 ng/mL. The interday and intraday coefficients of variation of controls were less than 5%. The lower limit of quantification was 30 ng/mL.
5
Quantitative Analysis of Bioactive Compounds
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The detection methods followed the Pharmacopeia of the People’s Republic of China. Periderm, phloem, and xylem samples were ground into powder (with three biological replicates for each sample), and each weighed sample of ground powder (0.2 g) was extracted with 50 mL of 75% methanol. After 1 h of heating reflux extraction, 75% methanol was added to complement and maintain a constant weight, and the sample was filtered through a 0.45-μm syringe filter. In addition, an LAB standard was dissolved with 75% methanol at a concentration of 140 mg/L. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) with a mobile phase of 30% methanol, 10% acetonitrile, 1% methanoic acid, and 59% H2O in a Waters UPLC system (Waters, USA). The detection wavelength was set to 286 nm.
6
Quantification of Phenolic Acids and Tanshinones
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The 5-month-old hairy roots were collected and dried to constant weight in an oven at 40 °C. The sample powder was divided into two parts. The first part was extracted with 2 mL of 75% methanol with 100 mg of powder for phenolic acid test; the second part was extracted with 5 mL of methanol with 1000 mg of powder for tanshinone test under ultrasonic treatment for 30 min. Then, the powder was centrifuged at 8000 × g for 10 min, and then filtered through a 0.22 µm microporous membrane filter. The Waters UPLC system (Waters, USA) equipped with a PDA detector was used to determine the content of phenolic acid and tanshinone. An ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 µm; Waters) was used. Phenolic and tanshinone were detected using 280 and 255 nm PDA wavelengths, respectively. Phenolic acid was detected by selective gradient elution using mobile phase acetonitrile (A)−0.5% formic acid (B) (volume percent, in deionized water). Linear gradients included 5%–25% A (0–10 min), 25%–40% A (10–20 min), 40%–90% A (20–25 min), and 90% A (25–30 min). The flow rate was 0.3 mL/min. Gradient elution using mobile phase methanol (A) -water (B) was used to detect tanshinone. The linear gradient included 20%−60% A (0 − 5 min), 60%−70% A (5 − 20 min), 70%−80% A (20−25 min), 80%−100% A (25−26 min), and 100% A (26–30 min). The flow rate was 0.25 mL/min.
7
Structural Characterization of Natural Compounds
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NMR spectra were recorded on a JEOL JEM-ECP NMR spectrometer. Chemical shifts δ are reported in ppm, using TMS as internal standard, and coupling constants (J) are in Hz. HRESIMS and ESIMS spectra were obtained from a Micromass Q-TOF spectrometer. Single-crystal data were measured on an Agilent Gemini Ultra diffractometer (Cu Kα radiation). UPLC-MS was performed on Waters UPLC® system (Waters Ltd., Milford, MA, USA) using a C18 column [(Waters Ltd.) ACQUITY UPLC® BEH C18, 2.1 × 50 mm, 1.7 μm; 0.5 mL/min] and ACQUITY QDa ESIMS scan from 150 to 1000 Da. Silica gel (Qing Dao Hai Yang Chemical Group Co., Qingdao, China; 200–300 mesh) and Sephadex LH-20 (Amersham Biosciences, Amersham, UK) were used for column chromatography (CC). TLC silica gel plates (Yan Tai Zi Fu Chemical Group Co., Yantai, China; G60, F-254) were used for thin-layer chromatography. Semi-preparative HPLC was operated on a Waters 1525 system using a semi-preparative C18 (Kromasil, 5 μm, 10 × 250 mm) column equipped with a Waters 2996 photodiode array detector, at a flow rate of 2.0 mL/min.
8
Mass Fragmentation of Isolated Compounds
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The mass fragmentation of isolated compounds (1 –9 ) was performed on a Waters UPLC system (Waters, Milford, MA, USA) coupled with Waters column and a Xevo G2-XS Q-TOF MS with electrospray ionization source (Waters, Milford, MA, USA). Triple TOF MS equipped with a DuosprayTM ion source was used to complete the high-resolution experiment. For mass detection, the instrument was operated in the negative ion electrospray mode, and the conditions of the MS/MS detector were as follows: the de-solvation gas was 800 L/h at 400 °C, the cone gas was 50 L/h, the source offset voltage was 80 eV, and the source temperature was 120 °C. A full scan was run in the negative mode with a mass range from m/z 50 to 1250 amu. The capillary voltage was 3.0 kV, and the sampling cone voltage was 40 eV. Sodium formate was used for mass spectrometer instrument calibration in the resolution mode. Leucine encephalin, which generated the reference ion (m/z 554.2615 [M-H]-), was used to ensure accuracy throughout the mass spectrometry analysis. All data acquisition and processes were performed for qualitative analysis by using MassLynx V4.1 and UNIFI V1.9 (Waters, Milford, MA, USA). The gradient elution of the mobile phase was conducted following the above established gradient elution.
9
SCFA Quantification in Plasma
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Stock solutions of the seven SCFAs standards were freshly and individually prepared in 50% aqueous acetonitrile. Then, a mixed standard solution containing 1 mM of each SCFA was made with the same solvent and diluted for calibration curves. For plasma samples, SCFAs from 20 ul plasma were extracted using precooled methanol/acetonitrile (2:1). After centrifugation, supernatant was used for derivatization with all calibration samples, and isotope-labeled internal standards of the seven SCFAs were used for normalization78 (link).
A Waters UPLC system (Waters, USA) was coupled to a QTRAP6500 (SCIEX, Canada) equipped with an ESI source and operated in the negative-ion mode. Chromatographic separations were performed on a Waters BEH C18 (2.1 × 50 mm, 1.7 mm) UPLC column, in which solvent A (water: formic acid; 100:0.01, v/v) and solvent B (acetonitrile: formic acid; 100:0.01, v/v) were used as the mobile phase for gradient elution. The column flow rate was 0.35 mL/min. All quantification data was processed using the MultiQuant 2.0 software (SCIEX, Canada).
A Waters UPLC system (Waters, USA) was coupled to a QTRAP6500 (SCIEX, Canada) equipped with an ESI source and operated in the negative-ion mode. Chromatographic separations were performed on a Waters BEH C18 (2.1 × 50 mm, 1.7 mm) UPLC column, in which solvent A (water: formic acid; 100:0.01, v/v) and solvent B (acetonitrile: formic acid; 100:0.01, v/v) were used as the mobile phase for gradient elution. The column flow rate was 0.35 mL/min. All quantification data was processed using the MultiQuant 2.0 software (SCIEX, Canada).
10
Characterization of Natural Products
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Optical rotations were measured on a JASCO P-1020 digital polarimeter (JASCO Ltd., Tokyo, Japan). IR spectra were recorded on a Nicolet-Nexus-470 spectrometer (Perkin Elmer Ltd., Boston, MA, USA) using KBr pellets. NMR spectra were recorded on a JEOL JEM-ECP NMR spectrometer (JEOL Ltd., Tokyo, Japan; 500 MHz for 1H and 125 MHz for 13C), using TMS as internal standard. The ESIMS spectra were obtained from a Micromass Q-TOF spectrometer (Waters Ltd., Boston, MA, USA). Semi-preparative HPLC was performed on a Hitachi L-2000 system (Hitachi Ltd., Tokyo, Japan) using a C18 column (Kromasil (Eka Ltd., Bohus, Sweden) 250 × 10 mm, 5 μm, 2.0 mL/min). UPLC-MS was performed on Waters UPLC® system (Waters Ltd., Boston, MA, USA) using a C18 column (ACQUITY UPLC® (Waters Ltd., Boston, MA, USA) BEH C18, 2.1 × 50 mm, 1.7 μm; 0.5 mL/min) and ACQUITY QDa ESIMS scan from 150 to 1000 Da. Silica gel (Qingdao Haiyang Chemical Group Co., Qingdao, China; 200–300 mesh), octadecylsilyl silica gel (YMC Co., Ltd., Tokyo, Japan; 45–60 μm), and Sephadex LH-20 (GE Ltd., Hartford, CT, USA) were used for column chromatography (CC). Precoated silica gel plates (Yantai Zhifu Chemical Group Co., Yantai, China; G60, F-254) were used for thin layer chromatography.
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