Waters acquity uplc beh amide column

Manufactured by Waters Corporation

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The Waters ACQUITY UPLC BEH Amide column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of polar and hydrophilic compounds. It features a 1.7 μm ethylene bridged hybrid (BEH) particle technology that enables efficient separations and high-resolution chromatography.

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7 protocols using waters acquity uplc beh amide column

UHPLC-MS/MS Quantification of Amino Acids

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The specific operation steps for UHPLC-MS/MS could refer to our previous work (20 (link)). Shortly described as follows: Equipped with a Waters ACQUITY UPLC BEH Amide column (100 × 2.1 mm, 1.7 μm; Waters Corporation, USA), an Agilent 1290 Infinity II series UHPLC system (Agilent Technologies, California, USA) was used for the UHPLC separation. Mobile phase A and B were respectively made up of 1% formic acid in water and 1% formic acid in acetonitrile. The column temperature was set to 35°C while the auto-sampler temperature was set to 4°C. For assay development, Agilent 6460 Triple Quadrupole mass spectrometer was connected with an Agilent Jet Stream electrospray ionization interface (Agilent Technologies, California, USA).
Isotope standards used for the quantifications were applied and the optimal Multiple Reaction Monitoring (MRM) parameters of the target metabolites were obtained. Agilent MassHunter Work Station Software (B.08.00, Agilent Technologies, California, USA) was used for the MRM data acquisition and processing. The raw data of amino acids concentration in PNI was in the Supplementary Table 2.

Fu Y., Ding L., Yang X., Ding Z., Huang X., Zhang L., Chen S., Hu Q, & Ni Y. (2021). Asparagine Synthetase-Mediated l-Asparagine Metabolism Disorder Promotes the Perineural Invasion of Oral Squamous Cell Carcinoma. Frontiers in Oncology, 11, 637226.

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Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry for Targeted Quantification

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Ultrahigh-performance liquid chromatography–tandem mass spectrometer targeted quantitative analysis specific operation steps refer to our previous report (15 (link)). The UHPLC separation was carried out using an Agilent 1290 Infinity II series UHPLC System (Agilent Technologies, California, USA), equipped with a Waters ACQUITY UPLC BEH Amide column (Waters Corporation, USA) (100 × 2.1 mm, 1.7 μm). Mobile phase A was 1% formic acid in water, and mobile phase B was 1% formic acid in acetonitrile. The column temperature and auto-sampler temperature were set at 35 and 4°C, respectively. An AJS–electrospray ionization interface was connected with Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies) for assay development. The MRM parameters of the target analytes are controlled by flowing injection of the standard solution of a single analyte.

Yang X.H., Jing Y., Wang S., Ding F., Zhang X.X., Chen S., Zhang L., Hu Q.G, & Ni Y.H. (2020). Integrated Non-targeted and Targeted Metabolomics Uncovers Amino Acid Markers of Oral Squamous Cell Carcinoma. Frontiers in Oncology, 10, 426.

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HILIC-QTOF-MS/MS Metabolite Profiling

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Data were acquired using the following chromatographic parameters, established as standard procedure in the Fiehn Laboratory. Analysis was done via HILIC-QTOF-MS/MS. The Waters Acquity UPLC BEH Amide Column (1.7 μm, 2.1 × 150 mm) and Waters Acquity and UPLC BEH Amide VanGuard Pre-Column (1.7 μm, 5 × 2.1 mm) were used at 40 °C and under a 0.4 mL/min flow rate. The injection volume was 3 μL for ESI (+), mass resolution was 10,000 for ESI (+) on an Agilent 6530 QTOF MS, and the scan range was m/z 60–1200 Da. The analytical ultra high-performance liquid chromatography (UHPLC) column was protected by a short guard column. This chromatography method yields excellent retention and separation of metabolite classes (biogenic amines, cationic compounds) and good within-series retention time reproducibility.

Fan S., Shahid M., Jin P., Asher A, & Kim J. (2020). Identification of Metabolic Alterations in Breast Cancer Using Mass Spectrometry-Based Metabolomic Analysis. Metabolites, 10(4), 170.

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UPLC-HRMS Metabolite Profiling Protocol

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Ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) data was acquired as described in Palmer et al. (Palmer et al., 2017 (link), Palmer et al., 2013 (link)). Briefly, data was obtained using two separate UPLC-HRMS systems, consisting of an Agilent 1290 Infinity LC system (Agilent Technologies) interfaced with either a Quadrupole Time-of-Flight (Q-TOF) mass spectrometer or a triple-quadrupole (QqQ) system (Agilent Technologies) with a Waters Acquity UPLC BEH Amide column (Waters, Milford, MA, USA) for chromatographic separation of metabolites. Data was collected over a 6.5-minute solvent gradient with 0.1% formic acid in water and 0.1% formic acid in acetonitrile.

Simms L., Rudd K., Palmer J., Czekala L., Yu F., Chapman F., Trelles Sticken E., Wieczorek R., Bode L.M., Stevenson M, & Walele T. (2020). The use of human induced pluripotent stem cells to screen for developmental toxicity potential indicates reduced potential for non-combusted products, when compared to cigarettes. Current Research in Toxicology, 1, 161-173.

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Label-free Targeted Metabolomics Analysis

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Agilent 1290 UHPLC and 6490 Triple Quadrupole (QqQ) Mass Spectrometer (LC–MS) were used for label-free targeted metabolomics analysis, as described previously (Lee et al., 2019 (link)). Agilent MassHunter Optimizer and Workstation Software LC–MS Data Acquisition for 6400 Series Triple Quadrupole B.08.00 was used for standard optimization and data acquisition. Agilent MassHunter Workstation Software Quantitative Analysis Version B.0700 for QqQ was used for initial raw data extraction and analysis. For RPLC, a Waters Acquity UPLC BEH TSS C18 column (2.1 × 100 mm, 1.7 µm) was used in the positive ionization mode. For HILIC, a Waters Acquity UPLC BEH amide column (2.1 × 100 mm, 1.7 µm) was used in the negative ionization mode. Further details are found in our previous study (Lee et al., 2019 (link)). The unprocessed metabolomics data are presented in Supplementary file 5.

Boyer S., Lee H.J., Steele N., Zhang L., Sajjakulnukit P., Andren A., Ward M.H., Singh R., Basrur V., Zhang Y., Nesvizhskii A.I., Pasca di Magliano M., Halbrook C.J, & Lyssiotis C.A. (2022). Multiomic characterization of pancreatic cancer-associated macrophage polarization reveals deregulated metabolic programs driven by the GM-CSF–PI3K pathway. eLife, 11, e73796.

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Serum Biogenic Amines Profiling by HILIC-QTOF MS/MS

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Serum biogenic amines were measured only in a subset of 10 randomly selected participants, due to budget restrictions. Biogenic amines, comprising acylcarnitines, TMAO, cholines, betaines, SAM, SAH, nucleotides and nucleosides and methylated and acetylated amines, were assessed by West Coast Metabolomics by HILIC-QTOF MS/MS, as previously described⁴². Hydrophilic interaction liquid chromatography (HILIC) method was used. Briefly, serum samples were extracted and derivatized. Five µL of re‐suspended samples were injected onto a Waters Acquity UPLC BEH Amide column (150 mm length × 2.1 mm id; 1.7 µm particle size) with an additional Waters Acquity VanGuard BEH Amide pre‐column (5 mm × 2.1 mm id; 1.7 µm particle size) coupled to an Agilent 1290 Infinity UHPLC. The mobile phases were prepared with 10 mm ammonium formate and 0.125% formic acid (Sigma‐Aldrich) in either 100% LC‐MS grade water for mobile phase (A) or acetonitrile: water (95:5, v/v) for mobile phase (B). Spectra were collected using Q Exactive HF mass spectrometer in positive ion mode. Quality control samples (Standard Reference Material NIST plasma samples) were included. For metabolite identification, HILIC spectral library of authentic standards was used in addition to m/z and retention time database. A total of 178 known and 2,946 unknown biogenic metabolites were measured.

Chang W.C., So J, & Lamon-Fava S. (2021). Differential and shared effects of eicosapentaenoic acid and docosahexaenoic acid on serum metabolome in subjects with chronic inflammation. Scientific Reports, 11, 16324.

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Quantifying GA and 2-KGA in Bacterial Cultures

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Total RNA extraction for qRT-PCR gene expression analysis was performed as described previously on 18-days-old seedlings [48] . Transcript levels were calculated relative to the reference gene At1g13320 [49] using the 2 ÀDCT method [50] .
Detection of GA and 2-KGA GA and 2-KGA concentrations in bacterial culture filtrates were determined using ultra-performance liquid chromatographymass spectrometry (UPLC-MS). Compounds were separated on a Waters Acquity UPLC BEH Amide Column (130A ˚, 1.7 mm particle size, 2.1 mm X 50 mm) by an Acquity UPLC system (Waters, Milford, MA, USA). The mobile phase A was 90% water, 10% acetonitrile, 0.1% formic acid and the mobile phase B was 100% acetonitrile, 0.1% formic acid. All solutions were ULC/MS grade from Biosolve BV (Valkenswaard, the Netherlands) The gradient was set from 10% to 90% with a flow rate of 0.25 mL min -1 . The run time was 6 min and the inject volume was 1 ml. Mass spectrometric detection was performed in negative ionization mode m/z 50 -1250 and SIR of 2 channels m/z 193 and m/z 195 on a Waters Acquity QDa detector (Waters, Milford, MA, USA). GA and 2-KGA was quantified by peak area obtained from standards for D-Gluconic acid sodium salt (Sigma-Aldrich, St. Louis, MO) and 2-Keto-D-gluconic acid hemi-calcium salt hydrate (Sigma-Aldrich, St. Louis, MO).

Yu K., Liu Y., Tichelaar R., Savant N., Lagendijk E., van Kuijk S.J.L., Stringlis I.A., van Dijken A.J.H., Pieterse C.M.J., Bakker P.A.H.M., Haney C.H, & Berendsen R.L. (2019). Rhizosphere-Associated Pseudomonas Suppress Local Root Immune Responses by Gluconic Acid-Mediated Lowering of Environmental pH. Current biology : CB, 29(22).

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