Acquity uplc beh amide column

Manufactured by Waters Corporation

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The ACQUITY UPLC BEH Amide column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of polar and hydrophilic compounds. The column features a proprietary hybrid organic/inorganic particle technology that provides excellent peak shape and resolution for a wide range of analytes.

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127 protocols using acquity uplc beh amide column

Quantification of Tetrodontoxin by UPLC-MS/MS

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TTX was measured on an ACQUITY ultra-performance liquid chromatography tandem triple quadrupole mass spectrometer (Waters Co., Milford, MA, USA). TTX was identified and quantified according to the method previously described [35 (link)]. The toxin was separated using an ACQUITY UPLC BEH Amide column (50 mm × 2.1 mm, 1.7 µm, Waters Co., Milford, MA, USA) at 40 °C with a sample volume of 10 µL. The initial mobile phase A was composed of 5 mmol/L aqueous ammonium acetate with 0.1% formic acid, and the initial mobile phase B was acetonitrile (A:B = 1:9). The elution was gradient elution (0–0.50 min, 10% A, 90% B; 1.50–4 min, 60% A, 40% B; 4.50–5 min, 10% A, 90% B) with a flow rate of 0.30 mL/min.
The electrospray ion source was used in positive ion mode (EMS+) and analyzed in Multiple Reaction Monitoring (MRM) mode with the following settings: TTX m/z 320→m/z 302; capillary voltage 3.50 kV; desolvation gas temperature 385 °C; ion source temperature 119 °C; cone gas orifice, high purity nitrogen; flow rate 55 L/h; desolvation gas flow rate 800 L/h; cone hole voltage 30 V; and collision energy 25 V.

Zhong Y., Zhang X., Yang Q, & Wang Q. (2023). Hepatorenal Toxicity after 7-Day Oral Administration of Low-Dose Tetrodotoxin and Its Distribution in the Main Tissues in Mice. Toxins, 15(9), 564.

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Quantification of 15N-labeled Glycans in Pancreatic Cancer Cells

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SU. 8686 and SW1990 pancreatic cancer cells were cultured for 72 h in L-glutamine-¹⁵N₂ (Sigma‒Aldrich, 490032). Cells cultured in unlabeled glutamine (Sigma‒Aldrich, Cat.# G3126) were used as controls. A total of 1 × 10⁷ cells were used for analysis. Before examination, a standard curve was built using 3'-Sialyl Lewis A (Carbosynth, Cat.# OS00745) diluted in methanol/water (1:1) at different concentrations (1, 5, 10, 50, 100, 500, 1000, 5000, 10000 nM). A total of 160 μL of methanol (precooled at − 20 °C) was added to each sample for metabolite extraction. Then, the samples were centrifuged at 18000 g and 4 °C for 20 min, and the clear supernatant was transferred to an autosampler vial for UHPLC‒MS/MS analysis. Ultra-performance liquid chromatography coupled to a tandem mass spectrometry system (ACQUITY UPLC I class-Xevo TQ-S system, Waters Corp., Milford, MA, USA) equipped with a Waters ACQUITY UPLC BEH Amide column (100 × 2.1 mm, 1.7 μm) was used to measure the metabolites. Mobile phase A was 10 mM ammonium acetate in water, and mobile phase B was acetonitrile. The elution gradient was set as follows: 0–1.0 min, 1% A; 1.0–3.0 min, 1–40% A; 3.0–5.0 min, 40% A; 5.0–5.1 min, 40–1% A; 5.1–6 min, 1% A. The injected volume was 5 μL. Data acquisition and metabolite quantification were performed using MassLynx 4.1 software (Waters Corp., Milford, MA, USA).

Liu C., Deng S., Xiao Z., Lu R., Cheng H., Feng J., Shen X., Ni Q., Wu W., Yu X, & Luo G. (2023). Glutamine is a substrate for glycosylation and CA19-9 biosynthesis through hexosamine biosynthetic pathway in pancreatic cancer. Discover. Oncology, 14, 20.

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Quantification of EntF and EntF* in Biological Samples

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Chromatographic separation was achieved on a Waters Acquity® UPLC BEH Amide Column (130 Å, 1.7 μm, 2.1 mm × 100 mm). Mobile phase composition, sample volume, flow rate, and MS settings were as described for the LC₁-MS₁ method. For EntF* quantification in mouse sera, the gradient program started with 10% of mobile phase A for 2 min, followed by a linear gradient to 40% of mobile phase A for 3.0 min. Gradient was then changed to 85% mobile phase A at 6 min, followed by a 1 min equilibration, before starting conditions were applied. EntF* retention time was 4.85–4.95 min. A sample was considered positive for the presence of EntF* when following criteria were met: correct retention time, both daughter fragments [b₂ (quantifier) and b₃ (qualifier) fragment ions] with a signal-to-noise ratio > 3.0, and quantifier/qualifier peak area ratio between 2.0 and 4.0.
For the quantification of EntF in the culture medium, the gradient program started with 100% of mobile phase B for 2 min, followed by a linear gradient to 40% of mobile phase B for 7 min, cleaning at 85% B and re-equilibration at starting conditions. Acquisition was done in the MRM mode. The selected precursor ion for EntF was m/z 667.1 with three selected product ions: m/z 129.0 (30 eV, b₂ fragment) and m/z 662.6 (22 eV, b₂₅ fragment), both as qualifier, and m/z 949.4 (22 eV, y₁₇ fragment) as quantifier.

Wynendaele E., Debunne N., Janssens Y., De Spiegeleer A., Verbeke F., Tack L., Van Welden S., Goossens E., Knappe D., Hoffmann R., Van De Wiele C., Laukens D., Van Eenoo P., Vereecke L., Van Immerseel F., De Wever O, & De Spiegeleer B. (2022). The quorum sensing peptide EntF* promotes colorectal cancer metastasis in mice: a new factor in the host-microbiome interaction. BMC Biology, 20, 151.

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UHPLC-MS/MS Analysis of Metabolites

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The UHPLC-MS/MS analysis was performed with Acquity^™ UPLC I-class system equipped with Xevo TQ-XS MS/MS system (Waters Corp. Milford, UK). The transitions for multiple reaction monitoring were previously reported(Nishizawa et al., 2020 (link)). The capillary voltages of electrospray ionization for positive and negative ion mode were 4.0 kV and 2.5 kV, respectively. The cone voltage, cone gas (nitrogen) flow rate, desolvation temperature, desolvation gas flow, collision gas flow and nebulization gas flow were 64 V, 150 L/hr, 600°C, 1000 L/hr, 0.15 ml/min 7.00 bar, respectively. The UHPLC condition was modified with the previous publication(Saigusa et al., 2016 (link)). LC separation was performed with an Acquity UPLC BEH Amide column (1.7 μm, 2.1 × 150 mm, Waters Corp.) kept at 20 °C with a gradient elution using solvent A (10 mmol/l NH₄HCO₃, adjusted to pH 9.2 using ammonia solution) and B (acetonitrile) at 0.2 ml/min. All data was analyzed by to the software (Traverse MS).

Rashad S., Al-Mesitef S., Mousa A., Zhou Y., Ando D., Sun G., Fukuuchi T., Iwasaki Y., Xiang J., Byrne S.R., Sun J., Maekawa M., Saigusa D., Begley T.J., Dedon P.C, & Niizuma K. (2024). Translational response to mitochondrial stresses is orchestrated by tRNA modifications. bioRxiv.

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Ileal Mucosal Metabolomic Profiling by LC-MS/MS

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The ileal mucosal metabolomic profile was analyzed by liquid chromatography (LC)- mass spectrometry (MS)/MS. Briefly, 100 mg ileal mucosa was homogenized in 200 μL double-distilled H₂O (ddH₂O), and mixed with 800 μL methanol–acetonitrile (1:1, vol:vol) and sonicated in an ice water-bath for 30 min. After incubation in −20 °C for 1 h, the mixture was centrifuged at 14,000 × g for 15 min at 4 °C. The supernatant was dried by speedvac, and resuspended in 100 μL methanol/ddH₂O (1:1, vol:vol) for LC-MS/MS analysis. An aliquot from each sample was pooled to create quality control samples that were used to evaluate the internal standards and instrument performance. The ultra-high-performance LC (UPLC; Agilent 1290 Infinity LC, USA) was performed on a 2.1 × 100 mm ACQUITY UPLC BEH Amide column (internal diameter 1.7 μm; Waters, USA). The column was warmed to 45 °C before use. The mobile phase for UPLC analysis consisted of 2 solutions: (A) 15 mmol/L ammonium acetate–H₂O and (B) acetonitrile. The MS/MS (5500 QTRAP, AB SCIEX, USA) spectra was set as follows: source temperature 450 °C; ion source gas 1, 45 ψ; ion source gas 2, 45 ψ; curtain gas, 30 ψ; ionSapary voltage floating, 4,500 V.

Wang J., Wang N., Qi M., Li J, & Tan B. (2021). Glutamine, glutamate, and aspartate differently modulate energy homeostasis of small intestine under normal or low energy status in piglets. Animal Nutrition, 8, 216-226.

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Serum Metabolite Analysis by LC-MS/MS

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The serum metabolite analysis was performed with an Agilent 1290 Infinity LC system (Agilent Technologies, Palo Alto, CA, USA), coupled with a Triple TOF 5600+ mass spectrometer (AB/Sciex, Foster City, CA, USA). Chromatographic separation of serum samples was performed on an ACQUITY UPLC BEH Amide column (1.7 µm, 2.1 mm × 100 mm, Waters, Milford, MA, USA), which was maintained at 25 °C, and a 2 μL aliquot of each sample was injected into the column. The mobile phase consisted of solvent A (25 mM ammonium acetate and 25 mM ammonia in water) and B (acetonitrile). The optimized gradient program was established as shown in Table 1. The samples were randomly placed in a 4 °C autosampler throughout the analysis to avoid instrument detection signal fluctuations and to monitor and evaluate the system stability and reliability of the experimental data. To detect the stability of the instruments and systems, the QC sample was run at the beginning, the middle and the end of the sample queue.

Huang M.Z., Cui D.A., Wu X.H., Hui W., Yan Z.T., Ding X.Z, & Wang S.Y. (2020). Serum Metabolomics Revealed the Differential Metabolic Pathway in Calves with Severe Clinical Diarrhea Symptoms. Animals : an Open Access Journal from MDPI, 10(5), 769.

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Fecal Metabolite Profiling by UHPLC-MS

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The fecal metabolites were analyzed with the Agilent 1,290 Infinity ultra high performance liquid chromatography (UHPLC) platform (Agilent, United States). An ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 µm, Waters, United States) was used for compound separation at 25°C. The eluents employed in both electrospray ionization positive (ESI +) and negative models (ESI −) were A (water containing 25 mM ammonium acetate and 25 mM ammonia) and B (acetonitrile). The elution gradient was set as 40–95% of the gradient of B. The mass spectral data were acquired by AB Triple TOF 6600 mass spectrometry (AB SCIEX, United States) in both ESI + and ESI − mode. And the MS-MS data were collected by information-dependent acquisition.

Xiong Y., Wu L., Shao L., Wang Y., Huang Z., Huang X., Li C., Wu A., Liu Z., Fan X, & Zhou P. (2022). Dynamic Alterations of the Gut Microbial Pyrimidine and Purine Metabolism in the Development of Liver Cirrhosis. Frontiers in Molecular Biosciences, 8, 811399.

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Targeted Metabolite Analysis by UHPLC-MS/MS

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With a Vanquish ultraperformance liquid chromatography system coupled to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific), an ACQUITY UPLC BEH Amide column (50 x 2.1 mm, 1.7 μm, Waters) was used for metabolite separation. Metabolites were separated with a 15 min gradient at a flow rate of 0.4 mL/min. Mobile phase A was H₂O with 10 mM NH₄COOH and mobile phase B was ACN. The gradient was set as follows: 0-1 min, 95% B; 1-7 min, 70% B; 7-10 min, 30% B, 10-12.5 min, 30% B, 12.5-13.0, 95% B, 13-15 min, 95% B. The above Chemicals and reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
The mass spectrometer was operated in electrospray ionization (ESI) positive ion mode. Analysis was performed in parallel reaction monitoring (PRM) scan mode. Relative quantification of metabolites was calculated by peak area. The instrument settings for the MS/MS mode were: 15,000 resolution, 1 × 10⁶ AGC, 100 ms maximum injection time, and dynamic collision energy of 20, 40, 60 ev. Source ionization parameters were: spray oltage set at 3.5 kV for positive ion mode, capillary temperature set at 320°C, sheath gas set at 25, and aux gas set at 5.

Shan D., Wang Y.Y., Chang Y., Cui H., Tao M., Sheng Y., Kang H., Jia P, & Song J. (2023). Dynamic cellular changes in acute kidney injury caused by different ischemia time. iScience, 26(5), 106646.

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Yeast Conditioned Medium Amino Acid Analysis

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Yeast conditioned medium was passed through 0.2 μm PVDF syringe filters and 3 kDa MWCO centrifugal filters (Millipore), diluted 1:10, then 1 μl was used for amino acid analysis on a liquid chromatography (Agilent 1290 Infinity) and tandem mass spectrometry (Agilent 6460) system, as described elsewhere (Mülleder et al., 2016a ). Method covered all proteinogenic amino acids (except cysteine), ornithine, citrulline, and α /γ-aminobutyric acid. In short, amino acids were separated by hydrophilic interaction chromatography with gradient elution on a Waters ACQUITY UPLC BEH Amide column (2.1 x 100 mm, 1.7 μm) using a binary solvent system of 50:50 acetonitrile:water and 95:5:5 acetonitrile:methanol:water, both containing 0.176% formic acid, 10 mM ammonium formate. The compounds were identified by comparing retention time and fragmentation pattern with analytical standards (Sigma-Aldrich). The obtained signals, operating the instrument in selected reaction monitoring mode, were processed and quantified by external calibration with Agilent MassHunter software.

Ponomarova O., Gabrielli N., Sévin D.C., Mülleder M., Zirngibl K., Bulyha K., Andrejev S., Kafkia E., Typas A., Sauer U., Ralser M, & Patil K.R. (2017). Yeast Creates a Niche for Symbiotic Lactic Acid Bacteria through Nitrogen Overflow. Cell Systems, 5(4), 345-357.e6.

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UPLC-MS/MS Quantification of Tetrodontoxin

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Chromatography separation was carried out using a 1290 Infinity ultra-high-performance liquid chromatography system coupled to an Agilent G6460C Triple Quadrupole mass spectrometer equipped with an Agilent Jet Stream ESI source (Agilent Tecnologies, Waldbronn, Germany). TTX identification and quantification was performed according to Rodriguez et al. [43 (link)]. Briefly, toxin was separated using an ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 µm, Waters, Manchester, UK) at 35 °C, with an injection volume of 5 µL. The composition of the mobile phase A was 10 mM ammonium formate and 0.1% formic acid in water, and mobile phase B was acetonitrile containing 0.1% formic acid and 2% 100 mM ammonium formate dissolved in water. Chromatographic separation was performed by gradient elution: starting with 95% B and reducing to 5% B over 11 min, then 5% B was held for 1 min, and increasing to 95% B over 1 min. Finally, 95% was held for 2 min until the next run (run time 15 min). The mass spectrometer was operated in positive mode, and analyses were performed in Multiple reaction monitoring (MRM) mode monitoring two transitions: m/z 320.0 > 161.9 (identification) and m/z 320.0 > 302.0 (quantification).

Boente-Juncal A., Vale C., Cifuentes M., Otero P., Camiña M., Rodriguez-Vieytes M, & Botana L.M. (2019). Chronic In Vivo Effects of Repeated Exposure to Low Oral Doses of Tetrodotoxin: Preliminary Evidence of Nephrotoxicity and Cardiotoxicity. Toxins, 11(2), 96.

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