Acquity ultra high performance liquid chromatography uplc system

Manufactured by Waters Corporation

Sourced in United States, United Kingdom

The Acquity ultra-high performance liquid chromatography (UPLC) system is a highly precise and efficient analytical instrument designed for the separation and identification of chemical compounds. It utilizes advanced technology to achieve superior chromatographic performance, enabling faster analysis times and increased resolution compared to traditional HPLC systems.

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11 protocols using acquity ultra high performance liquid chromatography uplc system

Quantification of Vitamin D Metabolites by UPLC-MS/MS

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Samples were analyzed using an Acquity UPLC ultra-high-performance liquid chromatography system coupled with Xevo TQ-S triple quadrupole mass spectrometer (Waters Crop, Manchester, UK). Ionization was performed in electrospray ionization (ESI) mode and the mass spectrometer was operated in the positive ion electrospray mode. The temperature of the electrospray source was maintained at 120 °C and a desolvation temperature of 500 °C. The capillary voltage was set 3.5 V. Multiple reaction monitoring (MRM) mode was used to monitor and quantify VD metabolites. The mass spectrometry conditions used for detecting the analytes are shown in Table 1.Table 1

MRM transitions, collision energies and cone voltages of vitamin D metabolites.

Compound	MRM transitions	Cone voltage(V)	Collision energy(eV)
25(OH)D₃	407.31 > 389.24	44	32
25(OH)D₃-d₃	410.50 > 392.26	77	22
1α,25(OH)₂D₃	423.26 > 369.25	49	22
1α,25(OH)₂D₃-d₆	429.36 > 374.36	60	25

Chromatographic separation was performed using a Waters Acquity UPLC BEH C₁₈ (100 × 2.1 mm, 1.7 µm), which was maintained at 40 °C in the column oven. The mobile phase was composed of lithium acetate (0.378 mM) aqueous solution (solvent A) and methanol (solvent B), with a total flow rate of 0.25 mL/min. The integration of the peak area and concentration calculation were done by the workstation UNIFI software (UNIFY 1.7.1.022).

Xu S., Ni R., Lv L., Chen R., Chen Y., Huang F, & Xu Z. (2022). Simultaneous determination of vitamin D metabolites 25(OH)D3 and 1α,25(OH)2D3 in human plasma using liquid chromatography tandem mass spectrometry. Journal of Mass Spectrometry and Advances in the Clinical Lab, 24, 65-79.

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Metabolomic Analysis by UPLC-MS

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Instrumentation and reagents employed in this study include the Triple TOF 5600⁺ mass spectrometer (SCIEX, Framingham, MA, USA), the ACQUITY UPLC ultra-high-performance liquid chromatography system (Waters, Milford, MA, USA), the Allegra X-30R high-speed centrifuge (Beckman Coulter, Pasadena, CA, USA), a vortex mixer (IKA, Staufen, Germany), a Milli-Q A10 ultrapure water system (Millipore, Boston, MA, USA), a programmable ultrasonic processor (Kunshan Ultrasonic Instruments Company, Suzhou, China), and the following specific chemicals: methionine sulfoximine (MSO, Sigma-Aldrich, St. Louis, MI, USA), tricaine methanesulfonate (MS-222, Sigma-Aldrich, St. Louis, MI, USA), sodium bicarbonate, sodium chloride (analytical grade, Tianjin Tiantai Chemical Factory, Tianjin, China), methanol, acetonitrile, formic acid (mass spectrometry grade, Merck, Darmstadt, Germany), physiological saline (Sichuan Kelun Pharmaceutical Company, Chengdu, China), and organic-phase filter membranes (13 mm, 0.22 μm; Anpu, Shanghai, China).

Sun Y., Geng C., Liu W., Liu Y., Ding L, & Wang P. (2024). Investigating the Impact of Disrupting the Glutamine Metabolism Pathway on Ammonia Excretion in Crucian Carp (Carassius auratus) under Carbonate Alkaline Stress Using Metabolomics Techniques. Antioxidants, 13(2), 170.

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Global Lipidomics of Murine Liver

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Liver tissue (lobe 1) was obtained from 10-day-old mice and subjected to exhaustive solvent extraction for lipids. Global lipidomics analysis was performed on a Xevo G2-S quadrupole time-of-flight (Q-TOF) mass spectrometer (MS) interfaced with an Acquity ultra-high performance liquid chromatography (UPLC) system (Waters, Milford, MA) operated in electrospray ionization mode. An Acquity CSH C18 UPLC column was used to chromatographically separate components over 20 minute gradient elution. Compounds were ionized with electrospray and positive and negative ions acquired over the mass range 50-1200 Daltons with high resolution. Deconvolution, peak alignment, and preliminary normalization were conducted on raw metabolomics data with Progenesis QI (Waters). Each compound ion feature was annotated by elution time with m/z. Raw data were normalized by total compound ion intensity and with a global scalar derived from logarithm ratio of each sample to the reference. Accurate molecular mass (m/z) was used to search against HMDB and lipid MAPS database for putative identification.

Carey A.N., Zhang W., Setchell K.D., Simmons J.E., Shi T., Lages C.S., Mullen M., Carroll K., Karns R., Bessho K., Sheridan R., Zhao X., Weber S.N, & Miethke A.G. (2017). Hepatic MDR3 expression impacts lipid homeostasis and susceptibility to inflammatory bile duct obstruction in neonates. Pediatric research, 82(1), 122-132.

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Global Lipidomics of Murine Liver

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UPLC-MS/MS Dapivirine Quantification

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Chromatographic separation of dapivirine and ²H₄-dapivirine was achieved using a Waters BEH C8, 50 × 2.1 mm UPLC column with a 1.7 μm particle size, on a Waters Acquity Ultra-High Performance Liquid Chromatography (UPLC) system (Waters Corporation, Milford, MA, USA). The mobile phase system used for separation consisted of water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The chromatographic method included a gradient elution from 30% to 50% mobile phase B over 2.0 min; dapivirine and its internal standard eluted at 1.35 min. The gradient was extended to 95% mobile phase B for 0.2 min, held at 95% mobile phase B for 0.4 min and the re-equilibrated to 30% mobile phase B over 0.1 min. The flow rate throughout the analytical run was 0.5 mL/min, and the analytical run time was 3.0 min.
Detection of dapivirine was performed using an API 5000 triple quadrupole mass analyzer (SCIEX, Redwood City, CA) using an ESI source operated in positive ionization and selective reaction monitoring (SRM) modes. Optimized declustering potential (170 V), collision energy (48 V) and exit potential (9 V) was determined for the analyte. Ion transitions monitored for dapivirine and ²H₄-dapivirine were 330.2→158.1 m/z and 334.3→119.0 m/z, respectively.

Manohar M, & Marzinke M.A. (2019). Validation and implementation of an ultrasensitive liquid chromatographic-tandem mass spectrometric (LC-MS/MS) assay for dapivirine quantitation in breast milk. Clinical biochemistry, 82, 66-72.

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Amino Acid Analysis Protocol

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Amino acid analysis was performed at the SPARC Biocentre (Sick Kids, Toronto ON). Samples were vacuum dried, suspended in 6N HCl containing 1% phenol, and hydrolyzed for 24 h at 110 °C under nitrogen. Following hydrolysis, samples were vacuum dried to remove excess HCl, resuspended in a re-drying solution of methanol: water: trimethylamine (2:2:1) and dried under vacuum for 15 min. Samples were derivatized for 20 min at room temperature in methanol: water: trimethylamine: phenylisothiocyanate (7:1:1:1). The derivatizing solution was removed under vacuum, and the samples were washed with re-drying solution and vacuum dried for an additional 15 min. Samples were then dissolved in diluent and injected into an ethylene bridged hybrid C18 column to run on a modified Pico-Tag gradient at 48 °C, with phenylthiohydantoin (PTH)-derivatized amino acid detection occurring at 254 nm. A Waters Acquity Ultra-High Performance Liquid Chromatography (UPLC) System was used to control the chromatography. Cysteine quantification for select samples was performed by performic acid oxidation prior to hydrolysis.

Tomalty H.E., Graham L.A., Eves R., Gruneberg A.K, & Davies P.L. (2019). Laboratory-Scale Isolation of Insect Antifreeze Protein for Cryobiology. Biomolecules, 9(5), 180.

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Amino Acid Analysis by UPLC

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Samples were vacuum dried, suspended in 6N HCl containing 1% phenol, and hydrolyzed for 24 h at 110 °C under N₂ stream. Following hydrolysis, samples were vacuum dried to remove excess HCl, resuspended in a methanol:water:trimethylamine (2:2:1) redrying solution and dried under vacuum for 15 min. Samples were derivatized for 20 min at room temperature in methanol:water:trimethylamine:phenylisothiocyanate (7:1:1:1) solution. This solution was then removed under vacuum, and the samples were washed with the redrying solution and vacuum dried for an additional 15 min. Chromatographic analysis was performed using a Waters Acquity Ultra-High Performance Liquid Chromatography (UPLC) System. Samples were dissolved and injected into an ethylene bridged hybrid C18 column, and a modified Pico-Tag gradient was used at 48 °C, with phenylthiohydantoin (PTH)-derivatized amino acid detection occurring at 254 nm. Cysteines were protected by performing acidic oxidation prior to hydrolysis. HPLC data correspond to the average of 4 replicates (Supplementary Table 11).

Poulhazan A., Arnold A.A., Mentink-Vigier F., Muszyński A., Azadi P., Halim A., Vakhrushev S.Y., Joshi H.J., Wang T., Warschawski D.E, & Marcotte I. (2024). Molecular-level architecture of Chlamydomonas reinhardtii’s glycoprotein-rich cell wall. Nature Communications, 15, 986.

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Brain Endothelial Cell Permeability Assay

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Example 10

The brain endothelial cell assay measures ability of compounds to cross a 2D brain microvascular layer. The assay is composed of two liquid-filled chambers separated by a semi-permeable membrane. Cells are propagated in endothelial cell growth media. The assay is performed in Hank's Balanced Salt Solution (HBSS).

The semi-permeable membrane is made of a polycarbonate membrane with pores of size 0.4 μM at a density of 1×10⁸pores/cm²on which human cerebral microvascular endothelial cells (HCMECs) are grown in a monolayer. Cells were obtained under Material Transfer Agreement from Cornell University. A compound is added to the liquid in contact with the human cerebral microvascular endothelial cells on the membrane, and ability to pass through the cells is measured at 37° C. for time points ranging from zero to two hours. Compound is measured by quantifying the material in solution using the Waters Acquity ultra-high performance liquid chromatography (UPLC) system.

This assay was similarly applied to LLNL-02, which demonstrated improved capacity for crossing brain endothelial cells relative to existing reactivators (FIG. 16).

US11186548B2. Compounds for central reactivation of organophosphorus-based compound-inhibited acetylcholinesterase and/or inactivation of organophosphorus-based acetylcholinesterase inhibitors and related compositions methods and systems for making and using them (2021-11-30). LAWRENCE LIVERMORE NATIONAL SECURITY, LLC [US]. Inventors: Carlos A. Valdez [US], Nicholas A. Be [US], Michael A. Malfatti [US], Heather Ann Enright [US], Brian J. Bennion [US], Timothy S. Carpenter [US], Saphon Hok [US], Hio Leong Lao [US], Tuan H. Nguyen [US].

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Characterization of Organic Compounds by NMR and MS

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¹H and 2D NMR experiments were recorded on a Varian Inova 750 MHz NMR spectrometer. ¹³C NMR spectra were recorded at 125 MHz in a Varian VNMRS-500-WB instrument (Agilent Technologies, Palo Alto, CA, USA). Chemical shifts (δ in ppm) are referenced to the carbon (δ_C 49.0) and residual proton (δ_H 3.31) signals of CD₃OD. Mass spectra were obtained from a Xevo TQ mass spectrometer (Waters, Manchester, UK) connected to an ACQUITY Ultra High Performance Liquid Chromatography (UPLC) system (Waters, Manchester, UK). Purification was performed on a Waters HPLC system: Waters 2767 sample manager, Waters system fluidics organizer, Waters 2545 binary solvent manager, Waters 515 post-column HPLC pump, Waters 3100 mass detector. HRMS was acquired by Ion Trap-Time of Flight (IT-TOF) mass spectrometer instrument from Shimadzu (Kyoto, Japan)

Boente-Juncal A., Álvarez M., Antelo Á., Rodríguez I., Calabro K., Vale C., Thomas O.P, & Botana L.M. (2019). Structure Elucidation and Biological Evaluation of Maitotoxin-3, a Homologue of Gambierone, from Gambierdiscus belizeanus. Toxins, 11(2), 79.

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UPLC-MS/MS Gradient Elution Protocol

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The UPLC-MS/MS system was equipped with an ACQUITY ultra-high performance liquid chromatography (UPLC) system (Waters Corporation, Milford, USA). The UPLC system was separated on a UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column (Temperature: 40 °C ). Acetonitrile (solution A) and 0.1% (v/v) formic acid in water (solution B) were selected as mobile phases for a created gradient elution. Gradient elution was performed at 5 µL injection volume and a flow rate of 0.35 mL/min for a total run time of 6 min. The specific gradient elution process is as follows: first, B runs at 95% for 0.8 min; second, linearly reduced from 95% B to 50% B for 0.8 to 3 min; third, linearly reducing from 50% B to 10% B for 4 to 5 min; and, finally, increasing linearity from 10% B to 95% B for 5.01 to 6 min.

Liu C., Han M., Wang H., Chen X., Tang Y., Zhang D., Li X, & Liu Y. (2023). Elimination of Cefquinome Sulfate Residue in Cow’s Milk after Intrauterine Infusion. Metabolites, 13(4), 492.

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