I class uplc system

Manufactured by Waters Corporation

Sourced in United States

The I-class UPLC system is a high-performance liquid chromatography (HPLC) instrument designed for ultra-high-performance liquid chromatography (UPLC) applications. The system is capable of delivering high-pressure mobile phases at flow rates and pressures suitable for the separation and analysis of a wide range of compounds.

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UPLC system (190 citations) I-Class UPLC (17 citations) I-Class (13 citations) I Class Acquity UPLC system (6 citations) I-class Acquity UPLC chromatography system (3 citations)

14 protocols using i class uplc system

UPLC-MS Metabolite Profiling Protocol

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Liquid chromatography was performed on a I-Class UPLC system (Waters Corporation, Milford, MA, USA) combining a binary pump, a FTN autosampler and a column oven. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C8 Column (100 mm × 2.1 mm, 1.7 μm) with binary solvent system at a flow rate of 450 μl/min. Mobile phase A was 0.1% acetic acid in water and B was 0.1% acetic acid in acetonitrile/isopropanol (1:1). The binary solvent gradient was as follow: 0.0–1.0 min at 0% B, 1.0–6.5 min from 0 to 100% B, 6.5–8.5 min 100% B, followed by 2 min of equilibration at initial conditions. Column oven temperature was set to 55°C and the autosampler injection volume to 1 μl.
High resolution mass spectrometric analysis was performed on a Q Exactive mass spectrometer (ThermoFisher Scientific, Bremen, Germany) operating in negative ionization mode over the mass range m/z 65–600 with a resolving power of 70,000 (at m/z = 200). Data was acquired in profile mode with an AGC target of 5e6 ions and a maximum injection time of 250 ms. The mass spectrometer was interfaced to the UPLC system using a HESI probe. The spray voltage was set to −4 kV. The heater and capillary temperatures were both set to 350°C. Sheath gas and auxiliary gas flow rate were set to 45 and 15 AU, respectively. The instrument was calibrated every 4 days according to manufacturer specifications.

Sonnay S., Chakrabarti A., Thevenet J., Wiederkehr A., Christinat N, & Masoodi M. (2019). Differential Metabolism of Medium-Chain Fatty Acids in Differentiated Human-Induced Pluripotent Stem Cell-Derived Astrocytes. Frontiers in Physiology, 10, 657.

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General Methods for Non-Aqueous Reactions

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Example 3

General methods: All non-aqueous reactions were performed in oven-dried glassware under an inert atmosphere of dry nitrogen. All the reagents and solvents were purchased from Aldrich (St. Louis, Mo.), Alfa-Aesar (Ward Hill, Mass.), Combi-Blocks (San Diego, Calif.), Ark Pharm (Libertyville, Ill.) and used without further purification. Analytical thin-layer chromatography was performed on Silica Gel GHLF 10×20 cm Analtech TLC Uniplates (Analtech, Newark, Del.) and were visualized by fluorescence quenching under UV light. Biotage SP1 Flash Chromatography Purification System (Charlotte, N.C.) (Biotage SNAP Cartridge, silica, 50 g & 100 g) was used to purify the compounds. 1H NMR and 13C NMR spectra were recorded on a Varian Inova-500 spectrometer (500 MHz) (Agilent Technologies, Santa Clara, Calif.) or a Bruker Ascend 400 (400 MHz) (Billerica, Mass.) spectrometer. Chemical shifts are reported in ppm on the δ scale and referenced to the appropriate solvent peak. Mass spectra were collected on a Brucker ESQUIRE electrospray/ion trap instrument in the positive and negative modes. High resolution mass spectrometer (HRMS) data were acquired on a Waters Xevo G2-S QTOF (Milford, Mass.) system equipped with an Acquity I-class UPLC system.

US11124490B2. Autotaxin inhibitors (2021-09-21). UNIVERSITY OF TENNESSEE RESEARCH FOUNDATION [US], UNIVERSITY OF MEMPHIS [US]. Inventors: Duane D. Miller [US], Gabor G. Tigyi [US], Souvik Banerjee [US], Abby L. Parrill-Baker [US].

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Differential 3-MH Expression in Myopathy

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Blood samples were taken at day 42 after separating the sampled broilers as myopathy (WB score > 1) and nonmyopathy (WB = 0) (n = 5 each group for each strain) groups. Plasma was separated from collected blood samples, and samples were analyzed for differential 3-MH expression. Targeted metabolomics of 3-MH was performed on a triple quadrupole MS coupled to an I-class UPLC system (Waters) for differential expression. Procedures for handling plasma samples in a laboratory, data processing, and bioinformatics were carried out as described in the article by Maharjan et al., 2020c (link).

Maharjan P., Weil J., Beitia A., Suesuttajit N., Hilton K., Caldas J., Umberson C., Martinez D., Owens C.M, & Coon C. (2020). In vivo collagen and mixed muscle protein turnover in 2 meat-type broiler strains in relation to woody breast myopathy. Poultry Science, 99(10), 5055-5064.

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Quantifying 3-Hydroxybutyric Acid via LC-MS

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Analysis of 3-hydroxybutyric acid (BHB) was done with LC-MS, performed on an I-Class UPLC system (Waters Corp., Milford, MA, USA) coupled to a Thermo Scientific Q Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany) operating in negative mode over the mass range 65–600 m/z at a resolving power of 35,000 (at m/z = 200), as previously described [15 (link)]. INS1E cells grown to confluence in 6-well plates were treated with the vehicle (DMSO), the GPR40 antagonist GW1100 (50 nM), the PLCβ inhibitor U73122 (2 μM), the IP3 receptor inhibitor (-) Xestospongin C (3 μM) or the l-type Ca²⁺ channel inhibitor Nifedipine (0.1 μM) for 15 min prior to incubation with 200 µM MCFA for 2 h in RPMI medium with 1 mM glucose. BHB was measured in the media by chromatographic separation performed on an Acquity UPLC BEH C8 column (1.7 mm, 100 × 2.1 mm; Waters Corporation, Milford, MA, USA). BHB was quantified using the generated 8-point external calibration curve with Xcalibur Software 4.0 (Thermo Scientific Inc., Waltham, MA, USA).

Pujol J.B., Christinat N., Ratinaud Y., Savoia C., Mitchell S.E, & Dioum E.H. (2018). Coordination of GPR40 and Ketogenesis Signaling by Medium Chain Fatty Acids Regulates Beta Cell Function. Nutrients, 10(4), 473.

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Carbohydrate Analysis of BMCC Hydrolysis

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Filtrate obtained after 72-h reaction with TrAA9A was centrifuged in 70% ethanol for 20 min at 4 °C to precipitate TrAA9A and supernatant was collected. Filtrate of BMCC incubated under the same condition without TrAA9A was also prepared in the same way for mass spectrometry analysis. Samples were analyzed on a Waters Xevo G2-XS Q-TOF system coupled to a Waters I-Class UPLC system. Carbohydrates were separated by an ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm) maintained at 40 °C, with the injection volume at 10 μL. Solvent A was 10-mM ammonium formate, and solvent B was 100% acetonitrile. The solutes were eluted at 0.2 mL/min starting at 95% B, followed by a linear gradient to 35% B over 14 min. The proportion of solvent B was maintained at 35% for 2 min and then increased back to 95% and kept for 4 min for re-equilibration.
The operation condition for mass spectrometer was capillary voltage of 3.0 kV, sample cone voltage of 80 V, source temperature of 100 °C, desolvation temperature of 350 °C, and desolvation gas flow of 600 L/h. Mass spectra were acquired in positive ion mode across the 50–2000-m/z range. Data processing was performed using the MassLynx software (version 4.1, Waters).

Song B., Li B., Wang X., Shen W., Park S., Collings C., Feng A., Smith S.J., Walton J.D, & Ding S.Y. (2018). Real-time imaging reveals that lytic polysaccharide monooxygenase promotes cellulase activity by increasing cellulose accessibility. Biotechnology for Biofuels, 11, 41.

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Targeted Metabolomics of Polar Metabolites

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Targeted metabolomics of polar, primary metabolites was performed on a TQ-XS triple quadrupole mass spectrometer (MS) coupled to an I-class UPLC system (Waters) for differential expression. Plasma was thawed on ice then 80 μL was aliquoted to a 1.5 mL Eppendorf tube on ice. Next, 240 μL of ice-cold methanol was added and samples were vortexed to mix. Samples were incubated for 30 min at −80° C, and then centrifuged at 16,000 × g at 4°C for 15 min. The supernatant was analyzed by LC-MS. Separations were carried out on a ZIC-pHILIC column (2.1 × 150 mm, 5 μM) (EMD Millipore). The mobile phases were (A) water with 15 mM ammonium bicarbonate adjusted to pH 9.6 with ammonium hydroxide and (B) acetonitrile. The flow rate was 200 μL/min and the column were held at 50°C. The injection volume was 1 μL. The gradient was as follows: 0 min, 90% B; 1.5 min, 90% B; 16 min, 20% B; 18 min, 20% B; 20 min, 90% B; 28 min, 90% B. The MS was operated in selected reaction monitoring (SRM) mode. Source and desolvation temperatures were 150 and 600°C, respectively. Desolvation gas was set to 1,100 L/h and cone gas to 150 L/h. Collision gas was set to 0.15 mL/min. All gases were nitrogen except the collision gas, which was argon. Capillary voltage was 1 kV in positive ion mode and 2 kV in negative ion mode.

Maharjan P., Hilton K., Weil J., Suesuttajit N., Beitia A., Owens C.M, & Coon C. (2020). Characterizing Woody Breast Myopathy in a Meat Broiler Line by Heat Production, Microbiota, and Plasma Metabolites. Frontiers in Veterinary Science, 6, 497.

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UPLC-Fluorescence Method for Compound Analysis

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The UPLC system consisted in an Acquity I CLASS UPLC System (Waters, Milford, CT, USA) coupled to an Acquity TUV and an Acquity Fluorescence Detector. Fluorescence detection was carried out at 224 and 304 nm for excitation and emission wavelengths, respectively.
The chromatography separation was performed with a Lichrospher RP-8 column (125 × 4 mm, 5 µm, Phenomenex). The method had the column at room temperature. The flow rate and the injection volume were 0.8 mL/min during 8 min and 10 µL, respectively. The mobile phase A consisted of 0.1 % of trifluoroacetic acid (TFA) in Milli-Q^® water and mobile phase B comprised methanol. The gradient elution program was: 50–50% B for 0–5 min; 100% B for 5–6 min; 50–50% B for 6–8 min.

Pérez-González N., Bozal-de Febrer N., Calpena-Campmany A.C., Nardi-Ricart A., Rodríguez-Lagunas M.J., Morales-Molina J.A., Soriano-Ruiz J.L., Fernández-Campos F, & Clares-Naveros B. (2021). New Formulations Loading Caspofungin for Topical Therapy of Vulvovaginal Candidiasis. Gels, 7(4), 259.

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Qingpi Metabolite Profiling by UPLC-QTOF-MS

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An ACQUITY I-class UPLC system equipped with a Xevo G2-XS Q-TOF MS instrument (Waters Corporation, Milford, MA, USA) was used to analyze the Qingpi samples. Compounds were separated using a Waters ACQUITY UPLC HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 μm; MA, USA). The injection volume was 4 μL and the flow rate was 0.4 mL/min. The temperature of column was set at 40 °C. The mobile phase was composed of water containing 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B). The gradient elution program was as follows: 0–15 min for 99–1% A, 15–17 min for 1% A, 17–17.10 min for 1–99% A, and 17.10–20 min for 99% A.
For the MS conditions, the positive mode of electrospray ionization (ESI) source was used, the mass range was m/z 50 to 1200, and the scanning time was 0.25 s. The voltage was set at +2.5 KV for capillary, 40 V for cone hole voltage, 80 V for ion source compensation, and +3.0 KV for spray voltage. The cone hole gas flow rate was 50 L/h. The desolvation gas temperature was 450 °C and its flow rate was 800 L/h. The low collision energy was set as 6 eV and the high collision energy increased from 15 to 50 eV. The MS^E data acquisition mode was used to collect data in real time.

Cheng Y., Wu C., Liu Z., Song P., Xu B, & Chao Z. (2023). Evaluation and Optimization of Quality Based on the Physicochemical Characteristics and Metabolites Changes of Qingpi during Storage. Foods, 12(3), 463.

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Warfarin Plasma Concentration Quantification

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The plasma concentration of total warfarin was measured by Ultra performance liquid chromatography (UPLC) equipped by Waters I-class-UPLC system (Waters, UK). The column used was a 1.7μm BEH C18 (2.1×50 mm).The mobile phases consisted of 60% 0.02mol/L NH₄Ac (PH = 3.0) and 40% acetonitrile with 0.3ml/min flow rate. The column temperature was maintained at 40°C and the detection wavelength was 308nm. 0.1ml internal standard, 0.5ml 1mol/L HCL and 3ml methyl tert-butyl ether (MTBE) were added to 0.5ml plasma sample. The mixture was thoroughly mixed for 5 minutes and then centrifuged for 3 minutes (2500r/min). The supernatant was separated and evaporated to dryness at 50°C. The residue was reconstituted with 100μl Mobile Phase and 4μl of the solution was then injected into the UPLC system. According to the peak height ratio of warfarin and internal standard, concentration quantitative analysis was performed in the standard curve.

Li S., Zou Y., Wang X., Huang X., Sun Y., Wang Y., Dong L, & Jiang H. (2015). Warfarin Dosage Response Related Pharmacogenetics in Chinese Population. PLoS ONE, 10(1), e0116463.

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Targeted Metabolomics of Primary Metabolites

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Targeted metabolomics of polar, primary metabolites was performed on a TQ-XS triple quadrupole mass spectrometer (Waters) coupled to an I-class UPLC system (Waters). Separations were carried out on a ZIC-pHILIC column (2.1 × 150 mm, 5 µM) (EMD Millipore, 150460). The mobile phases were (A) water with 15 mM ammonium bicarbonate adjusted to pH 9.6 with ammonium hydroxide and (B) acetonitrile. The flow rate was 200 µl/min and the column was held at 50 °C. The injection volume was 2 µl. The gradient was as follows for PfRAP01: 0 min, 90% B; 1.5 min, 90% B; 16 min, 20% B; 18 min, 20% B; 20 min, 90% B; 28 min, 90% B, and as follows for PfRAP21: 0 min, 90% B; 1.5 min, 90% B; 16 min, 10% B; 18 min, 10% B; 20 min, 90% B; 28 min, 90% B.
The MS was operated in selected reaction monitoring mode^{62 (link)}. Source and desolvation temperatures were 150 °C and 600 °C respectively. Desolvation gas was set to 1100 l/h and cone gas to 150 l/h. Collision gas was set to 0.15 ml/min. All gases were nitrogen except the collision gas, which was argon. Capillary voltage was 1 kV in positive ion mode and 2 kV in negative ion mode. System stability was monitored by analyzing a quality control sample (generated by pooling together equal volumes of all sample extracts) every 3 injections. Samples were analyzed in random order.

Hollin T., Abel S., Falla A., Pasaje C.F., Bhatia A., Hur M., Kirkwood J.S., Saraf A., Prudhomme J., De Souza A., Florens L., Niles J.C, & Le Roch K.G. (2022). Functional genomics of RAP proteins and their role in mitoribosome regulation in Plasmodium falciparum. Nature Communications, 13, 1275.

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