The UPLC-MS analysis was performed using a Waters Acquity H-Class UPLC which is connected to a PDA detector and a Waters Acquity TQD detector. The column used was a Waters Acquity UPLC BEH C18 1.7 μm, 2.1 × 50mm, with a Vanguard precolumn attached. Solvent A consisted of 90:10 water:acetonitrile with 0.01% formic acid and 0.1 mM hexylamine, while solvent B consisted of 90:10 acetonitrile:water with 0.01% formic acid with 0.1 mM hexylamine. A gradient run was performed such that solvent B was increased from 0% B to 100% B from time 0–7 mins, followed by 5 minute wash at 100% B and then a return and re-equilibration at 100% A in the next 3 mins. 10 μL of sample was injected per run. The eluent of the column was connected to a PDA UV detector which scanned from 250–350 nm and a 2D channel of 280 nm was set. The eluent was then introduced into the TQD detector. The TQD detector was set at positive ionization mode with a capillary voltage of 3.20 kV, cone voltage of 20 V, extractor voltage of 3 V, and RF lens voltage of 0.1 V. The source temperature was set at 150 °C, while the desolvation temperature was set at 350 °C and the desolvation and cone gas flows were set at 650 and 50 L/hr respectively. Scans were made from 100–700 m/z with scan duration of 0.5 seconds to obtain mass spectra at different time points.
H class uplc
Manufactured by Waters Corporation
Sourced in United States
The H-Class UPLC is an ultra-high-performance liquid chromatography system manufactured by Waters Corporation. It is designed to provide efficient and precise separation and analysis of a wide range of chemical compounds. The H-Class UPLC utilizes advanced technology to deliver high-resolution separations and enhanced sensitivity, making it a versatile tool for various analytical applications.
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Lab products found in correlation
Masslynx 4, by Waters Corporation (2 mentions)
Beh c18 column, by Waters Corporation (2 mentions)
Acquity uplc beh c18 reversed phase column, by Waters Corporation (2 mentions)
Empower 3, by Waters Corporation (1 mentions)
Duetta fluorescence and absorbance spectrometer, by Horiba (1 mentions)
Autoflex tof, by Bruker (1 mentions)
Beh amide, by Waters Corporation (1 mentions)
Acquity photodiode array pda detector, by Waters Corporation (1 mentions)
Xevo g2 s qtof ms, by Waters Corporation (1 mentions)
Quaternary pump, by Agilent Technologies (1 mentions)
Triple quadruple mass spectrometer, by Waters Corporation (1 mentions)
Agilent infinity 1100, by Agilent Technologies (1 mentions)
Acquity tqd, by Waters Corporation (1 mentions)
Agilent diode array detector, by Agilent Technologies (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
C18 column, by Waters Corporation (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Accq tag ultra derivatization kit, by Waters Corporation (1 mentions)
Accq tag ultra column, by Waters Corporation (1 mentions)
Accq tag ultra chemistry kit, by Waters Corporation (1 mentions)
33 protocols using h class uplc
1
UPLC-MS Analysis of Compounds
2
Quantification of MTD12813 in Plasma, Serum, and Bacteria
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MTD12813 (final concentration of 100 µg/ml) was added to either 90% human EDTA plasma or 90% human serum and incubated at 37 °C for 48 h. Samples were processed by addition of 10% HOAc and 5% ACN (final concentrations) at time 0 and 48 h, and MTD12813 was quantified by C18 RP-HLPC on an Acquity H-Class UPLC with an analytical PDA detector using Empower 3 software (Waters). All samples were performed with technical replicates and analyzed by UPLC in duplicate and the experiment repeated once. In separate experiments, MTD12813 (1.25 µg/ml final) was incubated with log phase Kp-1705 (5 × 107 CFU/ml) in 50 mM HEPES pH 7.4. A time 0 h sample was collected, and the remaining suspension incubated at 37 °C for 24 h. Samples were processed by addition of 5% formic acid/5% acetonitrile, vortexed, and clarified at 22,000×g for 10 min. Supernatant MTD12813 was then quantified by LC–MS/MS as described in PK section above. All samples were performed with technical replicates and analyzed by LC–MS/MS in duplicate.
3
Characterization of Silver Nanoclusters
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Absorbance spectra of nanoclusters were recorded using an Agilent Cary 60 UV-Vis absorbance spectrometer. EEM spectra were taken using the Horiba Duetta Fluorescence and Absorbance Spectrometer, and inner-filter effects and Rayleigh scattering were corrected using EzSpec™ software. Reverse-phase high performance liquid chromatography (RP-HPLC) was accomplished using a preparatory scale C18 column (Dim. 250 mm × 21.2 mm) using the Varian PS218 Solvent Delivery Module, as well as an analytical scale amide column (Waters BEH Amide; 2.1 mm × 100 mm; 1.7 μm particle size) on a Waters H-Class UPLC. The high-resolution mass spectra of AgNCs were recorded on a Bruker Autoflex TOF using laser desorption ionization (LDI) in negative ion mode with a linear detector. A total of 2000 laser shots were summed from 200 pulses of the 2 kHz SmartBeam laser set to a flat profile. The spectra were recorded in the mass range of 800–6000 m/z. Finally, thermogravimetric analysis (TGA) was performed with the TGA Q500 V6.7 Build 203 using a ramp of 5 °C min−1 and heating up to 900 °C under argon gas.
4
Quantification of Inhaled Anesthetics via UPLC/MS
5
Fungal Phenylpropanoid Quantification Protocol
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Fungal-derived phenylpropanoid concentrations were established in dried fungal cultures after 21 days of incubation (Supplementary Table S1 ), as previously described by Kulik et al. [28 (link),29 (link)] and Bilska et al. [30 (link)]. Alkaline and acid hydrolysis were conducted in sealed 17-mL culture tubes containing 0.2 g of fungal biomass. An ACQUITY H class UPLC (Ultra performance liquid chromatography) system equipped with a Waters ACQUITY Photodiode Array (PDA) detector (Waters, Milford, MA, USA) was used in the analysis. Concentrations of l -pyroglutamic acid were determined using an internal standard at a wavelength of λ = 310 nm. l -Pyroglutamic acid identification was based on a comparison of sample and standard retention times. A specific amount of the standard was added to the analyzed samples. In the next step, the analysis was repeated. The detection level was 1 μg/g. The retention time of l -pyroglutamic acid was 19.05 min.
6
UPLC-QTOF-MS Protocol for Compound Separation and Analysis
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UPLC was performed using an ACQUITY H-Class UPLC (Waters, Milford, MA, USA) according to the protocol reported by Lee et al. [32 (link)] with an ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 µm) held at 40 °C. Two microliters of sample were injected for separation, with a mobile phase consisting of Solvent A (5% acetonitrile, 0.1% formic acid) and Solvent B (95% acetonitrile, 0.1% formic acid) at a constant flow rate of 450 µL/min. Eluting compounds were detected with a Waters Xevo G2-S QTOF-MS (Waters) operating in positive and negative ion mode, with alternating high- and low-energy scans (MSE acquisition mode) and cone voltage 40 V, capillary voltage 3.0 kV, source temperature 120 °C, desolvation temperature 300 °C, cone gas flow 30 L/h, and desolvation gas flow 600 L/h. Accurate mass measurements were obtained by means of an automated calibration delivery system using leucine as internal reference (m/z 556.276 (ESI+), m/z 554.262 (ESI−)). Data were collected between m/z 100 and m/z 2000.
7
HPLC and LC/MS/MS Analysis of PD98059
8
Proteomic Workflow for Glycoprotein Analysis
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Samples were diluted with 300-μl denaturing buffer (6 M guanidine HCl, 360 mM Tris, 2 mM EDTA, pH 8.6), reduced using DTT (10 mM, 45 °C for 10 min), s-carboxymethylated with sodium iodoacetate (25 mM, 45 °C for 5 min), and quenched with DTT (50 mM, room temperature). Samples were then desalted using NAP-5 columns with an elution volume of 800-μl PBS, digested with 1:20 w/w ratio of protein:trypsin (37 °C for 1 h), deglycosylated with 0.15-μg PNGaseF (37 °C for 30 min), and quenched with 100% FA. Tryptic peptides (10 μg) were separated using a Waters H-Class UPLC with Waters Acquity UPLC CSH130 C-18 column (1.7 μm, 2.1 × 150 mm). Peptide separation occurred across a gradient from 100% solvent A (water and 0.1% FA) to 35% solvent B (acetonitrile and 0.1% FA) over 60 min at a flow rate of 0.3 ml/min and column temperature of 77 °C. MS analysis was performed with a Thermo Fisher Q Exactive mass spectrometer operating in a positive mode, performing MS2 on top-10 most abundant peaks in a data-dependent mode.
9
Quantification of Prodrug Motif Abundance
10
Proteomic LC-MS/MS Analysis of Samples
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LC–MS/MS analysis was performed as previously published58 (link). In brief, samples for LC–MS/MS analysis were denatured with guanidine, reduced with DTT and s-carboxymethylated with isodacitate. Samples were then desalted using NAP-5 columns and subjected to trypsinization followed by deglycosylation using PNGaseF. After quenching with formic acid, the tryptic peptides (10 µg) were separated using a Waters H-Class UPLC with a Waters Acquity UPLC CSH130 C18 column (1.7 µm, 2.1 × 150 mm), with a column temperature of 77 °C. Peptide separation occurred across a gradient from 100% solvent A (water and 0.1% formic acid) to 35% solvent B (acetonitrile and 0.1% formic acid) over 60 min at a flow rate of 0.3 ml min−1. MS analysis was performed with a Thermo Fisher Q Exactive operating in positive mode, performing MS2 on the top ten most abundant peaks in data-dependent mode.
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