The Waters H-Class UPLC was utilized for the Method development and validation study (Waters Corporation, Milford, MA). The entire study was conducted using empower software to acquire, process, and report chromatographic data (Waters Corporation, Milford, MA). A statistical tool, Design-Expert-13, was employed to screen and optimize the CMPs (Stat-Ease Inc, Minneapolis, USA). Various Acquity UPLC columns (100 mm Length × 2.1 mm ID), such as BEH C8, BEH C18, BEH Phenyl, HSS T3, and Protein BEH C4 were assessed for the separation of components (Waters Corporation, Milford, MA). The Waters Xevo G2-XS Quadrupole time-of-flight (Q-ToF) MS instrument with step wave ion optics and XS collision cell coupled with Waters I-Class UPLC was used to separate and identify unknown impurities (Waters Corporation, Milford, MA). UNIFI software was used to identify molecular and fragment ions and their molecular structures (Waters Corporation, Milford, MA).
Waters h class uplc
Manufactured by Waters Corporation
Sourced in United States
The Waters H-Class UPLC is an ultra-high performance liquid chromatography (UPLC) system designed for analytical applications. It features a high-pressure solvent delivery system, a temperature-controlled sample manager, and a detector to separate, identify, and quantify components within a sample. The system is capable of delivering solvent flow rates and operating pressures suitable for UPLC analysis.
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Lab products found in correlation
Hss t3, by Waters Corporation (1 mentions)
Unifi software, by Waters Corporation (1 mentions)
Beh c8, by Waters Corporation (1 mentions)
Beh phenyl, by Waters Corporation (1 mentions)
Waters 1 class uplc, by Waters Corporation (1 mentions)
Empower software, by Waters Corporation (1 mentions)
Beh c18, by Waters Corporation (1 mentions)
Cortecs t3 column, by Waters Corporation (1 mentions)
Exploris 480, by Thermo Fisher Scientific (1 mentions)
Christ rvc 2 25 cdplus, by Martin Christ (1 mentions)
5 protocols using waters h class uplc
1
UPLC-MS Method Development and Validation
2
UPLC Chromatographic Separation Protocol
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Waters H-class UPLC (Waters Corporation, America) have been carried for chromatography system equipped with a quaternary solvent delivery system, an on-line degasser, an auto-sampler, a column temperature controller and a photodiode array detector. All experimental data was collected and managed with Empower 2 software.
The chromatographic separation was achieved with a Waters Cortecs® T3 column (2.1 mm × 100 mm, 2.7 µm) with the temperature of 30 °C. Detection wavelength for determination was set at 210 nm. The mobile phase was 0.03% phosphoric acid in water (A) and acetonitrile (B) at a flow rate of 0.4 mL/min. The gradient elution program was as followed: 0–1% B in 0–3.5 min, 1%–2% B in 3.5–6.5 min, 2%–5% B in 6.5–10 min, 5%–10% B in 10–17 min, 17%–22% B in 10–13 min, 22%–26% B in 13–18 min, 26%–29% B in 18–20 min, 29%–32% B in 20–24 min and 24%–90% B in 32–35 min. The sample injection volume was 1 μL.
The chromatographic separation was achieved with a Waters Cortecs® T3 column (2.1 mm × 100 mm, 2.7 µm) with the temperature of 30 °C. Detection wavelength for determination was set at 210 nm. The mobile phase was 0.03% phosphoric acid in water (A) and acetonitrile (B) at a flow rate of 0.4 mL/min. The gradient elution program was as followed: 0–1% B in 0–3.5 min, 1%–2% B in 3.5–6.5 min, 2%–5% B in 6.5–10 min, 5%–10% B in 10–17 min, 17%–22% B in 10–13 min, 22%–26% B in 13–18 min, 26%–29% B in 18–20 min, 29%–32% B in 20–24 min and 24%–90% B in 32–35 min. The sample injection volume was 1 μL.
3
Urine Metabolomics Analysis Protocol
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Urine creatinine concentrations were measured using the Jaffe method. To reduce urine concentration variability for metabolomics analysis, all samples were diluted to one standard deviation below the mean creatinine concentration. Samples were then diluted 1:5 in acetonitrile/water/methanol (65/30/5) containing 10 µM α-aminopimelic acid as internal standard and injected onto a Waters H-Class ultra-performance liquid chromatography (UPLC) coupled to a Waters Xevo G2 quadrupole time of flight MS (QTOFMS) in positive and negative ionization mode, as previously described27 . Briefly, chromatographic separation was performed using an Acquity BEH amide column (2.1 × 50 mm) maintained at 40 °C and a flow rate of 0.4 mL/min in a Waters H-Class UPLC. Mobile phase consisted of 10 mM ammonium acetate in 90% acetonitrile (A, pH = 9.0) and 10 mM ammonium acetate in 10% acetonitrile (B, pH = 9.0). Accurate mass correction was performed using leucine-enkephalin. Urine metabolomics raw data files were processed using Progenesis QI software and normalized to creatinine. The resulting feature lists for positive and negative mode were combined after filtering features that ionize in both modes using previously published code27 .
4
High-pH Reversed-Phase Peptide Fractionation
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Pooled peptide samples of each group were separated by high-pH reverse phase LC columns (4.6×250 mm, C18, 3 µm; Waters Corporation) and then loaded onto the column in buffer A1 (H2O; pH 10) using a Waters H-class UPLC (Waters Corporation). The elution gradient was 5-30% buffer B1 [90% acetonitrile (ACN); pH 10; flow rate, 1 ml/min] over 30 min.
The eluted peptides were collected at one fraction per minute. After lyophilization using a CENTRIVAP (ChristRVC 2-25 CD plus; Martin Christ GmbH), the 30 fractions were resuspended in 0.1% formic acid and then concatenated into 10 fractions by combining fractions 1, 11, 21, etc. To generate the spectral library, the fractions from RPLC were analyzed in data-dependent acquisition (DDA) mode using an EXPLORIS 480 (Thermo Fisher Scientific, Inc.). The parameters were set as follows: the MS was recorded at 350-1,500 m/z at a resolution of 60,000 m/z; the maximum injection time was 50 msec, the auto gain control (AGC) was 1×106, and the cycle time was 3 sec. MS/MS scans were performed at a resolution of 15,000 with an isolation window of 1.6 Da and high-collision dissociation (HCD) collision energy of 32%; the AGC target was 50,000 and the maximum injection time was 30 msec.
The eluted peptides were collected at one fraction per minute. After lyophilization using a CENTRIVAP (ChristRVC 2-25 CD plus; Martin Christ GmbH), the 30 fractions were resuspended in 0.1% formic acid and then concatenated into 10 fractions by combining fractions 1, 11, 21, etc. To generate the spectral library, the fractions from RPLC were analyzed in data-dependent acquisition (DDA) mode using an EXPLORIS 480 (Thermo Fisher Scientific, Inc.). The parameters were set as follows: the MS was recorded at 350-1,500 m/z at a resolution of 60,000 m/z; the maximum injection time was 50 msec, the auto gain control (AGC) was 1×106, and the cycle time was 3 sec. MS/MS scans were performed at a resolution of 15,000 with an isolation window of 1.6 Da and high-collision dissociation (HCD) collision energy of 32%; the AGC target was 50,000 and the maximum injection time was 30 msec.
5
UPLC-HRMS Protocol for Bioanalysis
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Waters H-Class UPLC (Waters, United States), AB Sciex Triple TOF® 4600 high-resolution mass spectrum (SCIEX, United States), KQ-300 BD ultrasonic cleaning instrument (Kunshan Ultrasonic Instrument, China), Sigma 3K15 high-speed centrifuge (Sigma, United States), LNG-T98 centrifugal concentration dryer (Taicang Huamei, China), and R583S small animal anesthesia machine (RWD, China) were used for this study.
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