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).
Beh c8
Manufactured by Waters Corporation
Sourced in United States
The BEH C8 is a reversed-phase high-performance liquid chromatography (HPLC) column developed by Waters Corporation. It features a hybrid-silica particle design with a C8 alkyl stationary phase. The BEH C8 column is intended for the separation and analysis of a wide range of organic compounds.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc, by Waters Corporation (3 mentions)
Beh c18, by Waters Corporation (2 mentions)
Empower software, by Waters Corporation (1 mentions)
Hss t3, by Waters Corporation (1 mentions)
Waters 1 class uplc, by Waters Corporation (1 mentions)
Waters h class uplc, by Waters Corporation (1 mentions)
Beh phenyl, by Waters Corporation (1 mentions)
Unifi software, by Waters Corporation (1 mentions)
Acquity system, by Waters Corporation (1 mentions)
5 protocols using beh c8
1
UPLC-MS Method Development and Validation
2
Brain Metabolite Extraction and Analysis
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Metabolites were extracted from the frozen brain tissue powder by methanol: methyl-tert-butyl-ether (1:3 (v/v)) extraction26 (link)27 (link). In brief, 50 mg of frozen powdered PFC tissue was re-suspended in 1 ml extraction solution containing two internal standards (0.5 μg of corticosterone and 1.5 μg of 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC 34:0)). The samples were incubated for 10 min at 4 °C on an orbital shaker, before subjecting them to ultrasonication for 10 min in an ice-cooled bath-type sonicator. The insoluble tissue material (including proteins) was pelleted by a centrifugation step (5 min; 14,000g) and the supernatant was transferred to a fresh 2 ml Eppendorf tube. To separate the organic from the aqueous phase, 500 μl of an H2O:methanol mixture (3:1(v/v)) was added to the supernatant, mixed by vortexing and centrifuged (5 min; 14,000g). Five hundred microlitres of the upper methyl-tert-butyl-ether-phase was transferred to a 1.5-ml Eppendorf tube, concentrated in a speed vacuum and re-suspended in 100 μl of an acetonitrile:isopropanol mixture (7:3 (v/v)) before liquid chromatography–mass spectrometry analysis. For the analysis, 5 μl of lipid extract was injected onto the ultra performance liquid chromatography C8-reversed phase column (BEH C8, Waters), connected to an Orbitrap Exactive mass spectrometer26 (link)27 (link).
3
UPLC-UV Analysis of Bioactive Compounds
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The instrumentation consisted of a Waters ACQUITY UPLC (Milford, MA, USA) module equipped with a binary solvent manager system and an autosampler thermostatically controlled at 4 °C coupled to a Waters ACQUITY UPLC™ Tunable UV (TUV) detector. The analysis was performed on a Waters Acquity BEH C8 (2.1 mm × 100 mm, 1.7 μm) (Milford, MA, USA) analytical column. After optimization, the chromatographic conditions were; aq. ammonium formate 0.001 M as solvent A and methanol/acetonitrile 79/21 (v/v) as solvent B. The column temperature was maintained at 29 °C. The gradient mode was the same as the previously described program by our laboratory [19 (link)]. Briefly, the elution started with 15% B and was programmed to reach 80% B in 34 min at a flow rate of 0.15 mL min−1. The UV detector was set at 214 nm. The sample injection volume was 10 μL. Instrumental settings, data acquisition and processing were controlled via Empower 2 software.
4
MK4 Plasma Levels Quantification
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MK4 Plasma levels were determined at Admescope Ltd, in Oulu. The samples were prepared by protein precipitation (ratio 1:2) using acetonitrile containing of 1% of formic acid. The samples were then analysed with a UPLC-HRMS (Waters Acquity UPLC + Thermo Q-Exactive Focus Orbitrap MS) using a Waters BEH C8 (1.7 µm particle size, 50 × 2.1 mm) UPLC column with Methanol and 0.1% acetic acid as mobile phases for the chromatography and an APCI + ionization at the mass-spectrometer. The samples were quantified against standard samples prepared into mouse plasma by spiking blank matrix into concentrations ranging from 2 to 5000 ng/ml of analyte. Obtained accuracies ranged from 93.3 to 107.9%, with Snedecor precision of 11.7%. Quality control samples at mouse plasma concentrations of 30, 300 and 3000 ng/ml, were also included in the analysis. The Accuracy of the control samples ranged from 92.6 to 93.2%.
5
UPLC Method for Compound Separation
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Analytical UPLC runs were done using a Waters (Eschborn, Germany) Acquity system consisting of a sample manager FTN, quaternary solvent manager, PDA detector, column manager and column manager aux. The following Waters Acquity UPLC® columns were used: BEH C4 2.1 × 100 mm 300 Å 1.7 μm, BEH C8 2.1 × 100 mm 130 Å 1.7 μm, BEH C18 2.1 × 50 mm 130 Å 1.7 μm and BEH C18 2.1 × 100 mm 300 Å 1.7 μm. Separation was achieved using a gradient of solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in ACN) from 5% B to 95% B in 2.5 min at a flowrate of 0.613 ml/min for 50 mm columns or in 5 min at a flowrate of 0.5 ml/min for 100 mm columns.
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