The brains of mice injected intraperitoneally with A2ARPAM-1, optoA2ARPAM-1 or optoA2ARPAM-2 at 21:00 were collected 30 min after treatment. Each brain was combined with 300 μL of acetonitrile with 1% formic acid (v/v) and vortexed for 3 min under normal light conditions to convert all brain-penetrating optoA2ARPAM-1 or optoA2ARPAM-2 to A2ARPAM-1. The precipitated proteins were then removed by centrifugation (1000 × g for 5 min), and the supernatant was transferred to a HybridSPE-Phospholipid Ultra cartridge (Supelco) and eluted from the cartridge by applying a vacuum. The eluate was injected into a Waters ACQUITY UPLC-MS/MS system with an electrospray ionization interface and operated in the negative ion mode. An ACQUITY UPLC BEH C18 column (1.7 μm, 50 mm × 2.1 mm; Waters) with a graded acetonitrile/water mobile phase at a flow rate of 500 μL min−1 was used for UPLC separation. A2ARPAM-1 was detected by single ion (m/z 392) monitoring.
Acquity uplc ms ms system
Manufactured by Waters Corporation
Sourced in United States
The ACQUITY UPLC-MS/MS system is a liquid chromatography-tandem mass spectrometry (LC-MS/MS) solution designed for high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) applications. The system combines the ACQUITY UPLC instrument for sample separation and the Xevo TQ-S micro triple quadrupole mass spectrometer for sensitive and selective detection and quantification of analytes.
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Lab products found in correlation
Acquity uplc beh c18 column, by Waters Corporation (2 mentions)
Hybridspe phospholipid ultra cartridge, by Merck Group (2 mentions)
Xevo tq ms, by Waters Corporation (2 mentions)
Acquity 1.7um beh amide hilic column, by Waters Corporation (2 mentions)
Acquity beh c18 column, by Waters Corporation (1 mentions)
Abx pentra 400, by Horiba (1 mentions)
Robosep, by STEMCELL (1 mentions)
K2edta tubes, by BD (1 mentions)
Macsquant10 analyzer, by Miltenyi Biotec (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Tqd mass spectrometer, by Waters Corporation (1 mentions)
Tq s micro mass spectrometer, by Waters Corporation (1 mentions)
Acquity binary solvent manager, by Waters Corporation (1 mentions)
Triple quadrupole detector mass spectrometer, by Waters Corporation (1 mentions)
Spectroquant, by Merck Group (1 mentions)
16 protocols using acquity uplc ms ms system
1
Quantification of A2ARPAM-1 in Mouse Brains
2
A2ARPAM-1 Brain Distribution Analysis
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The brains of mice injected intraperitoneally with A2ARPAM-1 at 21:00 were collected 1 h after treatment. Each brain was combined with 300 μL of acetonitrile with 1% formic acid (v/v) and vortexed for 3 min. Then, the precipitated proteins were removed by centrifugation (3000 rpm for 5 min), and the supernatant was transferred to a HybridSPE-Phospholipid Ultra cartridge (Supelco) and eluted from the cartridge by applying a vacuum. The eluate was injected into a Waters ACQUITY UPLC-MS/MS system with an electrospray ionization interface operating in negative ion mode. An ACQUITY UPLC BEH C18 column (1.7 μm, 50 mm × 2.1 mm; Waters) with a graded acetonitrile/water mobile phase at a flow rate of 500 μL/min was used for UPLC separation. A2ARPAM-1 was detected by single-ion (m/z 392) monitoring.
3
Plasma EC Quantification Protocol
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For the analysis of plasma EC concentrations in experiment 1 and 2, blood samples were immediately placed on ice, centrifuged at 1570× g for 20 min at 4 °C, and stored at −80 °C. Samples were analyzed for AEA and 2-AG concentrations by the Research Core Unit Metabolomics at the Hannover Medical School using the cross-validated method as described recently [38 (link)]. Briefly, analyses were carried out using a Waters ACQUITY UPLC-MS/MS system with a tandem quadruple mass spectrometer XEVO TQ MS (Waters, Milford, MA, USA), using a Waters ACQUITY BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm particle size) for separation of analytes. Plasma-free, non-esterified fatty acid (NEFA) and glucose concentrations were analyzed spectrophotometrically at a semi-automatic analyzer (ABX Pentra 400, HORIBA Medical, Kyoto, Japan) using the respective NEFA-HR 91797 (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) and Glucose HK CP A11A01667 (HORIBA) kits.
4
Quantification of Tacrolimus Levels
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Blood samples were drawn pre-dose and 1.5, 48, 96, and 192 h after drug administration. The samples for whole blood PK measurement were collected in K2EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, US) and stored at −80 °C. PBMCs were collected using sodium heparin CPT tubes (Becton Dickinson), and T cells were isolated from heparinized whole blood by immunomagnetic cell sorting. The RoboSep human T cell isolation kit was used in combination with RoboSep (StemCell Technologies) to label unwanted cells with antibody complexes and magnetic particles, after which T cells were isolated by automated magnetic sorting. After PBMC and T cell isolation, the cells were washed, and the remaining red blood cells were removed using RBC lysis buffer (Thermo Fisher Scientific, Waltham, MA, US). PBMCs and T cells were counted with a MacsQuant 10 analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and stored in PBS at 20 × 106 cells/mL at −80 °C. The purity of the isolated T cell population was assessed with an anti-CD45-FITC and anti-CD3-VioGreen staining (Miltenyi Biotec).
Whole blood and intracellular tacrolimus concentrations were measured using a Waters Acquity UPLC–MS/MS system by the Department of Hospital Pharmacy, Erasmus Medical Center, as described previously [19 (link)].
Whole blood and intracellular tacrolimus concentrations were measured using a Waters Acquity UPLC–MS/MS system by the Department of Hospital Pharmacy, Erasmus Medical Center, as described previously [19 (link)].
5
Serum Metabolite Extraction and Analysis
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Venous blood samples were collected into Vacuette 454078 4-ml serum gel tubes (Greiner Bio-One GmbH, Rainbach im Mühlkreis, Austria). They were left at room temperature for 30 min, followed by 10 min of centrifugation at 2,400×g, +20°C, to separate serum. The serum samples were frozen at −80°C until analysed. The metabolites were extracted from the serum samples using acetonitrile:formic acid (99:1 v/v) as a solvent (1:4, sample:solvent) and analysed using an ACQUITY UPLC-MS/MS system (Waters Corporation, Milford, MA, USA). A detailed protocol and instrument conditions have been published elsewhere (19 (link)).
6
Bioanalytical Protocol for Abiraterone Quantification
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The drug concentration in plasma was
estimated using a validated
bioanalytical UPLC-MS/MS method for quantification of abiraterone
(active metabolite) and imatinib (internal standard), that is, ACQUITY
UPLC-MS/MS system (Waters Inc., Milford, U.S.A.) fitted with Zspray
Xevo TQD mass spectrometer. Chromatographic separation of the analyte
and internal standard in rat plasma was performed on a C18 column (100 mm, 1.7 μm particle size), using ammonium acetate
(2 mM) in water (pH 3.5) and acetonitrile with 0.1% formic acid as
the mobile phase mixture. The isocratic elution was used for the mobile
phase delivered at a flow rate of 0.4 mL/min, and run time was kept
at 5 min. Linear calibration plot of the drug spiked in rat plasma
was analyzed over the concentration ranging between 1 and 800 ng·mL–1. The run time was kept at 5 min, while retention
times for abiraterone and the internal standard were observed at 2.4
and 3.6 min, respectively. The detection was made by multiple reactions
monitoring of parent-to-daughter ion transition at m/z 350.1 → 156.15 for abiraterone and m/z 494.43 → 294.17 for the internal
standard. The plasma concentration data obtained at various time points
were subjected to fitting with various compartmental body models and
pharmacokinetic parameters (Cmax, Tmax, AUC0-t, MRT, Ka and t0.5) were calculated.
estimated using a validated
bioanalytical UPLC-MS/MS method for quantification of abiraterone
(active metabolite) and imatinib (internal standard), that is, ACQUITY
UPLC-MS/MS system (Waters Inc., Milford, U.S.A.) fitted with Zspray
Xevo TQD mass spectrometer. Chromatographic separation of the analyte
and internal standard in rat plasma was performed on a C18 column (100 mm, 1.7 μm particle size), using ammonium acetate
(2 mM) in water (pH 3.5) and acetonitrile with 0.1% formic acid as
the mobile phase mixture. The isocratic elution was used for the mobile
phase delivered at a flow rate of 0.4 mL/min, and run time was kept
at 5 min. Linear calibration plot of the drug spiked in rat plasma
was analyzed over the concentration ranging between 1 and 800 ng·mL–1. The run time was kept at 5 min, while retention
times for abiraterone and the internal standard were observed at 2.4
and 3.6 min, respectively. The detection was made by multiple reactions
monitoring of parent-to-daughter ion transition at m/z 350.1 → 156.15 for abiraterone and m/z 494.43 → 294.17 for the internal
standard. The plasma concentration data obtained at various time points
were subjected to fitting with various compartmental body models and
pharmacokinetic parameters (Cmax, Tmax, AUC0-t, MRT, Ka and t0.5) were calculated.
7
UPLC-MS/MS Analysis of Amino Acid Hydroxylation
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The products of the amino acid hydroxylation were analyzed using the Waters ACQUITY UPLC-MS/MS system. A Waters ACQUITY UPLC HSS C18 column (1.8 μm) was used. Gradient elution was performed for mobile phase A consisting of 20 mM NH4Ac-HAc buffer (pH 4.2) and mobile phase B consisting of acetonitrile. The flow rate was 200 μL/min, the column temperature was 25 °C, and the sample size was 5 μL.
8
Metabolomic Analysis of Plasma Samples
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Plasma was separated immediately after sampling and stored at −140°C. Metabolites were analyzed with ACQUITY UPLC‐MS/MS system (Waters Corporation) as in Khan et al (2014 ). The column was 2.1 × 100 mm Acquity 1.7um BEH amide HILIC column and the detection system Xevo® TQ‐S tandem triple‐quadrupole mass spectrometer (Waters Corporation). In order to analyze differences among groups, univariate analysis T‐test2 was performed on autoscaled data. In order to explain the separation among groups, unsupervised multivariate analysis, principal component analysis (PCA), was performed. Non‐transformed data were mean‐centered and autoscaled. Five components were used in all the PCA models. Dendrogram was plotted on normalized data using Ward's linkage clustering algorithm and Pearson's correlation similarity measure and visualized as heatmap.
9
Determination of Antidepressant Levels
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The levels of fluoxetine, norfluoxetine and paroxetine were determined by a Waters ACQUITY UPLC‒MS/MS system (Waters, Milford, MA, United States) according to our previous articles (Ji et al., 2015 (link); Fang et al., 2017 (link)).
10
Quantifying EXE Metabolites in HLM
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A 50‐μl incubation containing 50 μg of HLM in PBS (pH 7.4), 400 μmol/L EXE, and an NADPH regeneration system was placed in a 37°C water bath for 4 h before termination with 50 μL of cold acetonitrile. After a 15‐min refrigerated centrifugation at 13,200g, the supernatant was examined for phase I EXE metabolites. A 10‐min UPLC method was used to separate and detect EXE and the ten other reference compounds through multiple reaction monitoring with positive mode electrospray ionization on a Waters ACQUITY UPLC/MS/MS system (Milford, MA). The 1.7 μm ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, Ireland) used for these analyses was protected by a 0.2 μm in‐line filter. The UPLC gradient conditions used have previously been described (Platt et al. 2016 ). The fragmentation characteristics and retention time of suspected metabolite peaks were compared to compounds from the reference library.
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