According to the method previously described [15 (link), 28 (link)], LC/MS was carried out using a Nexera LC system (Shimadzu Co., Kyoto, Japan) equipped with two LC-30 AD pumps, a DGU-20As degasser, a SIL-30 AC autosampler, a CTO-20 AC column oven and a CBM-20A control module, coupled with an LCMS-8040 triple quadrupole mass spectrometer (Shimadzu Co.).
Nexera lc system
Manufactured by Shimadzu
Sourced in Japan
The Nexera LC system is a high-performance liquid chromatography (HPLC) platform designed for a wide range of analytical applications. It offers precise and reliable separation, detection, and quantification of various chemical compounds. The system features advanced technology to provide consistent and reproducible results, making it a versatile and efficient tool for analytical laboratories.
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Lab products found in correlation
Lcms 8040 triple quadrupole mass spectrometer, by Shimadzu (2 mentions)
Lcms 8050 triple quadrupole tandem mass spectrometer, by Shimadzu (1 mentions)
Inertsustain c18, by GL Sciences (1 mentions)
Compound discoverer 3, by Thermo Fisher Scientific (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Acquity uplc hss t3 column, by Waters Corporation (1 mentions)
Qtrap 5500 system, by AB Sciex (1 mentions)
Kinetex xb c18 column, by Phenomenex (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Qtrap 4500 mass spectrometry system, by AB Sciex (1 mentions)
Qtrap 5500 mass spectrometer, by AB Sciex (1 mentions)
Qtrap 4500, by AB Sciex (1 mentions)
5500 qtrap triple quadrupole mass spectrometer, by AB Sciex (1 mentions)
Synergi polar c18 column, by Phenomenex (1 mentions)
Kinetex c18 column, by Phenomenex (1 mentions)
Thermo q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
15 protocols using nexera lc system
1
LC/MS Analysis of Metabolites
2
LC-MS/MS Quantitative Analysis
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Samples were measured using online Nexera LC System coupled to LCMS-8050 triple quadrupole (QQQ) mass spectrometry (Shimadzu Corporation, Kyoto, Japan). Data were processed using LabSolutions LCMS version 5.65 software.
3
LC-MS/MS Analysis of Compounds
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LC–MS/MS analysis was performed on a Shimadzu Nexera LC system coupled with a Shimadzu LCMS-8050 triple quadrupole tandem mass spectrometer equipped with an ESI interface in positive ionization mode (Shimadzu, Kyoto, Japan). Chromatographic separation was performed on an ACQUITY UPLC C18 column (2.1 × 50 mm, 1.7 µm) maintained at 40 °C by gradient elution, using water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) as the mobile phase at a flow rate of 0.3 mL/min. The gradient program for compound identification was: 0–4.0 min, 20%–30% B, 4.0–8.0 min, 30%–90% B, 8.0–10.0 min, 20% B. The gradient program for compound quantitation was: 0–1.0 min 20% B, 1.0–3.5 min, 60% B, 3.5–4.0 min, 20% B. The MS conditions were set as follows: nebulizing gas flow, 3.0 L/min; drying gas flow, 10.0 L/min; heat-block temperature, 400 °C; desolvation line temperature, 250 °C; interface voltage, 4 kV. Product ion scan and MRM mode were used for identification and quantitation, respectively. The ion transitions and collision energies are listed in Table 6 . Data acquisition and processing were conducted using LabSolutions LCMS Ver.5.82 SP1 (Shimadzu, Kyoto, Japan).
4
Metabolite extraction from rhizosphere soil
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To extract primary metabolites, 10 g of fresh rhizosphere soil and root samples were ground in 1 ml 75%:25% v/v methanol:water solution using pestle and mortar. The mixture was transferred into a clean tube, vortexed briefly, then sonicated for 10 min before being centrifuged at 15,000 × g for 5 min at 4 °C. The supernatant (800 μl) was filtered through a 0.22-μm filter membrane syringe, before the filtrate was lyophilized using a freeze dryer, dissolved in water, and analyzed for key metabolites of central metabolic pathways, including organic acids, amino acids, nucleotides, vitamins and signalling molecules by UHPLC–MS/MS.
The UHPLC-MS/MS analyses were performed using a Nexera LC system (Shimadzu Corporation, Kyoto, Japan) coupled to a LCMS-8040 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan) using Shim-pack Velox® PFPP (pentafluorophenylpropyl) column (150 × 2.1 mm, 3 μm; Shimadzu Corporation, Kyoto, Japan) and an octadecylsilylated silica column (InertSustain C18, 150 × 2.1 mm, 3 μm; GL Sciences, Tokyo, Japan) for cationic and anionic analyses modes, respectively, as described previously [3 (link)].
The UHPLC-MS/MS analyses were performed using a Nexera LC system (Shimadzu Corporation, Kyoto, Japan) coupled to a LCMS-8040 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan) using Shim-pack Velox® PFPP (pentafluorophenylpropyl) column (150 × 2.1 mm, 3 μm; Shimadzu Corporation, Kyoto, Japan) and an octadecylsilylated silica column (InertSustain C18, 150 × 2.1 mm, 3 μm; GL Sciences, Tokyo, Japan) for cationic and anionic analyses modes, respectively, as described previously [3 (link)].
5
High-Resolution Mass Spectrometry Analysis
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Soluble fractions were dissolved in 50 μl of methanol/water (95:5, vol/vol). Flow injection analysis was performed using a Nexera LC system (Shimadzu Co., Kyoto, Japan) coupled with Q Exactive, a high performance bentchtop quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA), equipped with an electron spray ionization source. The flow injection conditions in the positive and negative ionization modes were described previously61 (link). Data mining procedure was described previously27 (link). In short, Compound Discoverer 3.0 software (Thermo Fisher Scientific) was used for data processing, involving peak detection, data grouping, and filling gaps. Differential analysis (fraction #54 vs. other fractions, fold change > 5) was performed using an XLSM extension developed with Visual Basic for applications in Microsoft Excel 2013. Among more than 20,000 ion peaks (including isotopes and adducts detected in the positive and negative ionization), 22 candidates were detected selectively and reproducibly present in fraction #54.
6
UHPLC-MS for Metabolite Profiling
7
LC-MS/MS Analysis of Platelet-Derived DiEpHEDE
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LC-MS/MS analysis of human platelet-derived and synthetic DiEpHEDE was performed on a Nexera LC system (Shimadzu) coupled by electrospray ionization to a tandem quadrupole ion trap mass spectrometer (4000 qTrap). Briefly, LC was performed at 40 °C using a C18 Spherisorb ODS2 (5 μm) 150 × 4.6-mm column (Waters) at a flow rate of 1 ml·min−1 over 75 min. Mobile phase A was water, 0.1% formic acid, and mobile phase B was acetonitrile, 0.1% formic acid. The following linear gradient for B was applied: 20% for 0.5 min, 20–42.5% over 50 min, 42.5–90% from 50 to 60 min, and held at 90% for 5 min followed by 10 min at initial condition for column re-equilibration. Injection volume was 10 μl. Ionization was performed using electrospray ionization in the negative mode (ESI−), monitoring parent ion to daughter ion m/z 351.2 → 165.1 (dwell time, 200 ms) with the following parameters: TEM, 650 °C; GS1, 70 p.s.i.; GS2, 55 p.s.i.; CUR, 40 p.s.i.; ESI spray voltage, −4.3 kV; DP, −53 V; EP, −10 V; CE, −26 V; and CXP at −7 V. Full-scan MS was carried out in negative mode, scanning Q3 from m/z 200 to 600 with total scan time (including pauses) over 4 s. Settings were: TEM, 650 °C; GS1, 60 p.s.i.; GS2, 30 p.s.i.; CUR, 35 p.s.i.; ESI spray voltage, −4.5 kV; DP, −55 V; EP, −10 V; CXP at −30 V; and IHE on.
8
LC-MS/MS Quantification of Analytes
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Blood serum samples were diluted 1000-fold with IS solution and centrifuged at 18,000×g for 5 min. The supernatant was transferred into a clean vial and 5 μL was injected into an LC-MS/MS system. LC analysis was performed using a Nexera LC system (Shimadzu, Kyoto, Japan). An L-column 2 ODS column (150 mm × 1.5 mm i.d.; 5 μm particle size; CERI, Tokyo, Japan), equipped with a guard column (OPTI-GUARD 1 mm C18; Optimize Technologies, Inc., Oregon City, OR, USA), was used for chromatographic separation. The mobile phase consisted of 10 mmol/L ammonium formate (95%) and methanol (5%) (solvent A), and 10 mmol/L ammonium formate (5%) and methanol (95%) (solvent B). The solvent gradient was increased linearly from 0 to 100% solvent B in 15 min and was maintained at this composition for 5 min. Subsequently, the gradient was changed to 0% solvent B and maintained for 10 min to re-equilibrate the column. The flow rate of the mobile phase was set at 0.1 mL/min and the column temperature was maintained at 40 °C. MS/MS detection was performed using a QTRAP 5500 system (SCIEX, Framingham, MA, USA). Quantitation was performed in SRM mode. The SRM transition 152 > 110 was used for quantitation, and the transition 152 > 65 was used as qualifier ions. The SRM transition 156 > 114 was used for IS (acetaminophen-D4). All experiments were conducted in positive ion mode.
9
UPLC-SRM Analysis of GDC-0152 in Brain Tumor Tissue
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Two pools of mice grafted with U87MG-iRFP and demonstrating brain tumors were used. Twenty sections from FFPE tissue blocks of GDC-0152 treated mice and of DMSO control mice were deparaffinized by incubating with 1 ml of xylen for 10 min at room temperature followed by centrifugation at 20 000 × g for 5 min. The supernatant was collected and transferred to a clean Eppendorf tube. The extract was dried in a concentrator. The dry extracts was then suspended in 20 μl of acetonitrile/0.1% formic acid (50 : 50).
UPLC-SRM was performed on a Nexera LC system (Shimadzu Corporation, Tokyo, Japan) coupled to a Triple quadripole 8040 mass spectrometry system (Shimadzu Corporation). Separation of GDC-0152 was carried out using a Kinetex XB-C18 column (150 × 2.1 mm), 2.6 μm particle size, Phenomenex with a column temperature of 30 °C. Elution was obtained by a linear gradient from 5 to 50% of acetonitrile in 0.1% formic acid over 6 min at a flow rate of 0.4 ml/min. Next, 5 μl of the extract was injected on the UPLC-SRM. After automatic optimization of collision energies for GDC-0152, the transitions were set to 499.2–>275 and 499.2–>70.1. Data acquisition and analysis were performed using Labsolution v5.6 software from Shimadzu Corporation.
UPLC-SRM was performed on a Nexera LC system (Shimadzu Corporation, Tokyo, Japan) coupled to a Triple quadripole 8040 mass spectrometry system (Shimadzu Corporation). Separation of GDC-0152 was carried out using a Kinetex XB-C18 column (150 × 2.1 mm), 2.6 μm particle size, Phenomenex with a column temperature of 30 °C. Elution was obtained by a linear gradient from 5 to 50% of acetonitrile in 0.1% formic acid over 6 min at a flow rate of 0.4 ml/min. Next, 5 μl of the extract was injected on the UPLC-SRM. After automatic optimization of collision energies for GDC-0152, the transitions were set to 499.2–>275 and 499.2–>70.1. Data acquisition and analysis were performed using Labsolution v5.6 software from Shimadzu Corporation.
10
Lipid Profiling by Charged Surface Hybrid HPLC-MS
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An Acquity charged surface hybrid technology C18 column (2.1 × 100 mm, 1.7 μm) and Acquity VanGuard charged surface hybrid technology C18 pre-column (2.1 × 5 mm, 1.7 μm) were used for lipid separation with a binary gradient elution as described in Supplementary Table S2 . A Shimadzu Nexera LC system (Kyoto, Japan) was utilized for the experiment. Lipid extracts were resuspended in methanol/toluene (9:1, v/v) and kept at 4 °C in an autosampler. The injected volume was ion-mode- and data-acquisition-dependent. From 200 μL of resuspended volume, 1 μL (scan profiling) and 2 μL (information-dependent acquisition and SWATH-based data-independent acquisition) were injected in positive ion mode; 3 μL (scan profiling) and 6 μL (information-dependent acquisition and SWATH) were injected in negative ion mode. The separated lipid ions were analyzed using an X500R QTOF with a Turbo V™ ion source with a TwinSpray probe (SCIEX, MA, USA). For the tandem MS analyses, either 45 eV (spread of 15 eV) or 25 eV (spread of 15 eV) were used. The MS parameters are shown in Supplementary Table S3 . Mass calibration was automatically performed after every fifth injection through the instrument’s CDS system, using X500R positive or negative calibration solutions.
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