Extracted samples (60 μl) were analysed on a Waters HPLC 2695 Separation Module using a Multospher 120 C18 column 125 × 2 mm, 5-μm particle size (CS-Chromatographie Service GmbH, Langerwehe, Germany) coupled with a guard column (20 × 2 mm, 5-μm particle size). The ceramides were resolved using a gradient starting from 6% mobile phase A (water containing 0.2% formic acid) at a flow rate of 0.15 ml/min to 100% mobile phase B (acetonitrile/2-propanol [60:40, v/v] containing 0.2% formic acid) over 15 min at a linear gradient, and then with 100% phase B for 5 min. The column was then reequilibrated for 5 min with 96% mobile phase B. The HPLC column effluent was introduced into a Micromass Quattro MicroTM API mass spectrometer (Waters, Milford, MA, USA) and analyzed using electrospray ionization in the positive mode and a single ion monitoring (SIM) modus: m/z 520.5 for C16-ceramide, m/z 575.5 for the C17-ceramide internal standard. The mass spectrometer and source parameters were set up as described previously [79 (link)]: capillary voltage, 3.0 kV; cone voltage, 40 V; source temperature, 120°C; desolvation temperature, 250°C, flow rate of desolvation gas, 700 l/h. Dwell and delay times were 0.1 and 0.05 s, respectively. The data were acquired using MassLynx software (version 4.1, Micromass Ltd, Manchester, UK). Ceramide levels were normalized to cellular protein.
Micromass quattro micro api mass spectrometer
Manufactured by Waters Corporation
Sourced in United States
The Micromass Quattro Micro API mass spectrometer is a high-performance liquid chromatography-mass spectrometry (LC-MS) system designed for analytical applications. It features a triple quadrupole mass analyzer that provides accurate mass measurements and sensitive detection of a wide range of analytes.
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Lab products found in correlation
Acquity uplc system, by Waters Corporation (2 mentions)
Ethylene bridged hybrid beh c18 column, by Waters Corporation (1 mentions)
Acquity uplc t3 c18 column, by Waters Corporation (1 mentions)
Masslynx v4, by Waters Corporation (1 mentions)
Acquity uplc separation module, by Waters Corporation (1 mentions)
Masslynx nt4, by Waters Corporation (1 mentions)
7 protocols using micromass quattro micro api mass spectrometer
1
Ceramide Quantification by HPLC-MS
2
HPLC-MS Analysis of YQFM Injection
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The preparation of YQFM injection and the method of HPLC-MS analysis were conducted as reported previously (Li et al., 2015 (link)). Triple-quadrupole tandem mass spectrometric detection was carried out on a Micromass Quattro microTM API mass spectrometer (Waters Corp, Milford, CT, United States) with an electrospray ionization (ESI) interface. The mobile phase consisted of acetonitrile (A) and water-0.1% acetic acid (B), and the gradient elution conditions were: 0–30 min, 8–18% A; 30–80 min, 18–50% A; 80–115 min, 50–100% A and then returned to the initial condition. The flow rate was 1.0 mL/min. Chromatographic separation was carried out at 30°C on an Alltima C18 column (4.6 mm × 250 mm, I.D., 5 μm, Serial No. 213040116, GRACE-Alltech, United States).
3
UPLC-MS/MS Metabolite Profiling Protocol
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Liquid chromatography was performed on a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA). Separation was achieved with a Waters ethylene-bridged hybrid (BEH) C18 column (50 mm × 2.1 mm, 1.7 μm) held at 30°C. Gradient elution with a mobile phase composed of water and acetonitrile with 0.1% formic acid was performed at a flow rate of 0.3 mL/min (Table 1 ).
Mass spectrometric detection was performed on a Micromass Quattro Micro API mass spectrometer (Waters Corp.) with an electrospray ionization interface and triple quadrupole mass analyzer. The electrospray ionization source was set in the positive mode. The following parameters were used: capillary voltage, 3.2 kV; cone voltage, 30 V; source temperature, 120°C; and desolvation temperature, 300°C. Nitrogen was used for desolvation and as the cone gas at flow rates of 600 L/h and 50 L/h, respectively. The full scan mode was used in a mass range of 100–1,000 amu. For MS/MS, argon was used as the collision gas, and the collision energy was set according to the metabolite composition. NaCsI was used for mass correction before the experiment was commenced. Data were collected in centroid mode.
Mass spectrometric detection was performed on a Micromass Quattro Micro API mass spectrometer (Waters Corp.) with an electrospray ionization interface and triple quadrupole mass analyzer. The electrospray ionization source was set in the positive mode. The following parameters were used: capillary voltage, 3.2 kV; cone voltage, 30 V; source temperature, 120°C; and desolvation temperature, 300°C. Nitrogen was used for desolvation and as the cone gas at flow rates of 600 L/h and 50 L/h, respectively. The full scan mode was used in a mass range of 100–1,000 amu. For MS/MS, argon was used as the collision gas, and the collision energy was set according to the metabolite composition. NaCsI was used for mass correction before the experiment was commenced. Data were collected in centroid mode.
4
UPLC-MS/MS Analysis of Compounds
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Analyses were performed on an ultraperformance liquid chromatography (UPLC) system (Waters Corp., Milford, MA, USA). An ACQUITY UPLC T3 C18 column (2.1 mm × 100 mm, internal diameter (i.d.), 1.8 μm) from Waters was used. The column temperature was maintained at 30°C. The standards and samples were separated using a gradient mobile phase consisting of water and acetonitrile with 0.1% formic acid. The flow rate was at 0.4 mL/min. The injection volume of sample was 2 μL. Mass spectrometry was carried out on a Micromass Quattro Micro API mass spectrometer (Waters Corp.) using an electrospray ionization source. The temperatures of source and desolvation were 120°C and 300°C, respectively. The flow rate of desolvation gas was set at 600 L/h.
5
LC-ESI-MS Analysis of Metabolites
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LC-ESI-MS analysis was performed on an ACQUITY UPLC separation module coupled with a Micromass Quattro Micro API mass spectrometer (Waters, Milford, MA, USA). The column was a Luna 5 μ C18 (150 × 4.6 mm) with a security guard cartridge (3.0 × 4.6 mm) and the mobile phases consisted of 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). The flow rate was 0.3 mL/min. The gradient method was the same as previously described for HPLC-PDA analysis. ESI-MS detection was performed under negative ion mode under the following conditions: capillary voltage: −3 kV; cone voltage: −50 V; source temperature: 120°C; desolvation temperature: 350°C; cone gas flow: 50 L/h; desolvation gas flow: 600 L/h. MS data collected were processed using MassLynx v.4.1 software (Waters).
6
Quantitative Analysis of Adagrasib in Biological Samples
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The concentrations of adagrasib in plasma and tissue homogenate were determined by Waters ACQUITY UPLC system (Waters, Milford, MA, USA) and Micro mass Quattro Micro API mass spectrometer (Waters, Milford, MA, USA). The electrospray ionization source interface was operated in positive mode. The capillary voltage was 3.0 kV, the source temperature was 150°C, and the desolvation temperature was 400°C. Desolvation gas flow rate was 800 L/h. The collision gas flow rate was 0.17 mL/min. The MS/MS parameters for adagrasib and Lapatinib-D4 are listed in Table 1 . The fragmentation patterns for adagrasib and the Lapatinib-D4 are shown in Figure 2 .
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Optimization of Mass Parameters of Adagrasib and Lapatinib-D4
| Compound | Precursor Ion (m/z) | Daughter Ion (m/z) | Cone Energy (V) | Collision Energy (V) |
|---|---|---|---|---|
| Adagrasib | 604.22 | 97.98 | 18 | 14 |
| Lapatinib-D4 (IS) | 585.21 | 365.10 | 50 | 35 |
Mass fragmentation pattern of adagrasib (
7
Quantitative Analysis of Trantinterol Metabolites
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The separation system consisted of an ACQUITY™ UPLC system (Milford, MA, USA), and an ACQUITY UPLC C8 BEH column (2.1 mm × 50 mm, 1.7 μm) was used for the chromatographic separation. The mobile phase consisted of methanol-0.2% formic acid (20 : 80, v/v) at a flow rate of 0.25 mL min−1 in an isocratic mode of elution.
A Waters Micromass® Quattro micro™ API mass spectrometer ((Milford, MA, USA)) equipped with an electrospray ionization (ESI) source was used for detection and the data was acquired and processed using MassLynx™ NT 4.1 software (Waters, Milford, MA, USA). The ESI source was operated in positive ionization mode, and the MS parameters were optimized with the capillary voltage set at 1.0 kV and source temperature set at 110 °C. Nitrogen was used as desolvation and cone gas with the flow rate at 500 and 30 L h−1, respectively. Quantification was performed using multiple reaction monitoring (MRM) mode with the following transitions: m/z 327 → m/z 254 for 4-hydroxylamine trantinterol (M1), m/z 327 → m/z 238 for tert-butyl hydroxylated trantinterol (M2) and m/z 277 → m/z 203 for the internal standard clenbuterol. The optimized cone voltages and collision energies for M1, M2 and IS were 15, 12, 12 V and 15, 14, 12, 12 eV, respectively.
A Waters Micromass® Quattro micro™ API mass spectrometer ((Milford, MA, USA)) equipped with an electrospray ionization (ESI) source was used for detection and the data was acquired and processed using MassLynx™ NT 4.1 software (Waters, Milford, MA, USA). The ESI source was operated in positive ionization mode, and the MS parameters were optimized with the capillary voltage set at 1.0 kV and source temperature set at 110 °C. Nitrogen was used as desolvation and cone gas with the flow rate at 500 and 30 L h−1, respectively. Quantification was performed using multiple reaction monitoring (MRM) mode with the following transitions: m/z 327 → m/z 254 for 4-hydroxylamine trantinterol (M1), m/z 327 → m/z 238 for tert-butyl hydroxylated trantinterol (M2) and m/z 277 → m/z 203 for the internal standard clenbuterol. The optimized cone voltages and collision energies for M1, M2 and IS were 15, 12, 12 V and 15, 14, 12, 12 eV, respectively.
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