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A liquid chromatography tandem mass spectrometry method (LC MS/MS) was developed for determining the twelve analytes using the multiple reaction monitoring mode (MRM).
Samples were homogenized in chloroform:methanol (1 2 (v/v)) and analyzed by direct flow injection on a triple-quadrupole mass spectrometer (API 4500 QTRAP MS/MS; Sciex Applied Biosystems, Toronto, ON, Canada) in the positive ionization mode using the multiple reaction monitoring (MRM) method.
Data were acquired using the multiple reaction monitoring (MRM) scan mode.
When coupled to a triple quadrupole tandem mass spectrometer (QQQ-MS/MS), it can achieve high sensitivity and selectivity by using the multiple reaction monitoring (MRM) scan mode without the baseline chromatographic separation of target analytes.
The elutes were used for HPLC-ESI-MS/MS separation in a HPLC (Agilent 1200, Agilent Technologies, CA) and then quantitation in a hybrid triple quadrupole/linear ion trap mass spectrometer (ABI 4000 Q-Trap, Applied Biosystems, CA) using the multiple reaction monitoring (MRM) and information dependent acquisition (IDA) mode.
Quantification was performed using the multiple reaction monitoring (MRM) mode, on the basis of parent → product ion transitions for HCTL (118.2 → 56) and homatropine (276.1 → 142).
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The mass spectrometer was operated using the multiple reaction-monitoring mode, and the analytes were detected and quantified using the most abundant transitions obtained during direct infusion of standards.
Electrospray ionization (ESI) interface operated in positive ionization mode was used for the multiple reaction monitoring (MRM).
Liquid chromatography was performed at 30°C using an Acquity UPLC BEH C18, 1.7 μm, 2.1 × 100 mm column (Waters, Milford, MA), and the Micromass Quattro Premier XE Tandem Mass Spectrometer (Waters) was used in the multiple reaction monitoring mode in the electrospray ionization positive mode.
Analysis was carried out using the dynamic multiple reaction monitoring mode and fast polarity switching.
The targeted mass spectrometry analysis was done using the MIDAS (Multiple Reaction Monitoring (MRM) initiated detection and sequence analysis) workflow [ 15] selecting a minimum of 3 peptides per protein, and three transitions per peptide based on information from in silico digestion.
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