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    Development of MRM Method - MRM and Acquity Water System

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    octavianistrate
    The document describes the development of an LC-MS/MS method using multiple reaction monitoring (MRM) to analyze compounds. It discusses optimizing the LC system using reverse phase chromatography with a C18 column, mobile phases of formic acid and methanol, and gradient elution. The mass spectrometer is operated in MRM mode to selectively monitor precursor-product ion transitions for targeted compounds to enable sensitive quantitation. Key parameters discussed are dwell time, duty cycle, and cycle time to ensure adequate sampling of chromatographic peaks.

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    Development of MRM Method - MRM and Acquity Water System

    Uploaded by

    octavianistrate
    142 views8 pages
    The document describes the development of an LC-MS/MS method using multiple reaction monitoring (MRM) to analyze compounds. It discusses optimizing the LC system using reverse phase chromatography with a C18 column, mobile phases of formic acid and methanol, and gradient elution. The mass spectrometer is operated in MRM mode to selectively monitor precursor-product ion transitions for targeted compounds to enable sensitive quantitation. Key parameters discussed are dwell time, duty cycle, and cycle time to ensure adequate sampling of chromatographic peaks.

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    mrm

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    Development of Mrm Method- Mrm and Acquity Water System

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    The document describes the development of an LC-MS/MS method using multiple reaction monitoring (MRM) to analyze compounds. It discusses optimizing the LC system using reverse phase chromatography with a C18 column, mobile phases of formic acid and methanol, and gradient elution. The mass spectrometer is operated in MRM mode to selectively monitor precursor-product ion transitions for targeted compounds to enable sensitive quantitation. Key parameters discussed are dwell time, duty cycle, and cycle time to ensure adequate sampling of chromatographic peaks.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    142 views8 pages

    Development of MRM Method - MRM and Acquity Water System

    Uploaded by

    octavianistrate
    The document describes the development of an LC-MS/MS method using multiple reaction monitoring (MRM) to analyze compounds. It discusses optimizing the LC system using reverse phase chromatography with a C18 column, mobile phases of formic acid and methanol, and gradient elution. The mass spectrometer is operated in MRM mode to selectively monitor precursor-product ion transitions for targeted compounds to enable sensitive quantitation. Key parameters discussed are dwell time, duty cycle, and cycle time to ensure adequate sampling of chromatographic peaks.

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    The document discusses the development of an MRM method for mass spectrometry analysis, including optimizing the LC system and UPLC conditions, using reverse phase chromatography, and key parameters of MRM such as dwell time, duty cycle, and cycle time.

    The LC system needs to be optimized to yield better separation and resolution of compounds being analyzed.

    Reverse phase chromatography (RP-LC) is being used, where the analyte partitions between a hydrophobic stationary phase and a polar mobile phase. RP-LC is useful for a range of analytes from moderately polar to rather hydrophobic.

    Development of an MRM method

    Previously optimized parameters (i.e. CE, DP, CXP,….etc) are used to construct the MRM
    method for mass spec analysis. The LC system is as equally important to be optimized to
    yield better separation and resolution.

    UPLC System (Acquity)


    • Binary Solvent Manager
    • Sample Manager
    • Column Manager
    • Detector

    Waters ACQUITY UPLC System


    The determination of optimum LC (Liquid Chromatography) is an important step in the
    development of LC/MRM method. For this demonstration, LC conditions has already been
    optimized and included in the method. Reverse Phase Chromatography (RP-LC) will be
    used in which the analyte partitions between a hydrophobic stationary phase and a polar
    mobile phase. Typical stationary phases are based on C18 hydrocarbon chains attached to
    silica particles through silyl-ether bonds: Si-O-CH2-R. The particle sizes in this application
    are 1.8 µm and permit much higher chromatographic resolution (UPLC). The mobile
    phase usually consists of acetonitrile-water mixtures or methanol-water mixtures.
    Reverse phase chromatography is useful for a range of analytes from moderately polar to
    rather hydrophobic.

    Column = Synergi hydro RP 2 X 250 mm


    Mobile Phase A: 0.1% Formic Acid
    Mobile Phase B: MeOH + 0.1 % Formic Acid
    Gradient: 0 min = 5% B
    8 min = 100% B
    9 min = 5% B
    12 min = Stop
    Flow Rate: 250 µl/min
    Column Temp: 55oC

    Line from UPLC

    UPLC Column

    Line from column to inlet


    MRM Analysis
    of mass spectrometer
    LC/MS/MS Operated in Multiple Reaction Monitoring (MRM)

    Fixed precursor ion Fragmentation Scanning product ion

    Features:
    • Selectivity and sensitivity for quantitation of targeted compounds
    • Simultaneous multiple compound identification

    Selected Reaction Monitoring (SRM) for one compound


    Key Parameters of Multiple Reaction Monitoring (MRM)

    • Dwell Time: Dwell time is the time spent acquiring a specific MRM transition during
    each cycle. A very short dwell times can be used (5 ms or less). However, longer dwell
    times are always desirable for better signal/noise and sensitivity,

    • Duty Cycle: Duty cycle is the total amount of time spent monitoring the chosen
    analytes. If chromatographic resolution requires a particular duty cycle time, then the
    dwell time for each analyte measured in the duty cycle is inversely related to the
    number of analytes. For example, if the duty cycle is 500 msec and there are 20 analytes
    to measure, the dwell time will be 25 msec.

    • Cycle Time: The duty cycle time for an MRM assay must take into consideration
    chromatographic peak shape. Ideally, the peak must be sampled 8-10 times as it is
    eluted to get an accurate measurement of its area. So, if a peak is 10 sec wide, then the
    sample time would be every 1 sec. For 25 msec dwell times, then 40 MRM transitions
    can be monitored.
    Ten to fifteen data points’ collection across the peak is
    considered standard for MRM.

    A large number of mass transitions can decrease sensitivity.

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