Agilent Measurement Journal - Issue 3 - 2007 - (Page 61)

in that it is optimized for homogenizing as little as 20 mg of material in a single-phase solvent and enables analysis by both LC/MS and GC/MS instruments. Analyzing extracted metabolites Figure 6A summarizes the instrument conditions for LC/TOF MS on an Agilent 1200 LC equipped with a ZORBAX SB-Aq column (2.1 x 150 mm), which was used to separate the rice extracts. An Agilent 6210 TOF LC/MS equipped with an ESI source was used to acquire proﬁling data. Figure 6B shows the conditions for LC/Q-TOF MS, which was performed on an Agilent 6510 Q-TOF LC/MS equipped with an ESI source and was used to acquire accurate mass MS/MS data for metabolite identiﬁcation. Examining the data analysis workﬂow Initial processing of the accurate-mass LC/TOF MS proﬁling data was done using MassHunter software (Figure 7). The feature extraction and correlation algorithms located all the co-variant ions in a TIC. Background was subtracted and charge state was set to 1. The algorithm identified the monoisotopic mass and retention time and calculated an empirical formula for each feature. This information was imported into GeneSpring MS software for subsequent statistical analysis. The workﬂow is summarized in Figure 8. Features were aligned and normalized and then checked for reproducibility of the sets of biological replicates in each class (condition) using hierarchical clustering analysis. To identify features with differential abundances across the different classes, we applied analysis of B Instrument conditions — LC/Q¯TOF MS/MS LC conditions Column: ZORBAX SB-Aq column 2.1 x 150 mm, 3.5 µm Mobile phase: A = 0.1% formic acid in water B = 0.1% formic acid in acetonitrile Gradient: 2% B at 0 min 98% B at 46 min 98% B at 54.9 min 2% B at 55min MS stop time: 54.9 min LC stop time: 55 min LC post time: 7 min Column temperature: 20° C Flow rate: 0.3 mL/min Injection volume: 2 µL MS conditions Ionization mode: Electrospray Ionization polarity: Positive ionization Drying gas ﬂow: 10 L/min Drying gas temperature: 250° C Nebulizer pressure: 40 psig Scan range MS: m/z 100-1000 at 250 ms/spectrum MS/MS: m/z 100-1000 at 250 ms/spectrum Collision energy: 5 x +10eV Isolation: medium Fragmentor voltage: 170 V Skimmer voltage: 65 V Octopole RF voltage: 750 V Capillary voltage: 4000 V Reference masses: m/z 121, 922 Reference mass ﬂow: 10 µL/min Reference nebulizer pressure: 15 psig A Instrument conditions — LC/TOF MS LC conditions Column: ZORBAX SB-Aq column 2.1 x 150 mm, 3.5 µm Mobile phase: A = 0.1% formic acid in water B = 0.1% formic acid in acetonitrile Gradient: 2% B at 0 min 98% B at 46 min 98% B at 54.9 min 2% B at 55min MS stop time: 54.9 min LC stop time: 55 min Column temperature: 20° C Flow rate: 0.3 mL/min Injection volume: 2 µL + 3 sec ﬂush MS conditions Ionization mode: Electrospray Ionization polarity: Positive ionization Drying gas ﬂow: 10 L/min Drying gas temperature: 250° C Nebulizer pressure: 35 psi Scan range: m/z 50-950 Fragmentor voltage: 170 V Capillary voltage: 4000 V Reference masses: m/z 121, 922 Reference mass ﬂow: 10 µL/min Figure 6. Summarizing the chromatographic and MS conditions used in the study, LC/TOF MS (A) was used for proﬁling and ﬁnding differential features and LC/Q-TOF MS (B) was used for identiﬁcation of metabolites from a targeted inclusion list of ions that was generated by LC/TOF MS. Agilent Measurement Journal 61

Table of Contents Feed for the Digital Edition of Agilent Measurement Journal - Issue 3 - 2007

Applying Ingenuity to the Measurement of Emerging Technologies Contents Emerging Innovations Department Delivering Bigger Benefits by Optimizing Customer Workflows Measuring Material Properties at the Nanoscale Utilizing In Situ Atomic Force Microscopy in Life Science, Pharmaceutical and other Bio-Related Applications WiMAX™: Plotting a New Path to Global Mobility Addressing the Triple Complexity of Triple-Play Networks What Next for Mobile Telephony? Exploring the Inner Workings of Tire-Pressure Monitoring Systems Developing, Assessing and Applying a High-Resolution Thin-Film Magnetic Probe Making Accurate Settling-Time Measurements Using a Vector Network Analyzer Applying Metabolomics Methods to the Study of Bacterial Leaf Blight in Rice Plants Measuring Stream Dynamics with Fiber Optics survey

Agilent Measurement Journal - Issue 3 - 2007

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