Agilent Measurement Journal - Issue 3 - 2007 - (Page 58)

The protocol must be reproducible and take into account the distribution of metabolites that are greatly affected by the extraction method. Therefore, variability between samples due to processing methodology must be minimized where possible. This includes numerous factors: methods for quenching metabolic turnover to prevent degradation or interconversion of metabolites; selection of appropriate extraction solvents and cosolvents, and adjustment of pH for optimal extraction; minimizing metabolite oxidation; sample storage temperature; protein precipitation methods; and processing-time considerations. Analytical reproducibility is very important for expression proﬁling. The combination of inherent biological sample variability and technical analytical variability determines the number of sample replicates required to verify the existence of statistically significant differences between sample sets. The smaller the analytical variance, the fewer replicates are required. Technical variability can be signiﬁcantly minimized by including appropriate internal standards along different parts of the extraction and analytical workﬂows. are gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), nuclear magnetic resonance (NMR) and capillary electrophoresis/mass spectrometry (CE/MS). Figure 2 shows some of the chromatographic and detection techniques that are included in the Agilent metabolomics laboratory for proﬁling, identiﬁcation and validation: GC/MS, LC, time-of-flight (TOF), quadrupole TOF (Q-TOF) and triple quadrupole (QQQ). GC/MS offers the highest routine separation power (great analyte peak capacity) and potential sample identiﬁcation with the use of electron impact (EI) ionization libraries. EI ionization has one big advantage relative to LC/MS ionization techniques: there is no ionization suppression effect. However, GC/MS analysis has four signiﬁcant disadvantages: • Most metabolites are nonvolatile and need to be chemically derivatized to become volatile prior to GC/MS analysis. • Some metabolites are not volatile even after derivatization and hence cannot be analyzed. • Thermally unstable metabolites lead to variable quantitation. Outlining analytical approaches for metabolomics Many methods can be used for metabolite proﬁling, and each one has advantages and disadvantages. Consequently, a combination of analytical techniques is often used to provide a more comprehensive view of the metabolome. The combinations used most often • They generally lack a molecular ion, which is extremely helpful in compound identiﬁcation. In contrast, LC/MS has the ability to analyze almost anything without the need for prior derivatization. This is because it operates in many ionization modes such as electrospray ionization (ESI) and 1200 LC Separation QQQ MS/MS quantification GC/MS quadrupole Separation, identification and quantification of volatile compounds Q-TOF Profiling and MS/MS accurate mass indentification TOF MS for accurate mass and profiling Figure 2. Agilent offers a complete set of instrumentation for metabolmics analysis.3 58 Agilent Measurement Journal

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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