Agilent Measurement Journal - Issue 3 - 2007 - (Page 64)

Developing new separation methods for metabolites One outcome of the rice metabolomics study was to identify gaps in our analytical workﬂow. Our C18 ZORBAX SB-Aq LC column had limited capacity to bind and separate polar metabolites. We also encountered signiﬁcant difﬁculties with GC separation and analysis of derivatized metabolites, resulting in MS source fouling and reduced column longevity. To address these issues, two new chromatographic separation methods are being developed. One uses a novel type-C silica material for separating metabolites by aqueous normal phase (ANP) LC that can be coupled to different sources for MS detection. The other builds a new metabolite derivatization module into the injection port of a GC oven, improving the analysis of labile metabolites by GC/MS. Binding and resolving polar metabolites At Agilent, we have recently been investigating new chromatographic materials for LC/MS analysis that can bind and resolve polar metabolites. Many endogenous metabolites are highly polar and unretained on standard reverse-phase high-pressure LC (HPLC) columns, even in a 100 percent aqueous mobile phase. Chromatographers have tried many approaches to solving this problem but only hydrophilic interaction chromatography (HILIC) can possibly address the chemical diversity of hydrophilic metabolites. Unfortunately, the available HILIC materials have three important limitations: They are slow to re-equilibrate; the chromatography has poor reproducibility; and they require high levels of salts or buffers, which can cause problems in metabolomics analysis. Type-C silica (Figure 10) is an ANP stationary phase with HILIC-like retention but without these disadvantages. The principle of ANP is analogous to that found in normal-phase chromatography but the mobile phase has water as part of the binary solvent. “Normal phase” implies that retention is greatest for polar solutes such as acids and bases. We used amino acids to demonstrate the utility of ANP to separate polar metabolites. Figure 11 shows the extracted ion chromatograms (EICs) for a mixture of 19 amino acids that were separated on a silica-hydride surface based on the polar properties of the individual amino acids. These would otherwise be unretained on a standard C18 column. All of the critical amino acid pairs — those that are isobaric or have masses within one mass unit — were separated under the gradient condition that was used (except for the leucine/isoleucine pair). Additional gradient formats and mobile phases are under investigation to address this issue. Table 3 summarizes the reproducibility of retention times for nine amino acids separated approximately between eight and 12.2 minutes at two temperatures, 15° C and 30° C. Four replicates were performed at each temperature. The reproducibility was 0.28 percent or better for the amino acids, an excellent result representing a signiﬁcant improvement over HILIC analyses. Improving the analysis of labile metabolites Many scientists who are proﬁcient — but not expert — users of GC/MS desire a turnkey solution that includes in situ (rather than ofﬂine) derivatization of the sample in the instrument. For in situ derivatization to be successful, it must be performed in the heated GC injection port. However, the sample solvent, derivatization agents and samples can contain a number of unwanted components that reduce sample throughput by degrading column or x103 5 Abundance 4 3 2 1 0 50 60 70 •CH2 56.05052 H •N + -CO 84.04484 H +N MS/MS spectrum of precursor ion m/z 130.0532 H +NH O C OH O -CH2O2 O 130.05320 (measured) 80 90 100 110 Mass-to charge ratio (m/z) 120 130 140 Figure 9. In this metabolite identiﬁcation, the MS/MS spectrum of the precursor ion at m/z 130.0532 shows a base peak representing the loss of formic acid (CH2O2) and a peak representing a subsequent loss of CO. Evaluation of this information against the structures of the six possible identities generated by a search of the Metlin database reduced the list of possible identities to two. 64 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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