Analytical strategies for LC-MS-based targeted metabolomics

Department of Chemistry, Princeton University, Princeton, New Jersey, United States
Journal of Chromatography B (Impact Factor: 2.73). 05/2008; 871(2):236-42. DOI: 10.1016/j.jchromb.2008.04.031
Source: PubMed


Recent advances in mass spectrometry are enabling improved analysis of endogenous metabolites. Here we discuss several issues relevant to developing liquid chromatography-electrospray ionization-mass spectrometry methods for targeted metabolomics (i.e., quantitative analysis of dozens to hundreds of specific metabolites). Sample preparation and liquid chromatography approaches are discussed, with an eye towards the challenge of dealing with a diversity of metabolite classes in parallel. Evidence is presented that heated electrospray ionization (ESI) generally gives improved signal compared to the more traditional unheated ESI. Applicability to targeted metabolomics of triple quadrupole mass spectrometry operating in multiple reaction monitoring (MRM) mode and high mass resolution full scan mass spectrometry (e.g., time-of-flight, Orbitrap) are described. We suggest that both are viable solutions, with MRM preferred when targeting a more limited number of analytes, and full scan preferred for its potential ability to bridge targeted and untargeted metabolomics.

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    • "The combination of SPE followed by LC-MS analysis is routinely used for metabolomics (Cheng and Mok 2004; Lu et al. 2008; Zhou et al. 2012). A similar procedure has been used to successfully quantify opioids and benzodiazepines from patient urine samples, using a triple quadrupole mass spectrometer. "

    12/2015; 6(1). DOI:10.1186/s40543-015-0057-2
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    • "compounds highly sensitive to oxidation or de-amidation). The finding of complex, compound-specific responses of organic N monomers to extraction method is consistent with studies of microbial cultures reporting that extraction methods are not universal but instead need to be tested for specific compound classes and purposes (Mashego et al., 2007; Lu et al., 2008; Winder et al., 2008). "
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    ABSTRACT: One way of investigating the function of soil is via the pool of low molecular weight organic compounds in the soil microbial biomass. This is because low molecular weight organic compounds have key roles in metabolism of soil microbes, can function in osmotic adjustment and other stress responses, and are intermediates in the breakdown of polymers to inorganic nutrients. Methods for measuring low molecular weight microbial metabolites in soil rely upon extracting total metabolites and then subtracting the contribution from metabolites in the soil extracellular matrix (i.e. microbial = total − extracellular). Recent studies have tested methods for extracting organic N monomers from the extracellular matrix of soil, but there has not been similar testing of methods for extracting total organic N monomers. The aims of this study were to examine methods for extracting total organic N monomers by a) contrasting chloroform gas fumigation with chloroform direct extraction, and b) examining whether it is possible to extract soil with two methods that combine quenching of metabolic activity with extraction, namely cold methanol/chloroform/water and hot aqueous ethanol. To evaluate methods, organic N compounds were extracted from soil and then capillary electrophoresis–mass spectrometry identified and quantified 42 organic N monomers including amino acids, quaternary ammonium compounds, nucleobases and nucleosides, amines and polyamines. Absolute concentrations of 32 out of the 42 quantified organic N monomers were significantly different between soil extracted by chloroform gas fumigation and chloroform direct extraction. These differences were probably a function of gains and losses of compounds due to oxidation, hydrolysis and deamidation during the two-day chloroform gas fumigation. Cold methanol/chloroform/water yielded large amounts of the extremely labile compound ergothioneine, probably because the extraction method rapidly quenched metabolic activity. The primary limitation of extraction with methanol/chloroform/water is that it was ineffective at extracting strongly cationic compounds (e.g. polyamines). Extraction with hot aqueous ethanol was unsuccessful with soil presumably because soil microbes are difficult to lyse. It is recommended that future studies examining organic N monomers in soil microbial biomass use chloroform direct extraction or cold methanol/chloroform/water rather than chloroform gas fumigation.
    Soil Biology and Biochemistry 11/2014; 81. DOI:10.1016/j.soilbio.2014.11.005 · 3.93 Impact Factor
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    • "Two separation modes capable of LCeMS of hydrophilic compounds without ion pairing or derivatization are hydrophilic interaction liquid chromatography (HILIC) (Alpert et al., 1994) and aqueous normal phase liquid chromatography (Pesek and Matyska, 2007). In recent times LCeMS with HILIC separations have been used by numerous groups for analysis of complex mixtures of hydrophilic compounds (Lu et al., 2008; Kato et al., 2009; Schiesel et al., 2010; Creek et al., 2011; Iwasaki et al., 2011; Rappold and Grant, 2011; Boudra et al., 2012; Buszewski and Noga, 2012; Chen et al., 2012; Fraser et al., 2012; Zhang et al., 2012) but it is unclear if methods developed for other biological samples (e.g. animals, plants, microbial cultures) can be directly applied to soil. "
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    ABSTRACT: The aim of this study was to develop an analytical procedure based on liquid chromatography-mass spectrometry (LC–MS) for analysis of monomeric organic N compounds in soil extracts. To benchmark the developed LC–MS method it was compared with a capillary electrophoresis–mass spectrometry (CE–MS) method recently used for analysis of small organic N monomers in soil. The separation was optimized and analytical performance assessed with 69 purified standards, then the LC–MS method was used to analyse soil extracts. Sixty-two out of 69 standards were analysable by LC–MS with separation on a hydrophilic interaction liquid chromatography column. The seven compounds that could not be analysed were strongly cationic polyamines. Limits of detection for a 5 μL injection ranged between 0.002 and 0.38 μmol L−1, with the majority (49 out of 62) having limits of detection better than 0.05 μmol L−1. The overall profile and concentration of small organic N monomers in soil extracts was broadly similar between LC–MS and CE–MS, with the notable exception of four ureides that were detected by LC–MS only. In soil extracts that had been concentrated ten-fold the detection and quantification of (some) organic N compounds was compromised by the presence of large amounts of inorganic salts. The developed LC–MS method offered advantages and disadvantages relative to CE–MS, and a combination of the two methods would achieve the broadest possible coverage of organic N in soil extracts.
    Soil Biology and Biochemistry 11/2014; 78:233–242. DOI:10.1016/j.soilbio.2014.08.008 · 3.93 Impact Factor
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