Tandem LC columns for the simultaneous retention of polar and nonpolar molecules in comprehensive metabolomics analysis

Chalcraft, Kenneth R.; McCarry, Brian E.

doi:10.1002/jssc.201300779

Cited by 49 publications

(26 citation statements)

References 46 publications

(47 reference statements)

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“…One approach has been to couple columns of orthogonal stationary phases directly in series so that the one column follows the other with either a single LC pump or two LC pumps (Chalcraft and McCarry 2013;Greco et al 2013). These methods have been shown to successfully fractionate both polar and non-polar metabolites in series and have been applied to metabolomics research (Chalcraft et al 2014;Rajab et al 2013).…”

Section: Introductionmentioning

confidence: 99%

A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

et al. 2015

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Liquid chromatography-mass spectrometry (LC-MS) methods using either aqueous normal phase (ANP) or reversed phase (RP) columns are routinely used in small molecule or metabolomic analyses. These stationary phases enable chromatographic fractionation of polar and non-polar compounds, respectively. The application of a single chromatographic stationary phase to a complex biological extract results in a significant proportion of compounds which elute in the non-retained fraction, where they are poorly detected because of a combination of ion suppression and the co-elution of isomeric compounds. Thus coverage of both polar and nonpolar components of the metabolome generally involves multiple analyses of the same sample, increasing the analysis time and complexity. In this study we describe a novel tandem in-line LC-MS method, in which compounds from one injection are sequentially separated in a single run on both ANP and RP LC-columns. This method is simple, robust, and enables the use of independent gradients customized for both RP and ANP columns. The MS signal is acquired in a single chromatogram which reduces instrument time and operator and data analysis errors. This method has been used to analyze a range of biological extracts, from plant and animal tissues, human serum and urine, microbial cell and culture supernatants. Optimized sample preparation protocols are described for this method as well as a library containing the retention times and accurate masses of 127 compounds.

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Section: Introductionmentioning

confidence: 99%

A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

et al. 2015

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“…Therefore, since HILIC can simultaneously separate polar and lipid metabolites, it was selected as the chromatographic method for high-throughput comprehensive metabolomic analyses. RPLC is often used to retain and separate nonpolar analytes [34]. Separation of polar compounds can also be achieved with RPLC with an ion-pairing agent in the mobile phase [35]; however, the ion-pairing reagents often lead to contamination in the MS instrument [36] and, therefore, were not preferred.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive and simultaneous coverage of lipid and polar metabolites for endogenous cellular metabolomics using HILIC-TOF-MS

2014

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The comprehensive metabolomic analyses using eukaryotic and prokaryotic cells are an effective way to identify biomarkers or biochemical pathways which can then be used to characterize disease states, differences between cell lines or inducers of cellular stress responses. One of the most commonly used extraction methods for comprehensive metabolomics is the Bligh and Dyer method (BD) which separates the metabolome into polar and nonpolar fractions. These fractions are then typically analysed separately using hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) liquid chromatography (LC), respectively. However, this method has low sample throughput and can also be biased to either polar or nonpolar metabolites. Here, we introduce a MeOH/EtOH/H2O extraction paired with HILIC-time-of-flight (TOF)-mass spectrometry (MS) for comprehensive and simultaneous detection of both polar and nonpolar metabolites that is compatible for a wide array of cellular species cultured in different growth media. This method has been shown to be capable of separating polar metabolites by a HILIC mechanism and classes of lipids by an adsorption-like mechanism. Furthermore, this method is scalable and offers a substantial increase in sample throughput compared to BD with comparable extraction efficiency. This method was able to cover 92.2 % of the detectable metabolome of Gram-negative bacterium Sinorhizobium meliloti, as compared to 91.6 % of the metabolome by a combination of BD polar (59.4 %) and BD nonpolar (53.9 %) fractions. This single-extraction HILIC approach was successfully used to characterize the endometabolism of Gram-negative and Gram-positive bacteria as well as mammalian macrophages.FigureThe extraction and ionization efficiency of MeOH/EtOH/H2O HILIC approach encompasses both the polar and nonpolar fractions from Bligh and Dyer extractionElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-7797-5) contains supplementary material, which is available to authorized users.

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“…al (40)), optimization of analytical gradients or the use of ion mobility MS to increase chemical coverage, and application of spectral de-convolution software such as CAMERA (52) or xMSannotator (K Uppal, DI Walker, DP Jones, unpublished) to identify isotopes and adducts associated with molecular ions. Application of complementary analytical methodologies and spectral de-convolution software have greatly increased chemical detection, the confidence of chemical matches and reduced chemical noise and artifacts from MS analysis (22, 46, 52, 53). …”

Section: Discussionmentioning

confidence: 99%

High-Resolution Metabolomics Assessment of Military Personnel

Liu

Walker

Uppal

et al. 2016

Journal of Occupational &Amp; Environmental Medicine

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Objective To maximize detection of serum metabolites with high-resolution metabolomics (HRM). Methods Department of Defense Serum Repository (DoDSR) samples were analyzed using ultra-high resolution mass spectrometry with three complementary chromatographic phases and four ionization modes. Chemical coverage was evaluated by number of ions detected and accurate mass matches to a human metabolomics database. Results Individual HRM platforms provided accurate mass matches for up to 58% of the KEGG metabolite database. Combining two analytical methods increased matches to 72%, and included metabolites in most major human metabolic pathways and chemical classes. Detection and feature quality varied by analytical configuration. Conclusions Dual chromatography HRM with positive and negative electrospray ionization provides an effective generalized method for metabolic assessment of military personnel.

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Tandem LC columns for the simultaneous retention of polar and nonpolar molecules in comprehensive metabolomics analysis

Cited by 49 publications

References 46 publications

A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

Comprehensive and simultaneous coverage of lipid and polar metabolites for endogenous cellular metabolomics using HILIC-TOF-MS

High-Resolution Metabolomics Assessment of Military Personnel

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