Serially coupling hydrophobic interaction and reversed-phase chromatography with simultaneous gradients provides greater coverage of the metabolome

Haggarty, Jennifer; Oppermann, Madalina; Dalby, Matthew J.; Burchmore, Richard; Cook, Ken; Weidt, Stefan; Burgess, Karl

doi:10.1007/s11306-014-0770-7

Cited by 36 publications

(15 citation statements)

References 14 publications

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“…Due to reduced volumetric flow rates and increased ionization efficiency, cLC couples particularly well with ESI-MS. 1–4 This has led to cLC’s dominance in the fields of proteomics, 5–10 metabolomics, 3,7,11–16 and in vivo determination of neuropeptides. 17–21 Combined with gradient elution, peak capacities exceeding 800 22,23 have been achieved.…”

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confidence: 99%

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

2016

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Capillary HPLC (cLC) with gradient elution is the separation method of choice for the fields of proteomics and metabolomics. This is due to the complementary nature of cLC flow rates and electrospray or nanospray ionization mass spectrometry (ESI-MS). The small column diameters result in good mass sensitivity. Good concentration sensitivity is also possible by injection of relatively large volumes of solution and relying on solvent-based solute focusing. However, if the injection volume is too large or solutes are poorly retained during injection, volume overload occurs which leads to altered peak shapes, decreased sensitivity, and lower peak capacity. Solutes that elute early even with the use of a solvent gradient are especially vulnerable to this problem. In this paper, we describe a simple, automated instrumental method, temperature-assisted on-column solute focusing (TASF), that is capable of focusing large volume injections of small molecules and peptides under gradient conditions. By injecting a large sample volume while cooling a short segment of the column inlet at subambient temperatures, solutes are concentrated into narrow bands at the head of the column. Rapidly raising the temperature of this segment of the column leads to separations with less peak broadening in comparison to solvent focusing alone. For large volume injections of both mixtures of small molecules and a bovine serum albumin tryptic digest, TASF improved the peak shape and resolution in chromatograms. TASF showed the most dramatic improvements with shallow gradients, which is particularly useful for biological applications. Results demonstrate the ability of TASF with gradient elution to improve the sensitivity, resolution, and peak capacity of volume overloaded samples beyond gradient compression alone. Additionally, we have developed and validated a double extrapolation method for predicting retention factors at extremes of temperature and mobile phase composition. Using this method, the effects of TASF can be predicted, allowing determination of the usefulness of this technique for a particular application.

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confidence: 99%

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

2016

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“…To overcome these problems, Haggarty and colleagues applied a method where a sample is injected onto both, RP and HILIC columns in tandem in a single injection [ 41 ]. To solve the problem with the solvent strength incompatibility between the columns, a high-aqueous mobile phase at low flow rate for the RPLC column and a high-acetonitrile mobile phase at high flow rate for the HILIC column were used [ 39 ].…”

Section: Data Acquisition and Analysismentioning

confidence: 99%

“…This tandem column approach was already successfully applied with quadrupole, time of flight (TOF), and orbitrap mass analyzers [ 39 , 41 , 42 , 43 ]. This method could significantly increase the amount of identified metabolites in a single analytical run, but one disadvantage is that isomers cannot be separated [ 39 ].…”

Section: Data Acquisition and Analysismentioning

confidence: 99%

Advantages and Pitfalls of Mass Spectrometry Based Metabolome Profiling in Systems Biology

Aretz

Meierhofer

2016

IJMS

130

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Mass spectrometry-based metabolome profiling became the method of choice in systems biology approaches and aims to enhance biological understanding of complex biological systems. Genomics, transcriptomics, and proteomics are well established technologies and are commonly used by many scientists. In comparison, metabolomics is an emerging field and has not reached such high-throughput, routine and coverage than other omics technologies. Nevertheless, substantial improvements were achieved during the last years. Integrated data derived from multi-omics approaches will provide a deeper understanding of entire biological systems. Metabolome profiling is mainly hampered by its diversity, variation of metabolite concentration by several orders of magnitude and biological data interpretation. Thus, multiple approaches are required to cover most of the metabolites. No software tool is capable of comprehensively translating all the data into a biologically meaningful context yet. In this review, we discuss the advantages of metabolome profiling and main obstacles limiting progress in systems biology.

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“…To overcome these limitations, several setups have been developed which combine RP chromatography and HILIC in a single run. A first type of setup uses serial coupling of HILIC and RP columns . Greco et al .…”

Section: Introductionmentioning

confidence: 99%

Tandem HILIC‐RP liquid chromatography for increased polarity coverage in food analysis

et al. 2018

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Comprehensive non-targeted analysis of food products normally requires two complementary chromatographic runs to achieve maximum compound coverage. In this study, we present a sensitive tandem-LC method, which combines RP and HILIC separation in a single run. The setup consists of a C18 trap column and two subsequently coupled analytical columns (HILIC and C18) which are operated in parallel. First, hydrophobic compounds are retained on the RP trap column while rather hydrophilic compounds are directly transferred onto a HILIC phase. Next, the pre-fractionated sample composition is analyzed by HILIC or RP chromatography, respectively. The presented setup allows individual and independent gradient elution as well as interfacing with mass spectrometry. The performance of the method has been proven by means of food relevant standards and analysis of complex food samples (e.g. red wine, meat extract). The simple and robust setup provides high flexibility in the selection of column combinations and does not require sophisticated instrumental setups or software. The method significantly increases the covered polarity range compared to classical one-dimensional chromatography. Our results indicate that tandem LC is a valuable and universal tool in the non-targeted screening of various types of complex food samples.

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Serially coupling hydrophobic interaction and reversed-phase chromatography with simultaneous gradients provides greater coverage of the metabolome

Cited by 36 publications

References 14 publications

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

Advantages and Pitfalls of Mass Spectrometry Based Metabolome Profiling in Systems Biology

Tandem HILIC‐RP liquid chromatography for increased polarity coverage in food analysis

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