Fractionation platform for target identification using off-line directed two-dimensional chromatography, mass spectrometry and nuclear magnetic resonance
Analytica Chimica Acta, vol. 1142, pp. 28–37
Abstract
The unambiguous identification of unknown compounds is of utmost importance in the field of metabolomics. However, current identification workflows often suffer from error-sensitive methodologies, which may lead to incorrect structure annotations of small molecules. Therefore, we have developed a comprehensive identification workflow including two highly complementary techniques, i.e. liquid chromatography (LC) combined with mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR), and used it to identify five taste-related retention time and m/z features in soy sauce. An off-line directed two-dimensional separation was performed in order to purify the features prior to the identification. Fractions collected during the first dimension separation (reversed phase low pH) were evaluated for the presence of remaining impurities next to the features of interest. Based on the separation between the feature and impurities, the most orthogonal second dimension chromatography (hydrophilic interaction chromatography or reversed phase high pH) was selected for further purification. Unknown compounds down to tens of micromolar concentrations were tentatively annotated by MS and structurally confirmed by MS and NMR. The mass (0.4-4.2 μg) and purity of the isolated compounds were sufficient for the acquisition of one and two-dimensional NMR spectra. The use of a directed two-dimensional chromatography allowed for a fractionation that was tailored to each feature and remaining impurities. This makes the fractionation more widely applicable to different sample matrices than one-dimensional or fixed two-dimensional chromatography. Five proline-based 2,5-diketopiperazines were successfully identified in soy sauce. These cyclic dipeptides might contribute to taste by giving a bitter flavour or indirectly enhancing umami flavour.
Authors 10
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Tom van der Laan Aachen Analytical Biosciences and Metabolomics Division of Systems Biomedicine and Pharmacology Leiden Academic Centre for Drug Research
Affiliation as printed
Analytical Biosciences and Metabolomics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Center for Drug Research, Leiden University, 2333 CC, Leiden, the Netherlands
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Hyung Lim Elfrink Aachen Analytical Biosciences and Metabolomics Division of Systems Biomedicine and Pharmacology Leiden Academic Centre for Drug Research
Affiliation as printed
Analytical Biosciences and Metabolomics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Center for Drug Research, Leiden University, 2333 CC, Leiden, the Netherlands
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Wageningen University & Research
Affiliation as printed
Agrotechnology and Food Sciences, Laboratory of BioNanoTechnology, Wageningen University, 6700 EK, Wageningen, the Netherlands
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Anne-Charlotte Dubbelman Aachen Analytical Biosciences and Metabolomics Division of Systems Biomedicine and Pharmacology Leiden Academic Centre for Drug Research
Affiliation as printed
Analytical Biosciences and Metabolomics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Center for Drug Research, Leiden University, 2333 CC, Leiden, the Netherlands
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Amy C. Harms Aachen Analytical Biosciences and Metabolomics Division of Systems Biomedicine and Pharmacology Leiden Academic Centre for Drug Research
Affiliation as printed
Analytical Biosciences and Metabolomics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Center for Drug Research, Leiden University, 2333 CC, Leiden, the Netherlands
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Unilever Foods Innovation Centre
Affiliation as printed
Unilever Global Food Innovation Centre, 6708 WH Wageningen, the Netherlands
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Affiliation as printed
Applied NMR Development and Hyphenation, BD-DE, Bruker Biospin, 76287, Rheinstetten, Germany
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Wageningen University & Research
Affiliation as printed
Agrotechnology and Food Sciences, Laboratory of BioNanoTechnology, Wageningen University, 6700 EK, Wageningen, the Netherlands
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Unilever Foods Innovation Centre · Wageningen University & Research
Affiliation as printed
Unilever Global Food Innovation Centre, 6708 WH Wageningen, the Netherlands; Laboratory of Biophysics, Wageningen University, 6708 WE, Wageningen, the Netherlands
Laboratory of Biophysics, Wageningen University, 6708 WE, Wageningen, the Netherlands
Unilever Global Food Innovation Centre, 6708 WH Wageningen, the Netherlands
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Thomas Hankemeier corresponding Aachen Analytical Biosciences and Metabolomics Division of Systems Biomedicine and Pharmacology Leiden Academic Centre for Drug Research
Affiliation as printed
Analytical Biosciences and Metabolomics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Center for Drug Research, Leiden University, 2333 CC, Leiden, the Netherlands. Electronic address: hankemeier@lacdr.leidenuniv.nl
Analytical Biosciences and Metabolomics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Center for Drug Research, Leiden University, 2333 CC, Leiden, the Netherlands
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