Fractionation platform for target identification using off-line directed two-dimensional chromatography, mass spectrometry and nuclear magnetic resonance

Tom van der Laan; Hyung Elfrink; Fatemeh Azadi-Chegeni; Anne-Charlotte Dubbelman; Amy C Harms; Doris M Jacobs; Ulrich Braumann; Aldrik H Velders; John van Duynhoven; Thomas Hankemeier

doi:10.1016/j.aca.2020.10.054

Fractionation platform for target identification using off-line directed two-dimensional chromatography, mass spectrometry and nuclear magnetic resonance

Anal Chim Acta. 2021 Jan 15:1142:28-37. doi: 10.1016/j.aca.2020.10.054. Epub 2020 Oct 30.

Affiliations

¹ Analytical Biosciences and Metabolomics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Center for Drug Research, Leiden University, 2333 CC, Leiden, the Netherlands.
² Agrotechnology and Food Sciences, Laboratory of BioNanoTechnology, Wageningen University, 6700 EK, Wageningen, the Netherlands.
³ Unilever Global Food Innovation Centre, 6708 WH Wageningen, the Netherlands.
⁴ Applied NMR Development and Hyphenation, BD-DE, Bruker Biospin, 76287, Rheinstetten, Germany.
⁵ Unilever Global Food Innovation Centre, 6708 WH Wageningen, the Netherlands; Laboratory of Biophysics, Wageningen University, 6708 WE, Wageningen, the Netherlands.
⁶ 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.

PMID: 33280701
DOI: 10.1016/j.aca.2020.10.054

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.

Keywords: Food; Identification; Mass spectrometry; Metabolomics; Nuclear magnetic resonance spectroscopy; Two-dimensional chromatography.

MeSH terms

Chemical Fractionation*
Chromatography, Liquid
Magnetic Resonance Spectroscopy
Mass Spectrometry
Metabolomics*