Analytical Chemistry

Ion-pairing chromatography and amine derivatization provide complementary approaches for the targeted UPLC-MS analysis of the polar metabolome

Authors

Abstract

Liquid chromatography coupled to mass spectrometry is a key metabolomics technology. Reversed-phase liquid chromatog-raphy (RPLC) is very widely used as a separation step, possessing excellent characteristics with respect to reproducibility and reliability, but typically has poor retention of highly polar metabolites. Here, we evaluated the combination of two alter-native methods for improving retention of polar metabolites based on 6-aminoquinoloyl-N-hydroxysuccinidimyl carbamate derivatization for amine groups, and ion-pairing chromatography (IPC) using tributylamine as an ion-pairing agent to retain acids. We compared both of these methods to RPLC and also to each other, for targeted analysis using a triple-quadrupole mass spectrometer, applied to a library of ca. 500 polar metabolites. IPC and derivatization were complementary in terms of their coverage: combined, they improved the proportion of metabolites with good retention to 91%, compared to just 39% for RPLC alone. We detected 132 metabolites for real biological samples (liver extracts) with good reproducibility (based on coefficients of variation in pooled biological quality control samples). Finally, we tested the combination of methods with real-world samples by analyzing a set of liver extracts from aged male and female mice that had been treated with the poly-phenol compound ampelopsin. Furthermore, we also compared the results of these LC-MS methods to 1H NMR spectrosco-py as an orthogonal method (also termed statistical heterospectroscopy (SHY)), and found a strong correlation between the results of these different analytical approaches. By these means, not only were a number of significantly changed metabolites detected, but also it could be shown that there was a clear interaction between ampelopsin treatment and sex, in that the di-rection of metabolite change was opposite for males and females.

Version notes

slight edits to text regarding comparison to HILIC methods

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