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Abstract
Electrospray ionization (ESI) is the most useful interface for mass spectrometry associated
with liquid chromatography (LC). However, analyses of polar metabolites become a challenge
because the high polarity impairs the separation of metabolites in reversed-phase
liquid chromatography and ionization of metabolites by ESI. In this article, we have
used cyanuric chloride to couple the amine-containing molecules with methylamine and
found that both resolution on LC chromatogram and ionization by ESI are greatly improved.
Derivatives would be obtained in 2-h coupling reactions, and then resolved by LC-ESI-MS
in 15 min for each sample. Most amino acids can be quantified with linear range from
1 nM to 1 μM and with an R(2) above 0.979. Although reversed-phase chromatography
is suitable for resolving the derivatives, phenyl columns with methanol elution provide
optimal separation and signal intensity. Moreover, most structural isomers are well
separated following cyanuric chloride and methylamine derivatization. Instead of synthesizing
a stable isotope-labeled cyanuric chloride, we can take advantage of using commercially
available methyl-d3-amine for a novel stable isotope-coded derivatization method.
Each metabolite can be directly quantified by the peak intensity ratio of each derivative
isotopologue pair in a single LC-MS analysis. The coupling reactions are relatively
easy and accessible to most investigators to generate multiple stable isotope-labeled
derivatives of amine-containing compounds for a differential metabolomic analysis.