Carboxylic
acids are central metabolites in bioenergetics, signal
transduction, and post-translation protein regulation. However, the
quantitative analysis of carboxylic acids as an indispensable part
of metabolomics is prohibitively challenging, particularly in trace
amounts of biosamples. Here we report a diazo-carboxyl/hydroxylamine-ketone
double click derivatization method for the sensitive analysis of hydrophilic,
low-molecular-weight carboxylic acids. In general, our method renders
a 5- to 2000-fold higher response in mass spectrometry along with
improved chromatographic separation. With this method, we presented
the near-single-cell analysis of carboxylic acid metabolites in 10
mouse egg cells before and after fertilization. Malate, fumarate,
and β-hydroxybutyrate were found to decrease after fertilization.
We also monitored the isotope labeling kinetics of carboxylic acids
inside adherent cells cultured in 96-well plates during drug treatment.
Finally, we applied this method to plasma or serum samples (5 μL)
collected from mice and humans under pathological and physiological
conditions. The double click derivatization method paves a way toward
single-cell metabolomics and bedside diagnostics.