UPLC-based profiling of secondary metabolites

Contrary to the analysis of lipids, which show a very systematic behaviour, the analysis of the highly complex secondary metabolites from plants, offers a number of challenges. Even though many secondary metabolites are grouped into distinct classes, only few can be validated on their class-specific and systematic retention time / m/z dependencies. One example for a systematically eluting secondary metabolite class can be illustrated for the methionine-derived aliphatic glucosinolates. These compounds are characterized by the addition of a CH2 groups, catalysed by a methylthioalkylmalate synthase, which leads to a steady increased retention time.

An additional way to validate compounds is to select frequently occurring ions (likely fragments of several different precursor molecules) and extract them from the MS and also the all-ion fragmentation spectra. A very straight forward example can be illustrated for the previously mentioned quercetin- or kaempferol-containing flavonoids. Based on the fact that quercetin- and kaempferol-based flavonoids are decorated with different numbers and kinds of sugars, it is possible to extract the exact m/z 287.05501 for the kaempferol- and the m/z of 303.04993 for the quercetin-containg peaks from the all-ion fragmentation spectra. This directly leads to the distribution of these flavonoids within the chromatogram. If now the retention time information and the isotope-labelling-derived proper mass-shift data are combined, the separated favonoids can be unambiguous annotated.