Post-column in-source derivatisation in LC-MS: a tool for natural products characterisation and metabolomics.

An efficient characterisation of metabolites is a crucial task in many aspects of basic research, such as the de-replication of crude extracts in natural products chemistry or the tentative identification of compounds in untargeted metabolomics. The goal of this work is the evaluation of the reaction with phenylhydrazine for the derivatisation post-column in situ of carbonyl-containing compounds in liquid chromatography-mass spectrometry (LC-MS). LC-MS was performed using electrospray, Atmospheric Pressure Chemical Ionisation (APCI) or Atmospheric Pressure Photoionization (APPI) as ionisation techniques. The post-column addition of phenylhydrazine was done through a syringe pump via a T-junction before entrance to the ion source. A variety of natural products having carbonyl groups, such as cycloartanes, steroids, cardenolides and other terpenoids, were analysed by this method. In the case of compounds with non-hindered aldehyde or keto groups, the main signals of the mass spectra were those corresponding to the phenylhydrazones. However, the spectra of compounds with hindered carbonyl groups displayed mainly those signals corresponding to the product of the nucleophilic addition adduct of phenylhydrazine to the carbonyl, which is the first step of the derivatisation process. Finally, those compounds with conjugated ketones did not react with phenylhydrazine. This methodology was applied in the analysis of crude natural extracts. The results show that in situ derivatisation of carbonyl compounds in the ionisation source was achieved, yielding the typical derivatives of carbonyl compounds with phenylhydrazine.