A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS

Abstract

Background: Aliphatic molecules containing free carboxyl groups are important intermediates in many metabolic and signalling reactions, however, they accumulate to low levels in tissues and are not efficiently ionized by electrospray ionization (ESI) compared to more polar substances. Quantification of aliphatic molecules becomes therefore difficult when small amounts of tissue are available for analysis. Traditional methods for analysis of these molecules require purification or enrichment steps, which are onerous when multiple samples need to be analyzed. In contrast to aliphatic molecules, more polar substances containing free carboxyl groups such as some phytohormones are efficiently ionized by ESI and suitable for analysis by LC-MS/MS. Thus, the development of a method with which aliphatic and polar molecules -which their unmodified forms differ dramatically in their efficiencies of ionization by ESI- can be simultaneously detected with similar sensitivities would substantially simplify the analysis of complex biological matrices.

Results: A simple, rapid, specific and sensitive method for the simultaneous detection and quantification of free aliphatic molecules (e.g., free fatty acids (FFA)) and small polar molecules (e.g., jasmonic acid (JA), salicylic acid (SA)) containing free carboxyl groups by direct derivatization of leaf extracts with Picolinyl reagent followed by LC-MS/MS analysis is presented. The presence of the N atom in the esterified pyridine moiety allowed the efficient ionization of 25 compounds tested irrespective of their chemical structure. The method was validated by comparing the results obtained after analysis of Nicotiana attenuata leaf material with previously described analytical methods.

Conclusion: The method presented was used to detect 16 compounds in leaf extracts of N. attenuata plants. Importantly, the method can be adapted based on the specific analytes of interest with the only consideration that the molecules must contain at least one free carboxyl group.

Figure 1

Examples of compounds analyzed as their Picolinyl ester derivatives by LC-MS/MS.

Figure 2

Reaction mechanisms for the formation of Picolinyl ester derivatives of carboxylic acids. The carboxyl group is first activated by reaction with 1,1'-carbidiimidazole to form the active amid 1. 1 is then transesterified with 3-(hydroxymethyl)-pyridine to form the Picolinyl ester derivative 2. After collision induced dissociation (CID), the major ion fragments obtained are m/z = 92 and m/z = 108.

Figure 3

Example of chromatograms of Picolinyl ester derivatives from a standard mixture and a N. attenuata leaf extract. A, Chromatogram (TIC) of a mix of derivatized commercial standards. B, Chromatogram (TIC) of a derivatized N. attenuata leaf extract after 60 min of FAC elicitation. *Analytes 11 to 13 are overlaid by a peak corresponding to an unknown compound in the leaf extract. Peaks are numbered according to Table 1.

Figure 4

Example of the linear correlation between the amounts of leaf tissue extracted from FAC elicited N. attenuata leaves and the amounts of compounds detected. Different amounts of leaf tissue (5 to 250 mg; fresh weight) from N. attenuata plants were collected after 60 min of FAC elicitation. Leaf material was extracted, derivatized and analyzed by LC-MS/MS. Two biological replicates were performed for each amount of tissue.