Analysis of highly polar compounds of plant origin: combination of hydrophilic interaction chromatography and electrospray ion trap mass spectrometry

The primary goal of metabolomic analysis is the unbiased relative quantification of every metabolite in a biological system. A number of different metabolite-profiling techniques must be combined to make this possible. Here we report the separation and analysis of highly polar compounds in a proof of concept study. Compounds were separated and analyzed using hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization (ESI) mass spectrometry. Two types of HILIC microbore columns (Polyhydroxyethyl A and TSK Gel Amide 80) were compared to normal phase silica HPLC columns. The best separations of standards mixtures and plant samples were achieved using the Amide 80 stationary phase. ESI enabled the detection of both positively and negatively charged metabolites, when coupled to a quadrupole ion trap mass spectrometer using continuous polarity switching. By stepwise mass spectrometric fragmentation of the most intense ions, unknown compounds could be identified and then included into a custom mass spectrometric library. This method was used to detect oligosaccharides, glycosides, amino sugars, amino acids, and sugar nucleotides in phloem exudates from petioles of fully expanded Cucurbita maxima leaves. Quantitative analysis was performed using external standards. The detection limit for stachyose was 0.5 ng per injection (Amide 80). The concentration of stachyose in investigated phloem samples was in the range of 1-7 mM depending on the plant.