Quantification and structural characterization of raffinose family oligosaccharides in Casuarina glauca plant tissues by porous graphitic carbon electrospray quadrupole ion trap mass spectrometry

Raffinose family oligosaccharides (RFOs) are non-structural, water-soluble carbohydrates widely distributed in the plant kingdom, and include the trisaccharide raffinose, the tetrasaccharide stachyose and the pentasaccharide verbascose. RFOs have been suggested to play a major role in storing carbohydrates in seeds and vegetative tissues, and in conferring osmoprotection against abiotic stresses, such as high salinity and drought. However, the high polarity nature of RFOs makes their analysis with typical reversed phase liquid chromatography (RP-LC) generally difficult, with these compounds eluting very close to the void volume with minimal retention. Moreover, carbohydrate-related compounds lack an inherent chromophore in the ultraviolet-visible (UV) region to make them suitable for UV detection. To overcome these issues, alternative analytical methods based on LC coupled to mass spectrometry (MS) have been developed and porous graphitic carbon (PGC) stationary phases described in the literature as good alternative to typical RP columns for retaining these highly polar compounds were used. In addition, PGC stationary phases allow the use of MS-compatible mobile phases for efficient on-line coupling with electrospray ionization (ESI), and when coupled to tandem MS (MSⁿ) techniques, powerful structural information can be obtained. The present work focuses on the application of a PGC-ESI-QIT-MSⁿ method for the quantitative analysis of RFOs in Casuariana glauca plant tissues under salt stress conditions. In addition, we describe the structural characterization of RFOs through collision induced dissociation (CID) and MSⁿ experiments using a quadrupole ion trap mass spectrometer (QIT-MS) in the ESI positive ion mode.