Development of a targeted LC-MS/MS method for the determination of different classes of grape lipids

Lipids are produced, transported and recognized by the combined action of many enzymes, which are genetically determined and influenced by environmental factors. The relative abundance of plant lipids are subsequently influenced by the genotype and phenotype. Despite being a very important class of plant metabolites, the knowledge about grape lipids is to date still very limited, with the exception of those located in the seeds, which are widely used for the production of edible oils. Few investigations on grape lipids have shown that the their profile depends on the grape maturity, variety and their localization in the berry (skin or pulp) [1,2]. Recently the advancement in liquid chromatography coupled to mass spectrometry has paved the way for faster analysis of lipids with minimal sample preparation. Here we describe a method validation for the extraction, identification and quantification of different classes of grape lipids: fatty acids, sterols, glycerolipids, glycerophospholipids, sphingolipids applying liquid chromatographic electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) using triple quadrupole ABsciex 5500. Optimal multiple reaction monitoring (MRM) transitions were selected for each lipid. Chromatography on an Ascentis Express C18 column (Sigma-Aldrich) was optimized to achieve separation of the compounds in a 30 min run using a polarity switching mode. The method was validated for 45 lipids, the linearity range (R2=0,990-0,999), LOQ (12,5-2500 ng/mL) and LOD (1,25-625 ng/mL), the intraday and interday repeatability were evaluated for each lipid. The developed lipid profiling method was successfully applied for the analysis of 20 grape samples (13 red grapes and 7 white grapes) from 4 different genetic groups: 11 Vitis vinifera, 2 Vitis non-vinifera, 4 Muscats and 2 Hybrids; 35 lipids were identified and 29 were quantified. This method, which can be easily expanded to include further compounds and other plant tissues, is the starting point for the analysis of the lipid profile in different grape tissues, an essential goal for a better understanding of the role of lipids in grape physiology