Derivatization for Lipidomics: Analysis of Free Fatty Acids, Neutral and Polar Lipids by UHPLC-FTMS Without Prior Fractionation
Wed 3:00 PM - Track 2: Discovery Metabolomics
Ivana Bobeldijk
TNO Quality of Life
Ivana Bobeldijk, TNO
Leon Coulier, TNO
Raymond Ramaker, TNO
Elwin Verheij, TNO
S. Wopereis, TNO
R. Kleemann, TNO
M. Bracht, University of Maastricht, The Netherlands

TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, the Netherlands
Changing concentrations of the different lipid classes known in plasma and tissues are important markers of life style associated diseases. Differences in lipid concentrations give mechanistic insight in onset, progression and/or status of the disease; lipid analysis is therefore often included in nutritional studies with genetically modified rodent models. In these types of studies mice are sampled several times during the course of the study, with only tens of microliters of plasma or serum available for each time point. In such cases it is important that the methods are sensitive and use a low volumes (or amount of tissue) of sample and provide as much information as possible.

With our method, lipids are extracted from low volumes of plasma (5 microliters), part of the extract is derivatised with a novel derivatisation method and analysed by UHPLC-Orbitrap. In ESI positive mode FFA, LPC, PC and SPM are detected, in APCI positive mode neutral lipids such as CER, MG, DG,TG, ChE are detected. This method combines quantitative analysis of free fatty acids with a comprehensive lipidomics approach. The introduction of a new polar group into the molecule greatly improves ionization in ESI+ and also separation to such extent that for unsaturated fatty acids the isomers with different position of the double bonds can be separated on reversed phase UHPLC column. The method enables high throughput. The derivatization method for the fatty acids was optimized and fully validated. The sensitivity of the method for free fatty acids is 10 pmol per ml, which is much more sensitive than conventional GC methods. As an example in human plasma, 28 FFA were detected, identified and quantified, 87 neutral lipids and 68 polar lipids were detected, and identified. The quality of the data is monitored in each study by using pooled study samples.