Rationale: Fatty acids and sterol lipids play crucial roles in several biological processes and several biological facts underline the interconnection between these lipid classes. Therefore, it is of interest to develop a comprehensive method analysing both classes in the form of their most favourable derivatives suitable for quantification and isotopologue analysis.
Methods: Lipids were derivatised by a sequential one-pot procedure using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MtBSTFA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). No clean-up or concentration steps were necessary. The prepared samples were directly available for gas chromatography-electron ionisation mass spectrometric (GC-EI-MS) analysis on a standard column. For quantification, the SIM mode was used and for isotopologue analysis scheduled scan mode was applied.
Results: Development of a sequential one-pot derivatisation for GC-EI-MS allowing comprehensive analysis of fatty acids and sterols as their most favourable derivatives. Validation carried out using human plasma, comparison with certified NIST plasma. LLOQ of usually 3.3 ng/mL achieved. Isotopologue analysis of 2-[(13)C]-acetate incorporation in HL-60 cells proving feasibility of method.
Conclusions: The presented method successfully combines two consecutive silylation reactions in one pot, enabling the analysis of both fatty acids and sterols in a comprehensive analytical method. The method has great potential for the quantification of lipids as well as the comprehensive study of both biochemical pathways, using [(13)C]-flux analysis.
Copyright © 2014 John Wiley & Sons, Ltd.