A fast GC-MS/MS method for the simultaneous measurement of key metabolites of peroxisomal beta-oxidation and ether lipid biosynthesis

Peroxisomes are subcellular compartments that host a variety of metabolic pathways, including the chain shortening of fatty acids (FAs) by beta-oxidation and certain steps in the formation of ether lipids. Here, we describe the development of a GC-MS/MS-based method for the simple simultaneous and reliable determination of key metabolites of these pathways, also including less common FA species related to peroxisomal metabolism that are typically not part of standard analytical methods. We for the first time utilize 1-chlorobutane for the extraction of FAs as an effective alternative to commonly used extraction solvents. 1-chlorobutane offers a broader polarity range than hexane and lower toxicity relative to chloroform with solvent consumption of less than 1 mL per sample. Six saturated long to very long-chain FAs, nine polyunsaturated FAs (PUFAs), two dicarboxylic FAs and 1-O-octadecyl glycerol (ODG, batyl alcohol) were extracted simultaneously. The method was validated using fibroblasts and for the majority of FA species accuracies ranged from 80 to 110% with coefficients of variations (CVs) from 6 to 20 %. For the estimation of the ether lipid synthesis rate the measurement of ODG was established as reliable marker. The suitability of the method was demonstrated by the analysis of primary human fibroblasts from controls and individuals with peroxisomal disorders. This cell type represents a commonly used model system for the investigation of peroxisomal metabolism and disease, thus rendering our protocol a valuable addition to the toolkit for studying peroxisomal pathways.