Very Long Chain Fatty Acid Analysis by UPLC-MS/MS for the Diagnosis of Inherited Peroxisomal Disorders
Mon 3:30 PM - Track 1: Small Molecule Analysis II
Aiping Liu
A. Liu 1, N. Longo 1,2,3, M. Pasquali 1,2,4

1 ARUP Institute for Clinical and Experimental Pathology®, Salt Lake City, UT 84108, USA
2 ARUP Laboratories, Salt Lake City, UT 84108, USA
3 Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA
4 Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA
Peroxisomes are organelles performing several cellular functions, including fatty acid alpha- and beta-oxidation and biosynthesis of polyunsaturated fatty acids, phospholipids, cholesterol and other isoprenoid compounds. Defects in one or more peroxisomal functions lead to severe clinical manifestations. The most severe peroxisomal disorder is Zellweger syndrome, in which the activity of multiple enzymes is defective and is characterized by neurological dysfunction, neurosensory defects, liver dysfunction, developmental delay/regression, hypotonia, and early death. At least 21 disorders involving peroxisomal function have been described. In more than half of the peroxisomal disorders with neurological involvement, there is accumulation of Very Long Chain Fatty Acids (VLCFA). Therefore the measurement of plasma VLCFA is used as the primary screen for peroxisomal disorders.

The analysis of VLCFA is typically performed on GC/MS. The capillary column can separate all the isomeric forms of saturated and unsaturated fatty acids. But the chromatographic separation requires a > 30 minute run time to resolve all the isomeric species. We have validated rapid analysis of VLCFA in plasma using UPLC-MS/MS. Fatty acids are released from lipids by acid hydrolysis and derivatized as trimethylaminoethyl iodide esters for analysis. The separation of VLCFAs, including straight-chain and branched-chain isomers and trans/cis unsaturated isomers, is achieved within 6 minutes using an Acquity BEH C18 column. VLCFAs are monitored in MRM mode for MS/MS analysis.

This new assay correctly identified patients with peroxisomal disorders and correlated well with existing methods of VLCFA analysis. The UPLC-MS/MS method significantly shortens the time required for VLCFA analysis and allows the simultaneous determination of clinically relevant fatty acids. This method can be extended to the analysis of essential fatty acids in plasma.