A new multiplex method for the diagnosis of peroxisomal disorders allowing simultaneous determination of plasma very-long-chain fatty acids, phytanic, pristanic, docosahexaenoic and bile acids by high-performance liquid chromatography-atmospheric pressure

• Quantification of VLCFA, DHA, phytanic, pristanic and bile acids by LC-APCI-MS/MS
• Simple and rapid sample preparation without the step of derivatization
• All metabolites are well separated in a single chromatographic run.
• The method allows the diagnosis of most peroxisomal disorders.

Peroxisomal disorders (PDs) present with wide phenotypic variability. An appropriate diagnosis requires a complete analysis of peroxisomal metabolites.We developed a multiplex LC-MS/MS method, using atmospheric pressure chemical ionization allowing the simultaneous determination in plasma of very-long-chain fatty acids, phytanic, pristanic, docosahexaenoic acids and di- and tri-hydroxycolestanoic bile acids.Two hundred microliters of plasma extracted with acetonitrile and 200 μl extracted with hexane after an acid hydrolysis were combined, evaporated, dissolved in 10 μl of methanol and analyzed.The acquisition was in negative-ion mode using multiple reaction monitoring.The method was validated analytically and clinically. Linearity was 0.1–200 μmol/l for docosanoic, cis-13-docosenoic, tetracosanoic, cis-15-tetracosenoic and phytanic acids; 0.01–10 μmol/l for hexacosanoic acid; 0.02–20 μmol/l for di-hydroxycolestanoic, tri-hydroxycolestanoic and pristanic acids; 0.3–300 μmol/l for docosahexaenoic acid. Intra-day and inter-day CVs were below 3.88 and 3.98 respectively for all compounds. Samples from patients with known peroxisomal disorders were compared with controls and the method allowed to confirm the diagnosis in all subjects with a 100% sensitivity.The advantage of this multiplex method is to allow in a single chromatographic run the simultaneous determination of a large number of peroxisome biomarkers with a simple preparative phase without derivatization.