title: 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 chemical ionization-tandem mass spectrometry.
abstract: 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 200μl extracted with hexane after an acid hydrolysis were combined, evaporated, dissolved in 10μl of methanol and analyzed. The acquisition was in negative-ion mode using multiple reaction monitoring. The method was validated analytically and clinically. Linearity was 0.1-200μmol/l for docosanoic, cis-13-docosenoic, tetracosanoic, cis-15-tetracosenoic and phytanic acids; 0.01-10μmol/l for hexacosanoic acid; 0.02-20μmol/l for di-hydroxycolestanoic, tri-hydroxycolestanoic and pristanic acids; 0.3-300μmol/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.
authors.affiliation: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Hospital, IRCCS (Institute for Treatment and Research), Rome, Italy. Electronic address: michela.semeraro@opbg.net.
authors.forename: Michela
authors.initials: M
authors.lastname: Semeraro
authors.affiliation: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Hospital, IRCCS (Institute for Treatment and Research), Rome, Italy.
authors.forename: Cristiano
authors.initials: C
authors.lastname: Rizzo
authors.affiliation: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Hospital, IRCCS (Institute for Treatment and Research), Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Boenzi
authors.affiliation: Endocrinology and Diabetic Unit, Bambino Gesù Children's Hospital, IRCCS (Institute for Treatment and Research), Rome, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Cappa
authors.affiliation: Unit of Neuromuscular and Neurodegenerative Disorders, Laboratory of Molecular Medicine, Bambino Gesù Children's Hospital, IRCCS (Institute for Treatment and Research), Rome, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Bertini
authors.affiliation: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Hospital, IRCCS (Institute for Treatment and Research), Rome, Italy.
authors.forename: Giacomo
authors.initials: G
authors.lastname: Antonetti
authors.affiliation: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Hospital, IRCCS (Institute for Treatment and Research), Rome, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Dionisi-Vici
mesh_terms: D001274:Atmospheric Pressure; D001647:Bile Acids and Salts / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004281:Docosahexaenoic Acids / Q000097:blood*; D005227:Fatty Acids / Q000097:blood*; D006801:Humans; D018901:Peroxisomal Disorders / Q000175:diagnosis*; D010831:Phytanic Acid / Q000097:blood*; D053719:Tandem Mass Spectrometry
chemical_list: D001647:Bile Acids and Salts; D005227:Fatty Acids; D010831:Phytanic Acid; D004281:Docosahexaenoic Acids; C002844:pristanic acid
keywords: Bile acids; Docosahexaenoic acid; Liquid chromatography-tandem mass spectrometry; Phytanic acid; Pristanic acid; Very-long-chain fatty acid