A clinically validated method to separate and quantify underivatized acylcarnitines and carnitine metabolic intermediates using mixed-mode chromatography with tandem mass spectrometry

• Validation of a simple LC-MS/MS protocol to analyze underivatized acylcarnitines. • Separation of clinically important isomeric acylcarnitine species. • Expanded coverage of free carnitine, carnitine metabolic precursors, and TMAO. • Novel application of mixed mode chromatography in a clinical setting. • Detection of peroxisome biomarkers long/very long chain dicarboxylic acylcarnitines. Acylcarnitines are intermediate metabolites of the mitochondria that serve as biomarkers for inherited disorders of fatty acid oxidation and amino acid metabolism. The prevailing clinical method used to quantify acylcarnitines involves flow-injection tandem mass spectrometry, an approach with a number of limitations; foremost the inability to separate and therefore distinguish key isobaric acylcarnitine species. To address these issues, we report a clinically validated liquid chromatography tandem mass spectrometry method to quantify acylcarnitines, free carnitine, and carnitine metabolic intermediates in human plasma. Importantly, this method resolves clinically relevant isobaric and isomeric acylcarnitine species in a single 22 min analysis without the use of ion pairing or derivatization reagents. This unique combination of features is not achievable by existing acylcarnitine methods and is made possible by the use of a novel mixed-mode chromatographic separation. Further clinical validation studies demonstrate excellent limits of quantification, linearity, accuracy, and inter-assay precision for analyses of 38 different calibrated analytes. An additional 28 analytes are semi-quantitatively analyzed using surrogate calibrators. The study of residual patient specimens confirms the clinical utility of this method and suggests expanded applicability to the diagnosis of peroxisomal disorders. In summary, we report a clinically validated acylcarnitine method that utilizes a novel mixed-mode chromatographic separation to provide a number of advantages in terms of specificity, accuracy, sample preparation time, and clinical utility.