title: Correcting false positive medium-chain acyl-CoA dehydrogenase deficiency results from newborn screening; synthesis, purification, and standardization of branched-chain C8 acylcarnitines for use in their selective and accurate absolute quantitation by UHPLC-MS/MS.
abstract: While selectively quantifying acylcarnitines in thousands of patient samples using UHPLC-MS/MS, we have occasionally observed unidentified branched-chain C8 acylcarnitines. Such observations are not possible using tandem MS methods, which generate pseudo-quantitative acylcarnitine "profiles". Since these "profiles" select for mass alone, they cannot distinguish authentic signal from isobaric and isomeric interferences. For example, some of the samples containing branched-chain C8 acylcarnitines were, in fact, expanded newborn screening false positive "profiles" for medium-chain acyl-CoA dehydrogenase deficiency (MCADD). Using our fast, highly selective, and quantitatively accurate UHPLC-MS/MS acylcarnitine determination method, we corrected the false positive tandem MS results and reported the sample results as normal for octanoylcarnitine (the marker for MCADD). From instances such as these, we decided to further investigate the presence of branched-chain C8 acylcarnitines in patient samples. To accomplish this, we synthesized and chromatographically characterized several branched-chain C8 acylcarnitines (in addition to valproylcarnitine): 2-methylheptanoylcarnitine, 6-methylheptanoylcarnitine, 2,2-dimethylhexanoylcarnitine, 3,3-dimethylhexanoylcarnitine, 3,5-dimethylhexanoylcarnitine, 2-ethylhexanoylcarnitine, and 2,4,4-trimethylpentanoylcarnitine. We then compared their behavior with branched-chain C8 acylcarnitines observed in patient samples and demonstrated our ability to chromographically resolve, and thus distinguish, octanoylcarnitine from branched-chain C8 acylcarnitines, correcting false positive MCADD results from expanded newborn screening.
authors.affiliation: Center for Mitochondrial Diseases, Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Minkler
authors.affiliation: Center for Mitochondrial Diseases, Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors.forename: Maria S K
authors.initials: MSK
authors.lastname: Stoll
authors.affiliation: Center for Mitochondrial Diseases, Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Ingalls
authors.affiliation: Center for Mitochondrial Diseases, Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA; Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Electronic address: charles.hoppel@case.edu.
authors.forename: Charles L
authors.initials: CL
authors.lastname: Hoppel
mesh_terms: D042964:Acyl-CoA Dehydrogenase / Q000172:deficiency*; D002331:Carnitine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005189:False Positive Reactions; D006801:Humans; D007231:Infant, Newborn; D008052:Lipid Metabolism, Inborn Errors / Q000175:diagnosis*; D015997:Neonatal Screening / Q000592:standards*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D042964:Acyl-CoA Dehydrogenase; C008698:octanoylcarnitine; D002331:Carnitine
keywords: Acylcarnitine; False positive; Medium-chain acyl-CoA dehydrogenase deficiency (MCADD); Newborn screening; Valproate; Valproylcarnitine