Abstract

OBJECTIVE: Newborn screening is used to diagnose inherited metabolic diseases (IEMs), many of which are diagnosed via flow injection analysis coupled with tandem mass spectrometry (FIA-MS/MS). Some metabolites are isobaric and thus are not distinguished. Second-tier tests have been developed to facilitate this separation, often involving liquid chromatography. Some isobaric acyl-carnitines are associated with IEMs characterized by the suppression of ketogenesis. These are acquired as a sum of signals at the first screening, as in the case of dicarboxylic and hydroxylated acyl-carnitines (C3DCC4OH, C4DCC5OH, C5DCC6OH). In this regard, a second-tier test based on FIA-MS/MS has been developed.

MATERIALS AND METHODS: Internal standards (ISs) of the NeoBase TM 2 non-derivatized MSMS kit (Revvity, ex PerkinElmer) were employed; for the isobaric couples, the corresponding ISs were 2H3-C4, 2H9-C5, and 2H6-C5DC. A derivatization step using 3N HCl in n-butanol was added to the normal sample preparation of the kit. Samples were analyzed by FIA-MS/MS through the RenataDX TM screening system coupled to a Xevo TM TQD IVD mass spectrometer.

RESULTS: This approach allowed rapid differentiation between isobaric couples without a chromatographic separation. The method is accurate and precise for routine diagnostics. In this context, a clinical suspect was identified between 3-methylcrotonylglycinuria, multiple carboxylase deficiencies, 3-methylglutaconic aciduria type I, and 3-hydroxy-3-methylglutaric aciduria in a newborn showing elevated methylmalonyl-carnitine (C4DC)/3-hydroxyisovaleryl-carnitine (C5OH) levels at the first screening. The second-tier test highlighted alterations in the levels of C5OH rather than C4DC.

CONCLUSIONS: For the clinical approach, the availability of this simple second-tier test supports a rapid differential diagnosis among IEMs that differ in the degree of metabolic decompensation through suppression of ketogenesis.