Newborn Screening of 57 Metabolites by FIA-MS/MS

The neonatal screening center of G. d’Annunzio University participates in an accredited national network of laboratories that adopts a uniform screening panel covering 57 clinically-relevant metabolites, including 14 amino acids, 2 nucleosides, free carnitine, 35 acyl-carnitines, 4 lysophosphatidylcholines and succinylacetone. Details of the metabolites evaluated and their reference values, deriving from the NeoBase™ 2 Non-derivatized MSMS kit (PerkinElmer Life and Analytical Sciences, Turku, Finland) are reported in Table S1. Metabolites were extracted from plasma specimens using a modified protocol based on the manufacturer’s workflow designed for newborn screening. Briefly, 10 µL of plasma were subjected to protein precipitation by incubation with 125 µL of internal standards (IS) solution (PerkinElmer) for 30 min at 45 °C, 700 rpm (Eppendorf ThermoMixer® C). Proteins were removed after centrifugation (at max speed in an Eppendorf 5424) and clear supernatants (125 µL) were transferred into new vials. An additional 1 h incubation step was required to derivatize succinylacetone (SA). Finally, 100 µL of centrifuged samples were transferred to 96-well plates for injection of 10 µL into the ion source. Acquisition, 1.2 min long injection-to-injection, was carried out on a FIA platform RenataDX Screening System, including a 3777 C IVD Sample Manager and an ACQUITY™ UPLC™ I-Class IVD Binary Solvent Manager, coupled to a Xevo™ TQD IVD tandem quadrupole mass spectrometer (both from Waters Corporation, Milford, MA, USA). The flow gradient for the mobile phase provided by the kit was set as follows: 0.15 mL/min from 0 to 0.170 min; 0.01 mL/min from 0.170 to 0.980 min; 0.7 mL/min from 0.980 to 1.180 min; 0.15 mL/min from 1.180 min to the end. Data were processed by MassLynx™ (IVD) Software V4.2 with NeoLynx™ Application Manager (Waters Corp). Mass spectrometry (MS) parameters and a complete list of the metabolites and their internal standards (ISs) are provided in Table S3. Metabolite extraction from dried venous or capillary blood spotted on filter paper was performed according to PerkinElmer’s protocol using the NeoBase™ 2 non-derivatized MSMS kit. As already described [62 (link)–65 (link)], samples were punched out into 3.2 mm disks for the extraction of amino acids, nucleosides, free carnitine, acyl-carnitines, lysophosphatidylcholines and succinylacetone. Finally, 10 µL of supernatant were analyzed on the same FIA-MS/MS platform described above, using the same parameters for injection, MS acquisition and raw data processing. The micromolar (µM) concentrations of all tested metabolites are presented in Table S7.