Neobase non derivatized kit

Amino acids, acylcarnitines and succinylacetone were extracted from a single 3.2 mm-diameter punch from each dried blood spot (DBS) using the NeoBase Non-derivatized kit and, from 2019, the NeoBase 2 Non-derivatized kit (PerkinElmer, Turku, Finland) and quantified by flow injection analysis with ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). These first-tier analyses were performed on two Acquity Xevo-TQS systems, an Acquity Xevo TQS micro or a Quattro Premier XE (Waters, Milford, MA, USA). The first-tier cut-off values used for the 20 IEMs are depicted in Table 1. In the case of an abnormal screening result in the first assessment, two new 3.2 mm diameter DBS punches were re-analyzed.
Biotinidase activity in DBSs was initially analyzed with a Victor Multilabel Plate Reader (PerkinElmer, Turku, Finland) and measured by a semi-quantitative method using biotin-6-amidoquinoline as a substrate [4 (link)]. From 2013, screening for biotinidase deficiency (BD) was performed using the Genetic Screening Processor (GSP^®) and the GSP Neonatal Biotinidase kit, both from PerkinElmer.

A targeted metabolome analysis was carried out, including glucose, free carnitine, acylcarnitines (AC2, AC3, AC4, AC5, AC5:1, AC6, AC8, AC8:1, AC10, AC10:1, AC10:2, AC12, AC12:1, AC14, AC14:1, AC14:2, AC14OH, AC16, AC16:1, AC16:1OH, AC16OH, AC18, AC18:1, AC18:1OH, AC18:2, and AC18OH), arginosuccinate (ASA), and 12 L-amino acids (alanine, glycine, arginine, methionine, proline, arginine, valine, leucine, phenylalanine, tyrosine, citrulline, and ornithine). The above was achieved by using a Quattro Micro API (MicroMass, Cary, NC, USA) tandem mass spectrometer (MS-MS). All procedures for sample preparation and MS-MS analysis were performed using a NeoBase Non-derivatized kit (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s protocol. The serum was dried in filter papers, and single disks were punched from each spot using a 3 mm punch. One disk was used per well. Using a multichannel pipette, 190 μL of extraction solution containing a mixture of the respective stable isotope-labeled internal standards was added to each well. The plate was covered with aluminum foil, shaken at 650× g, and incubated for 30 min at 30 °C. The plate was finally placed in the auto-sampler for analysis. All results were expressed in μM.

Using TQD HPLC-MS/MS system (Waters, MA, United States) and NeoBase non-derivatized kit (PerkinElmer, United States), a total of 43 metabolites in a DBS were measured, including 11 amino acids, 30 acyl-carnitines, free carnitine, and succinylacetone. In short, the assay consists of three operations. First, 100 μl extract solution containing internal standards was added into U bottom plates and incubated for 45 min at 45°C. Second, 75 μl extract solution was transferred into V bottom plates. Third, after 2 h stand at ambient temperature, 25 μl solution was used for metabolites analysis on tandem mass spectrometry. Three levels of internal quality controls including blank, low, and high levels of pertinent acylcarnitines in dried blood spot were used.