Objectives: Acylcarnitine and amino acid analyses of dried blood spot (DBS) samples using tandem mass spectrometry in newborn screening (NBS) programmes can generate false positive (FP) results. Therefore, implementation of second-tier tests (2TTs) using DBS samples has become increasingly important to avoid FPs. The most widely used 2TT metabolites include methylmalonic acid, 3-hydroxypropionic acid, methylcitric acid, and homocysteine.
Methods: We simultaneously measured 46 underivatised metabolites, including organic acids, acylglycine and acylcarnitine isomers, homocysteine, and orotic acid, in DBS samples using tandem mass spectrometry. To validate this method, we analysed samples from 147 healthy newborns, 160 patients with genetic disorders diagnosed via NBS, 20 patients with acquired vitamin B12 deficiency, 10 newborns receiving antibiotic treatment, and nine external quality control samples.
Results: The validation study revealed that 31 metabolites showed good analytical performance. Furthermore, this method detected key metabolites for all diseases associated with increased levels of the following acylcarnitines: C3, C4, C5, C4DC/C5OH, and C5DC. The sensitivity of this method to detect all diseases was 100 %, and the specificity was 74-99 %, except for glutaric aciduria type 1. This method can also be used to diagnose mitochondrial fatty acid β-oxidation disorders (FAODs) and urea cycle defects (UCDs).
Conclusions: We have described a 2TT panel of 31 metabolites in DBS samples based on an easy and rapid method without derivatisation. Its implementation allowed us to distinguish between different organic acidurias, some FAODs, and UCDs. This new strategy has increased the efficiency of our NBS programme by reducing FP and false negative results, second sample requests, and the time required for diagnosis.
Keywords: acylcarnitines; acylglycines; mass spectrometry; newborn screening; organic acids; second-tier test.
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