Background: Most first-tier newborn screening (NBS) biomarkers are evaluated by a 2-min flow injection analysis coupled to tandem mass spectrometry (FIA-MS/MS) assay. The absence of separation prior to MS/MS analysis can lead to false positives and inconclusive results due to interferences by nominal isobars and isomers. Therefore, many presumptive positive specimens require confirmation by a higher specificity second-tier assay employing separations, which require additional time and resources prior to patient follow-up.
Methods: A 3.2-mm punch was taken from dried blood spot (DBS) specimens and extracted using a solution containing isotopically labeled internal standards for quantification. Analyses were carried out in positive mode using a commercially available microfluidic capillary electrophoresis (CE) system coupled to a high-resolution mass spectrometer (HRMS).
Results: The CE-HRMS platform quantified 35 first- and second-tier biomarkers from a single injection in <2-min acquisition time, thus, successfully multiplexing first- and second-tier NBS for over 20 disorders in a single DBS punch. The CE-HRMS platform resolved problematic isobars and isomers that affect first-tier FIA-MS/MS assay specificity, while achieving similar quantitative results and assay linearity.
Conclusions: Our CE-HRMS assay is capable of multiplexing first- and second-tier NBS biomarkers into a single assay with an acquisition time of <2 min. Such an assay would reduce the volume of false positives and inconclusive specimens flagged for second-tier screening.
Keywords: acylcarnitines; amino acids; capillary electrophoresis; dried blood spots; high-resolution mass spectrometry; newborn screening.
Published by Oxford University Press on behalf of the American Association for Clinical Chemistry 2021. This work is written by US Government employees and is in the public domain in the US.