Improving the Throughput and Specificity for Small-Molecule Analysis During First-Tier Mass Spectrometry–Based Newborn Screening

Background/Objectives: Mass spectrometry-based newborn screening for small-molecule biomarkers typically employs a rapid first-tier screen that omits chromatographic separations before mass spectrometric analysis, followed, only for a subset of samples and disorders, by a longer, more specific second-tier assay that includes liquid chromatographic separation prior to mass spectrometry. The second-tier screen is used when the primary biomarker lacks sufficient specificity and may result in higher false-positive rates. The throughput and specificity of first-tier newborn screening assays have been relatively stagnant over the past two decades despite significant improvements in mass spectrometry instrumentation. With the continuous expansion of disorders added to the Recommended Uniform Screening Panel in the United States, newborn screening laboratories have a need for higher-throughput assays and improved specificity. Methods: We developed and evaluated two first-tier tandem mass spectrometry approaches using a modern dual-needle, dual-loop LC-MS/MS platform: (1) a 30-s flow injection analysis tandem mass spectrometry (FIA-MS/MS) assay and (2) a rapid first-tier liquid chromatography tandem mass spectrometry (LC-MS/MS) assay using a hydrophilic interaction chromatography (HILIC) guard column (1TH). Analytical performance was assessed using dried blood spot quality control and linearity materials, including evaluations of recovery, precision, linearity, and matrix effects. Results: The 30-s FIA-MS/MS assay quadrupled the throughput of current 2-min FIA-MS/MS assays used routinely in newborn screening laboratories. The throughput improvement was achievable due to increased scan speeds of the mass spectrometer as well as the dual needle/loop design of the autosampler. In addition, these instrumentation improvements made it possible to employ liquid chromatographic separations prior to MS/MS analysis without sacrificing the approximately 2-min sample-to-sample throughput of conventional FIA-MS/MS workflows. The 1TH LC-MS/MS method separated critical isobaric and isomeric biomarkers, reduced matrix effects, improved specificity and quantification accuracy, and demonstrated acceptable recovery, precision, and linearity for newborn screening applications. Conclusions: Recent advances in LC-MS/MS instrumentation can be leveraged to either substantially increase first-tier newborn screening throughput or improve analytical specificity while maintaining current workflow timelines. First-tier LC-MS/MS using a HILIC guard column provides improved specificity that can reduce the need for second-tier testing, thereby improving overall throughput and turnaround time of the newborn screening workflow. These approaches provide flexible solutions for newborn screening laboratories seeking to accommodate expanding screening panels without compromising analytical quality or efficiency.