The Changing Landscape of Mass Spectrometry-Based Newborn Screening

Konstantinos Petritis
Centers for Disease Control and Prevention, Atlanta, GA

Konstantinos Petritis, PhD (Presenter) Centers for Disease Control and Prevention

Presenter Bio: Dr. Petritis received his MSc and PhD in Analytical Chemistry from the University of Orleans, France. In 2002, he joined Pacific Northwest National Laboratory, in Richland, WA, as a post-doctoral fellow and later as a senior staff scientist where he worked in the field of mass spectrometry based proteomics. In 2009, he was hired as an associate professor and laboratory head at the Translational Genomics Research Institute in Phoenix, AZ to work on biomarker development. In 2014, he joined the Arizona office of newborn screening and Phoenix Children’s Hospital as a principal investigator where he led several federal public health and research grants before joining CDC in June 2017. He has worked on bioanalytical mass spectrometry, biomarker development, automation, predictive algorithms and proteomic research. His current interests include but not limited to advanced analytical methods development and validation for newborn screening, development of dried blood spots based quality assurance materials and calibrators, clinical assays harmonization and metabolomics.

Relevant Financial Disclosures (within past 24 months, reported on Jun 11, 2026) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Newborn screening is one of the earliest and most successful applications of mass spectrometry (MS) in clinical chemistry. MS enabled the rapid expansion of disorders screened at birth and can currently be used to detect over 85% of the 66 core and secondary conditions included in the U.S. Recommended Uniform Screening Panel (RUSP). The field is expected to undergo another major expansion driven by the introduction of advanced therapeutics, including gene therapies. Dozens of candidate disorders are currently under consideration, most of which are amenable to MS-based screening. However, this expansion presents several analytical challenges, including increased false-positive rates, limited analyte multiplexing capacity under flow-injection analysis tandem mass spectrometry (FIA-MS/MS), and limited specimen volume from dried blood spots (DBS).

METHODS:
DBS specimens were prepared by extracting a 3.2 mm punch with 100 µL mixtures of organic solvent and water containing application-specific organic acids. Multiple MS platforms were evaluated, including triple quadrupole instruments from Agilent Technologies, Waters, and SCIEX, as well as Thermo Q-Exactive Plus/HF systems. First-tier screening experiments were performed using FIA-MS/MS, direct infusion with the Advion Triversa Nanomate®, ultra-fast electrophoretic separations using the ZipChip® platform (908 Devices), and rapid chromatographic separations using HILIC guard columns. Second-tier screening was performed using a 150 mm HILIC column. Intelligent reflex-to-second-tier screening experiments were conducted on an Agilent Ultivo coupled to a 1290 Infinity II LC system using proprietary software.

RESULTS AND DISCUSSION:
Despite significant technological advances in LC-MS/MS instrumentation, the general format and throughput of MS/MS-based first-tier newborn screening have remained largely unchanged for more than three decades. This presentation will review the current state of newborn screening, the analytical challenges associated with expanding disorder panels, and the compromises currently required to balance throughput and clinical specificity.

One novel approach demonstrates a 4-fold increase in primary FIA-MS/MS screening throughput, reducing analysis time from 2 minutes to 0.5 minutes per specimen while simultaneously improving clinical specificity through the incorporation of more selective biomarkers, such as total homocysteine for the detection of homocystinuria, enabled through selective derivatization strategies.

Alternatively, ultra-fast electrophoretic or chromatographic separations can be implemented to resolve isomeric and isobaric compounds, enabling the use of more specific biomarkers and reducing interferences from existing biomarkers without compromising current screening throughput. The advantages and practical considerations of high-resolution mass spectrometry, with and without separations, for both targeted and untargeted metabolic profiling will also be discussed.

Another strategy to reduce false positives involves second-tier screening of presumptive positive specimens identified during first-tier analysis. Current second-tier approaches are typically limited to single disorders or provide only modest multiplexing capabilities. We present a novel highly multiplexed assay capable of simultaneously measuring dozens of second-tier analytes for the screening of more than 15 disorders.
Finally, we introduce the concept of intelligent reflex screening, in which software identifies presumptive positive specimens during first-tier analysis and automatically initiates confirmatory LC-MS/MS second-tier testing through reinjection of the same specimen without user intervention. This configuration enables same-day primary and confirmatory testing on a single instrument platform, which is particularly important for time-critical newborn disorders.