= Emerging. More than 5 years before clinical availability. (26.62%)
= Expected to be clinically available in 1 to 4 years. (38.91%)
= Clinically available now. (34.47%)
MSACL 2020 US : Pickens

MSACL 2020 US Abstract

Topic: Metabolomics

Podium Presentation in the Ether on Wednesday at 16:35 (Chair: Amy Caudy)

High Resolution Mass Spectrometry Newborn Screening Applications for Quantitative Analysis of Amino Acids and Acylcarnitines in Dried Blood Spots

Austin Pickens (Presenter)
Centers for Disease Control and Prevention

Presenter Bio(s): Austin Pickens received his doctorate in Human Nutrition from Michigan State University, investigating biomarkers discovery using targeted and high resolution untargeted mass spectrometry. He then joined a cancer informatics startup within the ORACLE Corporation as a data scientist. Austin joined the Biochemical Mass Spectrometry Laboratory (BMSL) at the Centers for Disease Control and Prevention in November 2017, where he develops methods, evaluates new technologies, and investigates metabolomics in newborn screening.

Authors: C. Austin Pickens, Konstantinos Petritis
Newborn Screening and Molecular Biology Branch, US Centers for Disease Control and Prevention, Atlanta, Georgia, USA


Introduction: Amino acid and acylcarnitine first-tier newborn screening typically employs derivatized or non-derivatized sample preparation methods followed by flow injection analysis (FIA) coupled to triple quadrupole (TQ) tandem mass spectrometers (MS/MS). The low resolving power of TQ instruments results in difficulties distinguishing nominal isobaric metabolites, especially those with identical quantifying product ions such as malonylcarnitine (C3DC) and 4-hydroxybutylcarnitine (C4OH). This limitation is often overcome by utilizing sample derivitization to shift masses for distinguishing nominal isobars such as C3DC and C4OH, however, this reaction increases sample preparation time, requires corrosive chemicals, and creates other isobars particularly with acylcarnitines such as acetylcarnitine and glutamic acid. The objective of our study was to develop and assess methodologies employing High Resolution (HR) MS for newborn screening assays to overcome these challenges.

Methods: The Centers for Disease Control and Prevention’s (CDC) Newborn Screening Quality Assurance Program (NSQAP) quality control dried blood spot cards were analyzed in this study. Samples were extracted following the non-derivatized protocol as previously described, and were resuspended and analyzed by FIA in 50:50 acetonitrile:water with 0.1% formic acid. HRMS analysis was performed on a hybrid quadrupole-orbitrap system using selected ion monitoring (SIM) across available resolving powers and parallel reaction monitoring (PRM) for MS/MS at mass resolution 17 000. Sample introduction of DBS extracts were conducted using a chip-based nano-ESI direct fusion system for FIA.

Results: Twenty-eight amino acids and acylcarnitines were analyzed by FIA-HRMS using MS acquisition methods including PRM, SIM across all available mass resolving powers, and a combined FIA-PRM/SIM hybrid. HRMS acquired data were compared to FIA-TQ-MS/MS and results were very similar for most analytes. Most notably, quantitation of C3DC and C4OH was successful by HRMS in non-derivatized sample extracts, thus, potentially eliminating sample derivatization requirements. Quantitation differed between SIM and PRM acquired data for several metabolites, and it was determined these quantitative differences were due to collision energy differences or kinetic isotope effects between the unlabeled metabolite and the corresponding labeled isotopologue internal standards. After optimization, a proof-of-concept hybrid PRM/SIM/Full Scan method and workflow were developed under FIA conditions which allowed accurate quantitation for analytes of interest. The addition of the full scan function allowed the combination of untargeted metabolomic screening combined with sensitive targeted quantitative screening in a single analysis.


Preliminary results indicate that HRMS not only is a viable alternative to TQ-MS/MS for newborn screening but presents several advantages over it.

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