Empower the Quantitation of Amino Acids and Acylcarnitine for Inborn Error of Metabolism Screening using Flow Injection – Orbitrap Mass Spectrometer

Importance of the Topic

Newborn screening for inborn errors of metabolism (IEM) represents one of the most effective disease prevention programs worldwide. Accurate quantitation of amino acids and acylcarnitines in dried blood spots (DBS) is essential for early detection of metabolic disorders. High-resolution accurate-mass (HRAM) mass spectrometry addresses limitations of traditional triple quadrupole methods by resolving isobaric interferences and providing full-MS and MS2 spectral confidence.

Study Objectives and Overview

This work aimed to develop and validate a flow injection analysis (FIA) method coupled with HRAM Orbitrap mass spectrometry to quantify 12 amino acids and 13 acylcarnitines in DBS using a commercial kit. The goals included demonstrating accuracy, intra- and inter-day precision, reproducibility of internal standards, and feasibility for routine IEM screening.

Methodology

Sample Preparation and Workflow:

• DBS discs (3.2 mm) from ClinChek control levels I & II were punched, extracted, and incubated with stable-isotope internal standards.
• Extracted supernatant transferred directly for analysis; no chromatographic separation was performed.
• Each sample was injected twice (5 µL) over three days for precision assessment.

Used Instrumentation

• Vanquish Flex UHPLC system for flow delivery.
• Thermo Scientific Orbitrap Exploris 120 mass spectrometer with OptaMax NG HESI source.
• TraceFinder software (v5.1 SP3 Clinical) for data processing, mzVault library matching, and isotope pattern matching.

Main Results and Discussion

• All 12 amino acids and 13 acylcarnitines were reliably quantified with targeted-MS2 (tMS2) except glycine, which utilized full-MS spectra due to low m/z.
• Intra- and inter-day precision (%RSD) for all analytes was below 12%; internal standard reproducibility (%CV) was below 9%.
• Measured concentrations fell within the kit’s defined target ranges, confirming method accuracy comparable to a benchmark triple quadrupole platform.
• TraceFinder interface demonstrated clear extracted ion chromatograms, library match scores, and isotope pattern fidelity, supporting high confidence in analyte identification.

Benefits and Practical Applications

• Elimination of chromatographic step accelerates sample throughput, ideal for high-volume screening laboratories.
• HRAM capability distinguishes isobaric compounds (e.g., leucine vs. 4-hydroxyproline) with >60,000 FWHM resolution, reducing false positives.
• Comprehensive MS2 spectra improve analyte specificity and support retrospective data mining.

Future Trends and Potential Uses

• Expansion of analyte panels to include additional biomarkers and lipids for broader metabolic profiling.
• Integration of machine learning algorithms for automated anomaly detection and quantitative review.
• Development of miniaturized or portable HRAM platforms for point-of-care metabolic screening.

Conclusion

The validated FIA-HRAM-MS/MS workflow provides a robust, high-throughput solution for quantitative analysis of amino acids and acylcarnitines in DBS. Its high mass accuracy, precision, and specificity make it well suited for first-tier newborn IEM screening and potential future expansions in clinical metabolomics.

Reference

1. Guo J., Song Y., Hassell K. Empower the Quantitation of Amino Acids and Acylcarnitine for Inborn Error of Metabolism Screening using Flow Injection–Orbitrap Mass Spectrometer. Thermo Fisher Scientific TN001293, 2023.