Determination of inborn errors of metabolism

Significance of the Topic

The profiling of amino acids and acylcarnitines in dried blood spots (DBS) is fundamental to newborn screening for inborn errors of metabolism. Early detection reduces mortality and morbidity through timely interventions. Mass spectrometry (MS/MS) offers high specificity and sensitivity, distinguishing true positives from false results in a single analytical run.

Objectives and Study Overview

This study aimed to implement a flow injection analysis–tandem mass spectrometry (FIA-MS/MS) method for simultaneous quantification of 13 amino acids and 13 acylcarnitines in DBS. The approach was designed for clinical research applications, emphasizing rapid throughput and analytical robustness.

Methodology

The sample preparation involved punching 3.2 mm DBS discs into a 96-well plate, adding 100 μL of extraction reagent containing isotopically labeled internal standards, and shaking at 600 rpm for 20 minutes. Extracts were transferred and analyzed by FIA using a binary UHPLC system with a 1.5 minute runtime. MS detection employed selected reaction monitoring (SRM) in positive electrospray mode, optimizing collision energies and tube lens settings for each analyte.

Instrumentation

• Thermo Scientific Vanquish Flex Binary UHPLC system for flow injection analysis
• Thermo Scientific TSQ Fortis triple-stage quadrupole mass spectrometer with heated electrospray ionization (HESI)
• Chromsystems MassChrom kit for reagents and internal standards

Main Results and Discussion

The method demonstrated excellent precision with intra‐assay CVs below 9% (except C5DC up to 14.9%) and inter‐assay CVs below 12.5% (C5DC up to 17.7%). Accuracy, expressed as % bias, ranged between –12.8% and 11.8% for all analytes, meeting supplier acceptance criteria. Representative SRM profiles confirmed clear detection of target transitions and stable internal standard responses.

Benefits and Practical Applications

• High‐throughput screening with 1.5 minute runtime per sample
• Simultaneous quantification reduces sample consumption and analysis time
• Robust reproducibility across multiple days supports clinical research workflows
• Non‐derivatized assay simplifies sample handling

Future Trends and Applications

Ongoing advances may include integration with automated DBS sampling, expanded panels covering additional biomarkers, and coupling with high‐resolution MS for improved specificity. Digital data management and AI‐driven interpretation can further enhance screening algorithms and predictive analytics.

Conclusion

The FIA‐MS/MS method using the MassChrom kit on Vanquish Flex and TSQ Fortis platforms provides a fast, reliable, and sensitive workflow for amino acid and acylcarnitine profiling in DBS. Its performance aligns with clinical research requirements, offering a practical solution for inborn error of metabolism studies.

References

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