Simultaneous Analysis of Amino Acids, Acylcarnitines, and Succinylacetone in Dried Blood Spots for Research Using Nonderivatized and Derivatized Methods

Importance of the Topic

This work addresses rapid and comprehensive screening of amino acids, acylcarnitines and succinylacetone in dried blood spots—a critical need in research on inborn errors of metabolism and newborn screening. Integrating nonderivatized and derivatized workflows simplifies sample handling, reduces use of corrosive reagents and leverages modern mass spectrometry sensitivity.

Aims and Study Overview

The study aimed to develop and compare two flow-injection analysis-tandem mass spectrometry (FIA-MS/MS) methods on a TSQ Endura instrument: one using derivatization (butyl ester formation) and one analyzing native free acids. Both approaches target 12 amino acids, 18 acylcarnitines and succinylacetone in a single extraction from dried blood spots.

Methodology

• Sample Preparation

• Nonderivatized: Single punch extraction with isotope-labeled standards, 45-minute shake, evaporation, reconstitution in 50:50:0.02 acetonitrile/water/formic acid.
• Derivatized: Same initial extraction, followed by butanol-HCl incubation at 65 °C, evaporation and reconstitution.

• Chromatography: Direct flow injection with a 1.5 min run time using a 50:50 acetonitrile/water/formic acid mobile phase.
• Mass Spectrometry: Heated electrospray ionization, positive mode, selected-reaction monitoring (SRM) for all analytes. Optimized collision energies and RF lens settings ensured high sensitivity and selectivity.
• Data Processing: Automated quantification using iRC PRO metacalculation software, reducing post-analytical processing to minutes.

Used Instrumentation

• TSQ Endura triple-quadrupole MS with heated electrospray ionization
• Dionex UltiMate HPG-3200 RS pump and UltiMate WPS-3000 TRS autosampler

Main Results and Discussion

• Precision: Both methods achieved intra-assay CVs below 10% and inter-assay CVs below 16% across low, intermediate and high QC levels.
• Sensitivity: SRM enabled reliable detection even for poorly ionizing analytes (succinylacetone, hydroxyacylcarnitines) in both workflows.
• Method Correlation: Quantitative differences between derivatized and nonderivatized approaches were low (average <5% for amino acids/succinylacetone and <15% for acylcarnitines), confirming strong agreement.

Benefits and Practical Applications

• Streamlined workflow: Single extraction for diverse analytes reduces handling time.
• Flexibility: Choice of nonderivatized method avoids corrosive reagents and simplifies lab operations.
• High throughput: 1.5 min per sample by FIA-MS/MS supports large-scale screening.
• Research utility: Suitable for metabolic profiling, newborn screening studies and biobank analyses.

Future Trends and Opportunities

• Integration with high-resolution MS to further improve selectivity.
• Expansion to additional biomarkers (e.g., organic acids, novel acylcarnitine variants).
• Automated sample preparation platforms for full walk-away operation.
• Application of machine learning in data processing for pattern recognition in metabolic disorders.

Conclusion

Both derivatized and nonderivatized FIA-MS/MS methods on the TSQ Endura deliver rapid, precise and highly correlated quantification of amino acids, acylcarnitines and succinylacetone from dried blood spots. The nonderivatized approach offers a greener, simpler alternative without compromising performance.

Reference

1. Chace DH et al. Clin. Chem. 2003, 49(11), 1797–1817.
2. Chace DH et al. Clin. Chem. 1993, 39(1), 66–71.
3. Millington DS et al. J. Inherit. Metab. Dis. 1990, 13(3), 321–324.
4. Dhillon KS et al. Clin. Chim. Acta. 2011, 412(11–12), 873–879.
5. De Jesus VR et al. Clin. Chim. Acta. 2010, 411(9–10), 684–689.