Simultaneous analysis of amino acids, acylcarnitines, and succinylacetone in dried blood spots for research using nonderivatized and derivatized methods

Importance of the Topic

Flow injection analysis coupled with tandem mass spectrometry (FIA‐MS/MS) has become a cornerstone technique for rapid, multiplexed quantification of metabolites in dried blood spots (DBS). Simultaneous measurement of amino acids, acylcarnitines, and succinylacetone supports newborn screening, inborn errors of metabolism research, and clinical studies. Simplifying sample preparation while maintaining analytical performance reduces turnaround time and hazardous reagent use in high‐throughput settings.

Objectives and Scope of the Study

This study aimed to develop and compare two FIA‐MS/MS workflows on a Thermo Scientific TSQ Endura triple quadrupole system for simultaneous detection of 12 amino acids, 18 acylcarnitines, and succinylacetone in DBS: one using derivatized (butyl esterification) sample preparation, the other employing nonderivatized extraction.

Methodology and Instrumentation

The core experimental approach and instrumentation included:

• Sample preparation: single-step extraction of a 1/8″ DBS punch with a mixture of stable isotope–labeled internal standards (12 amino acids, 12 acylcarnitines, succinylacetone) followed by butanol derivatization or direct reconstitution for nonderivatized analysis.
• Mass spectrometer: Thermo Scientific TSQ Endura triple quadrupole MS with heated electrospray ionization (HESI) in positive mode.
• Data acquisition: selected‐reaction monitoring (SRM) transitions optimized for each analyte and internal standard, collision energies and RF lens voltages tuned to maximize sensitivity and selectivity.
• Data processing: offline meta‐calculation software (iRC PRO) for automated peak integration, isotope ratio calculation, and quantification.

Key Results and Discussion

Both workflows delivered sharp FIA profiles and high signal‐to‐noise for all targets, including poorly ionizing analytes such as succinylacetone (SUAC) and C5DC.
Intra‐assay precision (n=10) for the derivatized method averaged 7.9%–8.0% for amino acids and 8.9%–9.0% for acylcarnitines across low, intermediate, and high concentrations. For the nonderivatized method it averaged 6.1%–7.2% for amino acids and 7.6%–8.2% for acylcarnitines.
Inter‐assay precision (n=70) remained under 15% for both approaches: derivatized assays showed 12.9%–13.5% for amino acids and 15.0%–16.1% for acylcarnitines; nonderivatized assays achieved 12.6%–12.8% for amino acids and 10.5%–12.7% for acylcarnitines.
Quantitative comparison between derivatized and nonderivatized workflows revealed strong correlation. Average method differences were ≤5% for amino acids and ≤15% for acylcarnitines, indicating equivalent accuracy.

Benefits and Practical Applications

Key advantages of these FIA-MS/MS methods include:

• Sub–2-minute run times and minimal sample cleanup for high throughput.
• Elimination of corrosive reagents in the nonderivatized workflow.
• Robust quantification supported by SRM and stable isotope internal standards.
• Broad applicability for newborn screening research, clinical validation, and QA/QC labs.

Future Trends and Potential Applications

Emerging trends likely to enhance FIA‐MS/MS platforms include integration of high‐resolution mass spectrometry, expanded panels for lipids and other small molecules, automated sample handling robots, and cloud‐based data analytics. Application areas will grow in metabolomics, pharmacokinetics, epidemiological studies, and point-of-care testing.

Conclusion

Both derivatized and nonderivatized FIA‐MS/MS approaches on the TSQ Endura system enable rapid, precise, and accurate simultaneous analysis of amino acids, acylcarnitines, and succinylacetone in DBS. The nonderivatized method simplifies workflow and reduces reagent hazards without sacrificing performance.

References

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