Rapid Analysis of Acylcarnitinesin Dried Blood Spots Using Fast Chromatography with Ion Mobility High Resolution Mass Spectrometry

Importance of the Topic

Acylcarnitines serve as essential intermediates in fatty acid transport and oxidation, making their accurate analysis crucial for diagnosing metabolic disorders such as those monitored in newborn screening programs.
The presence of isobaric acylcarnitines—molecules sharing identical nominal masses but differing in structure—poses significant analytical challenges, requiring orthogonal separation techniques to ensure precise identification and quantitation.

Study Objectives and Overview

This work presents the development and demonstration of a rapid one-minute chromatographic method combined with ion mobility high-resolution mass spectrometry (LC-IM-QTOF) for profiling acylcarnitines directly from dried blood spots (DBS).
The primary goal was to achieve baseline separation and confident identification of two critical isobaric biomarkers, malonylcarnitine (C3DC) and 3-hydroxybutyrylcarnitine (C4OH), in a high-throughput workflow.

Methodology and Instrumentation

Sample Preparation:

• DBS punch: 3 mm disc; incubated with 100 µL internal standard solution for 45 min with shaking.
• No derivatization or extract drying; supernatant directly analyzed to minimize hands-on time.

Chromatography and Detection:

• Column: 100 mm short C18.
• Mobile phase: 70:30 to 95:5 acetonitrile:water gradient over 0.5 min (total run time 1 min), containing ammonium modifier.
• Instrument: Agilent 6560 IM-QTOF with ion mobility spectrometry (IMS).

Key Results and Discussion

The nominal mass difference between C3DC (m/z 248.1134) and C4OH (m/z 248.1498) of 0.03 Da was resolvable by the high-resolution mass analyzer.
Ion mobility separation provided an additional drift time distinction (~4 Ų difference in collisional cross section), allowing clear discrimination of the isobaric pair in under one minute.
Extracted ion chromatograms and calibration curves demonstrated robust detection sensitivity even at differing abundance levels; it was recommended to increase punch size (e.g., two 3 mm or a single 5 mm) to boost analyte recovery and signal quality when necessary.

Benefits and Practical Applications

This rapid LC-IM-QTOF approach enables:

• High-throughput screening with sub-minute analysis times.
• Direct analysis from DBS without extensive sample work-up.
• Confident differentiation and quantitation of isobaric acylcarnitines relevant to newborn and metabolic disorder screening.

Future Trends and Potential Applications

Future work may explore:

• Optimization of punch size and sample loading to further enhance sensitivity for low-abundance markers.
• Expansion to larger biomarker panels for comprehensive metabolic profiling.
• Integration of machine learning tools to predict collision cross sections and streamline method development.

Conclusion

The presented one-minute LC-IM-QTOF method delivers rapid, reliable separation and identification of challenging isobaric acylcarnitines directly from dried blood spots, offering a powerful tool for high-throughput metabolic investigations and clinical screening applications.

Reference

1. Plante P-L et al., ‘‘Predicting Ion Mobility Collision Cross-Sections Using a Deep Neural Network: DeepCCS,’’ Analytical Chemistry, 2019, 91(8):5191–5199.