Measurement of Amino Acids and Acylcarnitines in Dried Bloodspots by Flow-Injection Analysis/Tandem Mass Spectrometry (FIA-MS/MS) for Clinical Research Use

Importance of the Topic

Dried blood spot (DBS) sampling offers a minimally invasive, stable and transport-friendly way to collect biological specimens for clinical research, epidemiology and therapeutic monitoring. Quantification of amino acids and acylcarnitines in DBS supports newborn screening, metabolic disorder studies and drug discovery by enabling high-throughput analysis of key biomarkers from small volumes.

Objectives and Study Overview

The primary goal was to demonstrate a rapid, cost-effective workflow for simultaneous measurement of amino acids and acylcarnitines extracted from DBS controls. Key aims included maximizing sample throughput, minimizing solvent consumption and reducing hands-on time while ensuring analytical precision suitable for clinical research use.

Methodology and Instrumentation

• Sample preparation: Commercial non-derivatized DBS control cards (RECIPE ClinSpot®) were extracted by adding solvent, followed by centrifugal filtration into a collection plate.
• Flow-Injection Analysis/Tandem MS: A variable-flow FIA method on a Waters ACQUITY UPLC/Xevo TQD IVD System under MassLynx 4.2 control with NeoLynx™ 4.2 Application Manager.
• MRM acquisition: 70 transitions covering 13 amino acids, 31 acylcarnitines and 26 isotopic internal standards.
• Injection cycle: Approximately 1.8 minutes per sample, leveraging load-ahead functionality and active needle wash to achieve a batch of 192 samples in under 6 hours, consuming <200 mL solvent.

Main Results and Discussion

Between-batch imprecision (five batches) for amino acids ranged from 4.0 % to 12.5 % CV; within-batch (n = 10) was <15 % CV for all targets. Acylcarnitine precision varied between 4.1 % and 6.3 % CV; low-abundance analytes met detection limits (~S/N 3:1). Variable flow rates enhanced dwell time and sensitivity without chromatographic separation. NeoLynx Application Manager enabled automated data processing, real-time technical review, encrypted result files and an audit-trail for full traceability.

Benefits and Practical Applications

This workflow delivers:

• High throughput: nearly 200 samples per run in under 6 hours.
• Low solvent use: <200 mL per batch, reducing operational costs.
• Integrated data management: automated report generation, hands-free review and secure data handling.
• Robust precision: CVs well within acceptable limits for clinical research.

Future Trends and Possibilities

Advancements may include expanded analyte panels for broader metabolomic profiling, further miniaturization of sampling devices, integration with automated sample-handling platforms, real-time cloud-based data analytics and coupling with other omics technologies. Adoption of ultra-high-throughput workflows and machine-learning-driven data interpretation will enhance large-scale screening and longitudinal studies.

Conclusion

The demonstrated FIA-MS/MS method on the Waters ACQUITY UPLC/Xevo TQD IVD System achieves rapid, precise quantification of amino acids and acylcarnitines from DBS. The combination of variable flow injection, minimal solvent consumption and streamlined data processing meets the demands of high-volume clinical research and supports reliable biomarker analysis.

Reference

RECIPE Chemicals + Instruments GmbH ClinSpot® DBS control kit; Waters Corporation ACQUITY UPLC/Xevo TQD IVD System and NeoLynx™ 4.2 Application Manager.