Quantification of amino acids and acylcarnitines in dried blood spots by FIA-MS/MS

Significance of the topic

Early and accurate measurement of amino acids and acylcarnitines in dried blood spots is critical for newborn screening and the diagnosis of inborn errors of metabolism. The non-derivatized FIA-MS/MS approach streamlines sample preparation, reduces analysis time, and enables high-throughput workflows in clinical research and diagnostic laboratories.

Objectives and overview

The study aimed to implement and evaluate a flow injection analysis-tandem mass spectrometry (FIA-MS/MS) method for simultaneous quantification of 11 amino acids and 13 acylcarnitines in dried blood spots. Using a NeoBase™ Complete Kit and Thermo Scientific instrumentation, the method was optimized for selectivity, sensitivity, and speed.

Methodology and instrumentation

The workflow comprised the following key steps and instruments:

• Sample preparation: 3.2 mm DBS punches extracted with PerkinElmer reagent containing isotopically labeled internal standards; incubation at 45 °C for 45 min with shaking.
• Flow injection analysis: Vanquish™ Flex Binary UHPLC system; 10 µL injection; total run time 1.5 min; mobile phase supplied by PerkinElmer.
• Mass spectrometry: TSQ Fortis™ triple-stage quadrupole with heated electrospray ionization in positive mode; selected reaction monitoring (SRM) transitions optimized for each analyte; data acquired and processed with TraceFinder™ 4.1.

Main results and discussion

The method demonstrated excellent precision and accuracy:

• Intra-assay precision (n=5) yielded median CVs below 6% for most analytes and a maximum of 10.1% for glutarylcarnitine (C5DC).
• Inter-assay precision (n=15) showed CVs below 10.3% across analytes.
• Accuracy, expressed as bias between nominal and measured values, ranged from –8.6% to +8.8% within supplier acceptance criteria.

The use of surrogate controls enabled quantification of arginine and ornithine despite their absence in native controls. Representative FIA-MS/MS profiles confirmed baseline separation and consistent internal standard response.

Benefits and practical applications of the method

This non-derivatized FIA-MS/MS assay delivers rapid, high-throughput screening of critical metabolic biomarkers in a 1.5-minute runtime. Its robust performance makes it suitable for clinical research, newborn screening programs, and quality control in drug development.

Future trends and applications

Advancements may include expansion to additional metabolites, automation of sample handling, integration with laboratory information management systems, and incorporation of machine-learning algorithms for data interpretation. Miniaturized sampling devices and multiplexed detection strategies will further enhance throughput and accessibility.

Conclusion

The implemented FIA-MS/MS method combined with the NeoBase™ kit on Thermo Scientific instrumentation achieves high selectivity, sensitivity, and reproducibility. It meets clinical research laboratory requirements for precision, accuracy, and speed, supporting reliable metabolic screening and monitoring.

Reference

No additional literature references were provided in the source document.