Integration of amino acid, acylcarnitine and steroids analysis in single FIA/LC-MS/MS platform

Importance of the topic

Dried blood spot (DBS) screening using mass spectrometry enables minimally invasive sampling and high-throughput detection of biochemical markers crucial for newborn screening and clinical diagnostics. Combining amino acids, acylcarnitines, and steroids in a single platform reduces sample volume, streamlines workflows, and enhances diagnostic accuracy.

Objectives and Study Overview

This work aimed to integrate flow injection analysis (FIA) of amino acids and acylcarnitines with liquid chromatography-mass spectrometry (LC-MS/MS) analysis of key steroids on a single system. The study evaluated analytical performance, sensitivity, and throughput for monitoring 8 amino acids, 17 acylcarnitines, and 5 steroids from one DBS punch.

Methodology and Workflow

Sample Preparation:

• 5 mm DBS punches extracted in 180 µL of 80% acetonitrile-water with isotopically labeled internal standards for each analyte class
• Ultrasonic extraction for 30 min in 96-well plates

Chromatographic Setup:

• Two‐position, six‐port high‐pressure switching valve to direct flow between FIA and LC paths
• FIA mode for rapid AA and AC quantification: direct injection at 1 µL, isocratic conditions (0.1% formic acid in water/methanol), 74 s runtime per sample
• LC mode for steroids: Phenomenex Kinetex XB-C18 column (50 × 2.1 mm, 2.6 µm), 5 min gradient (50–90% methanol, 0.1% formic acid)

Mass Spectrometry:

• Heated electrospray ionization on a triple quadrupole (LCMS-8050)
• Optimized multiple reaction monitoring (MRM) transitions for all target analytes and internal standards

Instrumentation

• Nexera UHPLC system (SIL-30AC autosampler, LC-30AD pumps)
• High‐pressure switching valve (FCV-12AH)
• Guard columns for desalting (GL Sciences cartridge)
• Triple quadrupole mass spectrometer (Shimadzu LCMS-8050)

Main Results and Discussion

Analytical performance demonstrated:

• Precision: intra- and inter-day CVs generally <10% across all analytes
• Sensitivity: LOD for 17-hydroxyprogesterone at 0.19 ng/mL; other steroids 0.23–0.54 ng/mL; amino acids and acylcarnitines in sub-ng/mL range
• Throughput: full panel analysis in two injections within 7 min
• Linear response across clinically relevant concentration ranges

Integration of FIA and LC modes minimized false positives and improved quantitative accuracy compared to immunoassays.

Benefits and Practical Applications

The combined platform offers:

• Reduced sample volume to 1 µL per injection
• Streamlined workflow for newborn screening labs and clinical research
• Enhanced screening capability for metabolic disorders and corticosteroid profiling
• Simplified logistics: DBS storage, transport, and lower biohazard risk

Future Trends and Applications

Potential developments include:

• Expansion to additional biomarkers (e.g., vitamins, lipids)
• Automation and coupling with microfluidics for point-of-care testing
• High-resolution mass spectrometry for broader analyte coverage
• Integration with data analytics and AI for rapid clinical decision support

Conclusion

This single-platform FIA/LC-MS/MS method delivers fast, sensitive, and reliable multi-class analysis of amino acids, acylcarnitines, and steroids from DBS. It meets the demands of high-throughput clinical screening with minimal sample consumption.

Reference

Tanigawa T, Minohata T; ASMS 2015 ThP-251, Shimadzu Corporation