A Novel LC-MS/MS Quantification Method for Amino Acids in Human Plasma, Including Alloisoleucine, without Ion Pairing or Derivatization

Importance of the Topic

Amino acid profiling in human plasma is critical for diagnosing inherited metabolic disorders such as maple syrup urine disease. Conventional methods often require derivatization or ion-pair reagents, increasing complexity, cost and analysis time. A streamlined LC-MS/MS approach that quantifies a broad panel of amino acids—including the key marker alloisoleucine—in a single run addresses these limitations and supports rapid clinical decision-making.

Objectives and Study Overview

This study aimed to develop and validate a novel LC-MS/MS method for simultaneous quantification of 47 amino acids in human plasma without derivatization or ion pairing. Key goals included:

• Achieving chromatographic separation of isobaric leucine, isoleucine and alloisoleucine in one injection.
• Simplifying sample preparation to protein precipitation with direct injection.
• Reducing per-sample cost from ~13 € to < 2 €.
• Demonstrating accuracy, precision and throughput suitable for clinical laboratories.

Materials and Methods

Human plasma samples were protein-precipitated with organic solvent containing internal standards, centrifuged and the supernatant directly injected (1 µL). Gradient elution on a mixed-mode Intrada Amino Acid column enabled retention of polar analytes without derivatization.

Instrumental Setup

• LC System: Shimadzu Nexera X2 HPLC
• Mass Spectrometer: Shimadzu LCMS-8050 triple quadrupole
• Column: Imtakt Intrada Amino Acid 150 × 2 mm
• Mobile Phase A: Acetonitrile/THF with 0.3% formic acid in ammonium formate buffer
• Mobile Phase B: Acetonitrile with ammonium formate buffer
• Gradient: 25 min total run time
• Detection: MRM transitions optimized for each amino acid, including specific transitions for isobaric pairs.

Results and Discussion

The method achieved baseline separation of leucine, isoleucine and alloisoleucine within 25 min. Calibration with certified plasma controls yielded accuracies of 80–120% and RSD < 15% (intra- and inter-day). Direct quantification of alloisoleucine enhances sensitivity for MSUD diagnosis. Sample throughput and reproducibility meet clinical requirements, while matrix effects are minimized by low injection volume.

Practical Benefits and Applications

This approach reduces cost, labor and turnaround time, making it suitable for newborn screening, clinical diagnostics, QA/QC in pharmaceutical research and metabolic monitoring in industrial settings.

Future Trends and Opportunities

Future developments may include automation of sample prep, expansion of the amino acid panel to non-proteinogenic and modified derivatives, integration with high-throughput platforms and use in pharmacometabolomic studies to track therapy impact on metabolic pathways.

Conclusion

A robust LC-MS/MS method was established for high-throughput, cost-effective quantification of 47 amino acids in plasma without derivatization or ion pairing. Its performance supports clinical diagnostics, especially MSUD screening, and offers a scalable workflow for research and industrial laboratories.

References

• A. Jaffuel and M. Levi, “A novel fast quantification method for amino acids in human plasma by LC-MS/MS, without ion pairing or derivatization,” poster at the 3rd MSACL EU Conference, Salzburg, Austria, 2015.