Reliable Quantitation of 52 L-Amino Acids in Human Plasma for Clinical Research by LC-MS/MS

Importance of the Topic

Amino acids serve as fundamental building blocks of proteins and key intermediates in metabolic pathways. Accurate quantitation of these compounds in human plasma is essential for clinical research, biomarker discovery, and monitoring metabolic disorders. A rapid, robust method that avoids derivatization can streamline laboratory workflows and improve throughput.

Objectives and Study Overview

The study aimed to develop and validate an LC–MS/MS workflow to quantify 52 L-amino acids in human plasma within 18 minutes. Key goals included simplifying sample preparation by eliminating derivatization, addressing carryover and retention time shifts, and achieving reliable calibration across a wide concentration range (0.1–500 µM).

Methodology and Instrumentation

Sample Preparation:

• Offline protein precipitation: 100 µL plasma mixed with 100 µL precipitation reagent, vortexed, refrigerated, centrifuged.
• Supernatant diluted with internal standard mix before injection.

Liquid Chromatography:

• Trap column to remove basic amino acid carryover.
• Guard column for extended lifetime.
• Analytical reverse-phase column at 30 °C.
• 18-minute aqueous/organic gradient, injection volume 4 µL.

Mass Spectrometry:

• Thermo Scientific Vanquish Flex UHPLC coupled to TSQ Quantis triple quadrupole.
• HESI source in positive mode: spray voltage 3.5 kV, sheath gas 45, aux gas 15, capillary temp 270 °C.
• SRM acquisition with optimized Q1/Q3 resolutions (0.7 FWHM), cycle time 0.4 s, collision gas 1.5 mTorr.

Main Results and Discussion

Calibration and Sensitivity:

• Eight calibration standards (0.1–500 µM) with internal or external calibration.
• LLOQ defined by <20 % inaccuracy and <20 % CV for triplicate injections; R² values >0.98 for most analytes.

Carryover and Retention Time Stability:

• Cation exchange trap column effectively removed basic amino acid carryover.
• pH control limited batch-to-batch retention shifts to <0.1 min.

Repeatability:

• Mid and high level QCs showed %RSD <5 % for key amino acids in plasma.

Overall, the method demonstrated high reproducibility, broad dynamic range, and rapid analysis time.

Benefits and Practical Applications

• Derivatization-free sample prep reduces time, cost, and complexity.
• Simultaneous quantitation of 52 amino acids in a single 18-minute run.
• Suitable for high-throughput clinical studies, nutritional research, and metabolic profiling.

Future Trends and Potential Applications

• Further validation for all 52 amino acids across diverse clinical matrices.
• Integration with automated sample preparation platforms.
• Expansion into comprehensive metabolomics panels combining amino acids with other biomarkers.
• Application in personalized medicine and real-time metabolic monitoring.

Conclusion

The presented LC–MS/MS method offers a streamlined, reliable approach for quantifying 52 L-amino acids in human plasma. By removing derivatization, optimizing chromatographic conditions, and employing a robust triple quadrupole platform, the workflow achieves high sensitivity, reproducibility, and throughput, making it well suited for clinical research applications.

References

• Samra S, Thibert V, Netter C. Reliable Quantitation of 52 L-Amino Acids in Human Plasma for Clinical Research by LC-MS/MS. Thermo Fisher Scientific, 2019.