Direct Determination of Plasma Free Amino Acids by Combined MRM-SIM Method on LC/MS/MS

Significance of the Topic

Free amino acid profiling in blood plasma and serum underpins diagnosis of metabolic disorders and biomarker discovery in oncology and other diseases. Rapid and derivatization-free analysis enhances throughput and reduces sample preparation complexity.

Objectives and Study Overview

This study aims to develop and validate a direct LC-MS/MS method for quantifying twenty free amino acids in human plasma and serum without chemical derivatization. A combined MRM-SIM acquisition strategy is implemented to boost detection sensitivity for small amino acids such as glycine.

Methodology and Instrumentation

• Instrumentation: Shimadzu LCMS-8040 triple quadrupole mass spectrometer coupled with UFLCXR
• Column: Combined-mode Amino Acid column (100 × 3 mm, 3 µm)
• Mobile phases: A: ACN/THF/25 mM ammonium formate/formic acid (9/75/16/0.3); B: ACN/100 mM ammonium formate (20/80)
• Gradient: 0 % B (0–3 min), 0–17 % B (3–9 min), 17–100 % B (9–16 min), 0 % B (19.5 min)
• Flow rate 0.6 mL/min, column temperature 35 °C, injection volume 2 µL
• Ionization: ESI positive mode; data acquisition: 19 MRM channels plus one SIM channel for glycine
• Sample prep: Protein precipitation of plasma/serum with methanol:acetonitrile (50:50) at 3:1 ratio, centrifugation, filtration

Key Results and Discussion

• Calibration: Linear dynamic range of 10–100 µM with R² > 0.992 for all amino acids
• Sensitivity: MRM LOQs ranged from 0.16 to 13.7 µM; glycine LOQ improved from 528 µM (MRM) to 17.6 µM (SIM)
• Reproducibility: Replicate analyses of pooled plasma and serum showed consistent quantitation for 19 amino acids by MRM and for glycine by SIM
• Mode comparison: SIM acquisition offered superior sensitivity and accuracy for glycine, whereas MRM provided adequate performance for the other amino acids
• Biological levels: Plasma and serum profiles exhibited high glutamate and alanine, low methionine and cystine, consistent with literature trends

Benefits and Practical Applications

• Eliminates derivatization steps, reducing analysis time and potential artifacts
• Enables direct, high-throughput monitoring of amino acid profiles in clinical research and quality control
• Improves sensitivity for small analytes such as glycine through combined MRM-SIM
• Applicable to biomarker discovery and metabolic profiling in disease studies

Future Trends and Potential Applications

• Extension to include biogenic amines and related metabolites on the same platform
• Integration with high-resolution mass spectrometry for untargeted profiling alongside targeted quantitation
• Automation of sample preparation to support large-scale clinical studies
• Application to other biofluids and tissues for comprehensive metabolomics workflows

Conclusion

The combined MRM-SIM LC-MS/MS method on the LCMS-8040 with a novel amino acid column provides a robust, sensitive and derivatization-free platform for quantifying twenty free amino acids in human plasma and serum. The approach offers high linearity, low detection limits and streamlined sample preparation suitable for clinical and research applications.

Reference

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