High-throughput comprehensive analysis of trace D- and L- amino acids using extra-facile chiral separation and column switching

Significance of the Topic

Accurate determination of trace levels of D- and L-amino acids is vital in fields ranging from food quality assessment to biomedical research. D-enantiomers, though present at low concentrations, serve as emerging biomarkers in neurological studies, fermented food profiling and nutritional evaluation. Fast, sensitive and derivatization-free analytical protocols enhance laboratory throughput and support large-scale screening efforts.

Study Objectives and Overview

This study aimed to develop a high-throughput LC-MS/MS method capable of simultaneous chiral separation and quantitation of trace D- and L-amino acids within a 10-minute runtime. By integrating facile crown ether-based columns with automated column-switching technology, the workflow targets comprehensive enantiomer profiling in complex matrices such as black vinegar and yogurt extracts.

Methodology and Sample Preparation

Enantiomeric separation was achieved under isocratic conditions using CROWNPAK CR-I(+) and CR-I(–) columns (3 mm I.D. × 150 mm, 5 µm) at 20 °C. The mobile phase comprised acetonitrile, ethanol, water and trifluoroacetic acid (80/15/5/0.5 v/v) at 0.6 mL/min. A valve-switching unit alternated the flow between the two chiral columns automatically, enabling dual injections per vial.

Sample pre-treatment involved liquid-liquid extraction of 100 µL homogenized food samples with methanol, water and chloroform, followed by protein precipitation and phase separation. The final supernatant was diluted with mobile phase prior to injection.

Used Instrumentation

• HPLC: Shimadzu Prominence/Nexera X2 system with FCV valves and SIL-30AC autosampler
• Chiral columns: DAICEL CROWNPAK CR-I(+) and CR-I(–)
• MS detection: Triple quadrupole LCMS-8050/8060 (ESI positive mode)
• Ion source settings: Nebulizing gas 3.0 L/min, drying gas 15 L/min, interface 250 °C, DL 250 °C, heat block 300 °C

Main Results and Discussion

The method successfully separated and quantified 22 D/L-amino acids in a single analytical run. In black vinegars and yogurts, all samples contained detectable levels of D-enantiomers. Notably, yogurt samples exhibited D-glutamic acid levels over 40-fold higher than their L-forms. D-alanine and D-lysine were also abundant in certain fermented products. Co-eluting isomers such as threonine and allo-threonine were resolved by column switching, yielding distinct MRM chromatograms for each enantiomer.

Benefits and Practical Applications

The presented workflow achieves:

• Rapid enantiomeric separation within 10 minutes without derivatization
• High sensitivity for trace-level D-amino acids
• Automated column switching for continuous batch analysis
• Data integration into a single acquisition file for streamlined processing

This approach supports quality control of fermented foods, nutritional studies and biomarker discovery in clinical research.

Future Trends and Applications

Emerging trends include coupling chiral LC-MS/MS with high-resolution mass spectrometry for broader enantiomer libraries, integrating microfluidic sample prep for enhanced throughput, and applying machine learning algorithms to predict enantiomer distribution in biological systems. Expansion to other chiral metabolites and in vivo metabolic flux analysis is anticipated.

Conclusion

A fully automated LC-MS/MS platform employing crown ether columns and valve switching offers a robust solution for rapid, sensitive and comprehensive D/L-amino acid profiling. This high-throughput method enables detailed enantiomeric analysis in complex food and biological matrices, facilitating advancements in food science, clinical diagnostics and metabolomics.

References

• Nakano Y., Konya Y., Taniguchi M., Fukusaki E. Journal of Bioscience and Bioengineering, 123, 134–138 (2017).