Fast UHPLC Methods for Analysis of Amino Acids

Significance of the topic

Amino acids serve as key neurotransmitters and neuromodulators in the central nervous system. Rapid and sensitive quantification of neuroactive amino acids is essential in neurochemical research, enabling high temporal resolution in microdialysis studies and detailed profiling of enantiomeric forms that influence receptor function and hormone regulation.

Objectives and study overview

This work presents two fast UHPLC methods for amino acid analysis. The first is an isocratic UHPLC-EC method for eleven neuroactive amino acids. The second is a gradient UHPLC-fluorescence method for simultaneous determination of D- and L-aspartic acid and D- and L-serine. Both methods are applied to rat brain tissue homogenates to assess region-specific amino acid levels and the effect of amphetamine treatment.

Methodology

Pre-column derivatization with o-phthaldialdehyde (OPA) combined with β-mercaptoethanol (βME) enabled electrochemical detection. A separate chiral derivatization using OPA and N-acetyl-L-cysteine (NAC) provided fluorescence detection of D- and L-enantiomers. Brain samples were homogenized in perchloric acid, diluted, and mixed online with derivatizing reagent prior to injection. Gradient conditions and mobile phases were optimized for separation speed and sensitivity.

Instrumentation

• UHPLC system: Thermo Scientific™ Dionex™ UltiMate™ 3000 series modules (pump, autosampler, column thermostat).
• Detector for isocratic method: Electrochemical detector ECD-3000RS with 6011RS cell (E1=150 mV, E2=550 mV).
• Detector for gradient method: Fluorescence detector FLD-3400RS (excitation 340 nm, emission 450 nm).
• Columns: Accucore™ C18 (2.6 µm, 100×3 mm) for EC; Hypersil GOLD™ (1.9 µm, 200×2.1 mm) for fluorescence.

Main results and discussion

The isocratic UHPLC-EC method separated 11 amino acids in 10 minutes with detection limits of 10–40 pg on-column. The fluorescence method resolved D- and L-Asp and Ser enantiomers in 20 minutes with sub-pg sensitivity (200–400 fg). Application to eight rat brain regions revealed amphetamine-induced increases in glutamate and GABA (>200%) and region-specific changes in aspartate. Enantiomer analysis showed a 440% rise in D-Asp and up to 8500% rise in D-Ser in brain stem; hippocampus exhibited 875–958% increases.

Benefits and practical applications

• High throughput: analysis times reduced by up to 50% compared to conventional HPLC.
• High sensitivity: low-pg to sub-pg limits enable analysis of trace levels in microdialysis and tissue samples.
• Selective enantiomer resolution: simultaneous chiral analysis of D- and L-amino acids.
• Automated online derivatization improves reproducibility and sample throughput.

Future trends and applications

Expanding these methods to include additional amino acid enantiomers and coupling to mass spectrometry could further enhance specificity. Integration with real-time microdialysis sampling and in vivo monitoring will support dynamic neurochemical studies. Emerging UHPLC column chemistries and miniaturized detectors may drive further reductions in analysis time and sample volume.

Conclusion

The developed UHPLC-EC and UHPLC-fluorescence methods provide fast, sensitive, and robust tools for neuroactive amino acid quantification, including critical D- and L-enantiomer profiling. The methods demonstrated excellent performance in rat brain studies, revealing amphetamine-induced regional neurochemical changes and offering broad applicability in neuroscience and bioanalytical research.

References

1. DʼAniello A. D-Aspartic acid: An endogenous amino acid with an important neuroendocrine role. Brain Res. Rev. 2007, 53, 216–234.
2. Bruckner H., Haasmann S. et al. Liquid chromatographic determination of D- and L-amino acids by derivatization with o-phthaldialdehyde and chiral thiols. J. Chromatogr. A 1994, 611, 259–273.
3. Bruckner H., Wittner R. Automated enantioseparation of amino acids by derivatization with o-phthaldialdehyde and N-acetylated cysteines. J. Chromatogr. 1989, 476, 73–82.