Sensitive and derivatization-free amino acid analysis using liquid chromatography-Raman spectroscopy

Liquid chromatography-Raman spectroscopy with post-column spotting and solvent evaporation achieved nanogram-level detection of amino acids without derivatization and demonstrated the feasibility of quantification at this level for phenylalanine and glutamine. All 20 amino acids were successfully detected regardless of chromophore presence. Lysine modifications were directly discriminated via distinct vibrational fingerprints. Co-eluting compounds were resolved by MCR-ALS-based spectral deconvolution, demonstrating that LC-Raman spectroscopy can add a spectral separation axis to chromatographic analysis. Furthermore, LC-Raman spectroscopy was also applied to quantitative analysis. For phenylalanine and glutamine, the calibration curves exhibited excellent linearity (R² = 0.9925 for phenylalanine, and R² = 0.9994 for glutamine). The quantitative results of these amino acids in the culture medium obtained via LC-Raman spectroscopy (65.9 ± 3.5 µg mL^-1 (n = 3, %RSD = 5.2%) for phenylalanine, and 576.3 ± 17.5 µg mL^-1 (n = 3, %RSD = 3.0%) for glutamine) were in good agreement with those determined by conventional detectors (66.1 µg mL^-1 for phenylalanine, and 584 µg mL^-1 for glutamine). This label-free approach establishes LC-Raman spectroscopy as a structural detection mode for LC, enabling chromatographic analysis to be coupled directly with molecular fingerprinting.