Optimal excitation and emission wavelengths to analyze amino acids and optimize neurotransmitters quantification using precolumn OPA-derivatization by HPLC

• Published in: Amino acids 2015-05, Vol.47 (5), p.963-973
• Main Authors: Perucho, J., Gonzalo-Gobernado, R., Bazan, E., Casarejos, M. J., Jiménez-Escrig, A., Asensio, M. J., Herranz, A. S.
• Format: Article
• Language: English
• Subjects: Amino acids; Analytical Chemistry; Animals; Aspartates; Aspartic Acid - chemistry; Aspartic Acid - isolation & purification; Biochemical Engineering; Biochemistry; Biomedical and Life Sciences; Cerebellum - chemistry; Cerebral Cortex - chemistry; Chromatography, High Pressure Liquid; Emission analysis; Excitation; gamma-Aminobutyric Acid - chemistry; gamma-Aminobutyric Acid - isolation & purification; Glutamates; Glutamic Acid - chemistry; Glutamic Acid - isolation & purification; Glutamine - chemistry; Glutamine - isolation & purification; Glycine; Life Sciences; Male; Neurobiology; Neurotransmitter Agents - chemistry; Neurotransmitter Agents - isolation & purification; o-Phthalaldehyde - chemistry; Original; Original Article; Proteomics; Rats; Rats, Sprague-Dawley; Separation; Taurine - chemistry; Taurine - isolation & purification; Wavelengths
• ISSN: 0939-4451; 1438-2199
• DOI: 10.1007/s00726-015-1925-1

We describe an analytical methodology to obtain high sensitivity and better resolution through the study of fluorometric excitation ( λ ex ) and emission ( λ em ) spectrum wavelengths of OPA–amino acids. The spectrum emission study revealed a maximum signal peak at 450 nm for aspartate and glutamine. For glycine, taurine, and GABA, the maximum signal peak was at 448 and for glutamate at 452 nm. The remaining amino acids analyzed showed a maximum emission around 450 nm. The best signal obtained within the spectrum excitation experiments was using 229- to 450-nm λ ex – λ em . The drawbacks observed at these wavelengths were a baseline drift and negative peaks occurrence. Thus, the excitation wavelength of 240 nm was chosen (240- to 450-nm λ ex – λ em ) as a compromise between a very good signal response and a baseline stability to resolve the 18 amino acids studied. Furthermore, this protocol was properly validated. On the other hand, the elution gradient program used for neuroactive amino acids (aspartate, glutamate, glycine, taurine and GABA) showed separation to the baseline, in a 15-min run in all of them. Other amino acids, up to 18, also exhibited a very good separation in a 25-min run. In conclusion, we propose the use of 240- to 450-nm λ ex – λ em wavelengths, in OPA–amino acids analysis, as the most suitable protocol to obtain the best signal response, maintaining an optimum chromatographic resolution.