Spectrophotometric Study of Excitatory Amino Acids with Ortho-Phthalaldehyde
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Abstract
Background: Understanding the concentration of amino acids in biological samples is pivotal for numerous biochemical and clinical studies. Aspartic acid and glutamic acid, being excitatory amino acids, play crucial roles in the neurotransmission processes of the mammalian brain. Their precise quantification is essential for advancing our knowledge in neurochemistry and for the diagnosis of related disorders.
Objective: This study aimed to develop a robust, accurate, and sensitive spectrophotometric method for quantifying aspartic and glutamic acids in solution, both in their native forms and when complexed with ortho-phthalaldehyde (OPA), a derivatization agent that enhances their absorbance properties.
Methods: Utilizing a double-beam Hitachi 220 Spectrophotometer equipped with dual 1 cm silica cuvettes, we performed spectrophotometric analyses across a wavelength range of 165 to 1000 nm. Calibration curves for aspartic acid and glutamic acid, with and without OPA, were generated by plotting absorbance against concentration. Various solvents including methanol, ethanol, acetone, and acetonitrile were evaluated for their efficacy in dissolving the amino acids and their complexes. Linear regression analysis was employed to establish the relationship between concentration and absorbance, and the method's precision and accuracy were validated through replicate measurements at multiple concentrations.
Results: The calibration curves exhibited excellent linearity for both amino acids, with and without OPA, across the tested concentration ranges. For aspartic acid, absorption maxima were observed at 201 nm (106,333.33 L mol^-1 cm^-1 molar absorptivity) and 229 nm (71,250 L mol^-1 cm^-1) for its OPA derivative. Glutamic acid showed absorption maxima at 198 nm (84,333.33 L mol^-1 cm^-1) and 216 nm (128,857.14 L mol^-1 cm^-1) for its OPA derivative. The correlation coefficients (R²) for the calibration curves ranged from 0.9954 to 0.9982, indicating a high degree of linearity and reliability of the method.
Conclusion: The developed spectrophotometric method provides a reliable, precise, and sensitive approach for quantifying aspartic and glutamic acids. Its application is significant for biochemical analysis and has potential implications in clinical diagnostics, offering a promising tool for neurochemical studies.
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