Dynamic pH junction-sweeping technique for on-line concentration of acidic amino acids in human serum by capillary electrophoresis with indirect UV detection.

Abstract

Glutamic acid (Glu) and aspartic acid (Asp), as two important neurotransmitters, have been the focus of increasingly intense research over the past several years. Glu and Asp are present in biological fluids such as serum at trace levels, but complex components in biological matrices make it difficult to determine them in biological samples. In this paper, a sensitive and simple method coupled with indirect UV detection, using benzoic acid (BA) as the UV-absorbing probe, was developed and validated for the quantitative determination of Glu and Asp in human serum and Compound Amino Acid Injection-18 AA. The method combines a dynamic pH junction with a sweeping technique using β-cyclodextrin (β-CD) as the complexing agent for sweeping. Employing this proposed method, low detection limits of 0.061μg/mL for Glu and 0.032μg/mL for Asp were obtained. The sensitivity was improved 30- and 55-fold for Glu and Asp compared to conventional CE method. Standard curves were linear (r>0.999) over the concentration range of 0.1-8.0μg/mL. To further improve the resolution of Asp from interfering substances in human serum, 6% (v/v) methanol was added to the sample matrix, and resulted in the detection limits of 0.125μg/mL for Glu and 0.057μg/mL for Asp. With a simple precipitation of protein, the method has been successfully applied to the analysis of human serum, and the recoveries (82% for Glu and 87% for Asp) were achieved with relative standard deviations of 1.9% and 2.0%, respectively.