Optimization and validation of a CE-LIF method for amino acid determination in biological samples.

There is increasing evidence that amino acids and related compounds are among the most important metabolites for living systems and their quantitative analysis is a continuous challenge for clinical and biochemical laboratories. The lack of chromophore group and their high polarity make the analytical process more difficult. A simple, rapid and inexpensive procedure based on CE-LIF has been optimised for biological samples such as urine and hippocampus tissue in terms of sample treatment, separation and quantitation. Around 30min were required for derivatization and determination by CE-LIF of l-alanine, l-aspartate, l-β-aminoisobutyrate, d-β-aminoisobutyrate, glycine, l-glutamate, l-glutamine, l-histidine, l-isoleucine, l-leucine, l-methionine, l-ornithine, l-phenylalanine, 4-hydroxy-l-proline, l-proline, l-serine, d-serine, taurine, threonine and l-valine. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) is the labelling agent used for obtaining fluorescence derivatives stables in urine up to 12h and up to 24h in hippocampus extracts in refrigerated conditions. Electrophoretic conditions were: 90mM borate buffer at pH 10.25 prepared with 12.5mM native β-cyclodextrin. Applied voltage was +21kV. The method was validated for a representative group of amino acids in urine: l-phenylalanine, glycine, l-serine, d-serine and taurine. In hippocampus tissue the method was validated for the neurotransmitters: gamma-aminobutyric acid, l-glutamine, glycine, l-glutamate and l-aspartate. The method has been successfully applied to real samples, seven amino acids were quantified in 16 urine samples from healthy and type I diabetic children living in Spain, aged 8-11, and the results were statistically compared. They were in accordance to published reference values. Moreover neurotransmitters in hippocampus extracts were determined in samples of control mice and reported results were in accordance with previous references.