Trace analysis of oxidized, nitrated, and chlorinated aromatic amino acids by capillary electrophoresis with electroosmotic flow modification allowing large-volume sample stacking

Abstract

A capillary electrophoresis method has been developed for the simultaneous analysis of the oxidized, nitrated, and chlorinated aromatic amino acids, as well as their parent compounds. These modifications of the aromatic amino acids in proteins or free form are induced by the attack of reactive, mainly free radical species generated during cell stress, and these stable products may serve as biomarkers of cell damage. The analytes tyrosine, phenylalanine, dihydroxyphenylalanine, tryptophan, 3-nitrotyrosine, 3-chlorotyrosine, ortho-tyrosine, meta-tyrosine, 3-hydroxyphenylacetic acid (internal standard 1), and alpha-methyltyrosine (internal standard 2) were separated in their anionic forms in alkaline borate buffer. The polyamine spermine was used as electroosmotic flow (EOF) modifier. Adsorbing to the capillary wall, spermine can either suppress or even reverse the EOF depending on its concentration and the pH. The effects of the pH of the separation buffer, the spermine concentration, the temperature, and the applied field strength on the separation were examined. The modified aromatic amino acids are present in biological fluids in a much lower concentration than their parent compounds, thus high detection sensitivity of the analytical method is required. To achieve good detection sensitivity, field-amplified sample stacking of large injection volumes was applied. Omitting polyamine from the sample buffer allowed local reversal of the EOF, thus removal of the low conductivity sample buffer at the capillary inlet. In this way, 100% of the capillary to the detection window could be filled with the sample, and the detection limits achieved for the modified aromatic amino acids were in the range of 2.5-10 nM.

Contemporary sample stacking in CE: A sophisticated tool based on simple principles

“…The result was a 44-to 90-fold improvement of sensitivity when compared with CE without stacking. Trace analysis of oxidized, nitrated, and chlorinated aromatic amino acids, biomarkers of cell damage, in the presence of EOF was described by Tábi et al [10]. They used borate buffer (pH 8.8) and spermine as EOF modifier and achieved a gain in concentration detection sensitivity higher than two orders of magnitude.…”

“…It is a well-known fact in CE that this method, when used with current commercial instrumentation with optical detectors, suffers from poor sensitivity and analyses below 10 25 M are already difficult. Simple injection of a larger volume of a sample does not always help since a long injected zone is detrimental for separation efficiency and resolution.…”

Progress in capillary electrophoresis of biomarkers and metabolites between 2002 and 2005

“…Recently, Tabi et al [74] developed a CE method for the analysis of oxidized, nitrated, and chlorinated aromatic amino acids, as well as their parent compounds. These modifications of the aromatic amino acids in proteins or free form are induced by the attack of reactive species generated during cell stress, and these stable products may serve as biomarkers of cell damage.…”

Recent advances in amino acid analysis by capillary electrophoresis

“…Omitting polyamine from the sample buffer allowed local reversal of the EOF, thus allowing for removal of the low-conductivity sample buffer at the capillary inlet. In this way, 100% of the capillary to the detection window could be filled with the sample, and the LODs achieved for the modified aromatic amino acids were in the range of 2.5-10 nM using UV detection [50].…”