3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde labeling for direct analysis of amino acids in plasma is not suitable for simultaneous quantification of tryptophan, tyrosine, valine, and isoleucine by CE/fluorescence.

Abstract

Capillary electrophoresis coupled to LED-induced fluorescence detection is a robust and sensitive technique used for amino acids (AA) analysis in biological media, after labeling with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA). We wanted to quantitate in plasma tryptophan (Trp), tyrosine (Tyr), valine (Val), and isoleucine (Ile). Among the different labeled AA-CBQCA, Trp has the lowest fluorescence yield, which makes its detection and quantification very difficult in biological samples such as plasma. We tried to improve Trp analysis by CE/LED-induced fluorescence detection to its maximal sensitivity by using large volume sample stacking as a preconcentration step in our analytical protocol. At pH 9.5, this step caused a drop in resolution during the separation of the four AAs and it was therefore necessary to work at pH 10. We have found that Tyr, Val, Ile, and Trp are detected and well separated from the other AAs, but Trp cannot be quantified in plasma samples, mainly because of the low fluorescence yield of the Trp-CBQCA derivative. The recorded LOD is 0.18μM for Trp-CBQCA in standard solution with a resolution between Trp and Tyr of 1.2, while the LOD is 6μM in plasma with the same resolution. Trp, Tyr, Val, and Ile are, however, efficiently quantified when using a 3 M acetic acid electrolyte and CE associated with capacitively coupled contactless conductivity detection, which also has the advantage of not requiring derivatization or large volume sample stacking. This article demonstrates, for the CE user, that quantitative analysis of these four AA in mouse plasma can be performed by CE-fluorescence after CBQCA labeling, with the exception of Trp. It can be advantageously replaced by CE/capacitively coupled contactless conductivity detection, the only efficient one for Trp, Tyr, Val, and Ile quantification. In this case, the LOD for Trp is 2μM. The four AAs are separated with resolution with neighbors above 1.5.