Determination of desmosine, isodesmosine, and other amino acids by liquid chromatography with electrochemical detection following precolumn derivatization with naphthalenedialdehyde/cyanide

Abstract

Naphthalenedialdehyde (NDA) in the presence of cyanide (CN) reacts with primary amines to produce fluorescent cyano[ f]benzoisoindole (CBI) derivatives. These derivatives have been shown to be substantially more stable than the corresponding o-phthalaldehyde derivatives. However, one drawback of this method is that compounds derivatized at more than one site exhibit quenching, precluding the use of fluorescence detection. The CBI derivatives have been found to be electroactive and are oxidized at a modest oxidation potential (+750 mV). Electrochemical detection is especially useful for the analysis of compounds containing more than one primary amine site because the response is not attenuated as it is in fluorescence detection. Desmosine and isodesmosine were of particular interest because of their importance in elastic fiber and the lack of highly sensitive HPLC methods for the determination of these compounds. Both of these compounds react with NDA CN to produce electrochemically active derivatives. The combination of derivatization with NDA CN and electrochemical detection was found to be linear over three orders of magnitude. Detection limits for CBI-lysine and CBI-desmosine were 100 fmol at a S N of 2. Amino acids in elastin were quantitated using this method. The results correlate well with what has been reported previously in the literature. A significant advantage of the use of liquid chromatography with electrochemical detection with precolumn derivatization with NDA CN for the analysis of desmosine and isodesmosine is that they can be separated and quantitated individually using this method. In addition, the unique voltammetry of multiderivatized CBI-amino acids can be used to verify peak purity.