High-performance liquid chromatographic analysis of fluorescent derivatives of adenine and adenosine and its nucleotides : Optimization of derivatization with chloroacetaldehyde and chromatographic procedures

The use of chloroacetaldehyde (CAA) as a potential precolumn fluorimetric labelling reagent for adenine compounds was examined in detail. The reaction kinetics was greatly influenced by parameters such as the pH, temperature and CAA concentration. These parameters were optimized with regard to the reaction yield. The resulting procedure for CAA derivatization of adenine compounds was found to be excellent for quantitative analysis. Because the CAA derivatives of the adenine compounds studied were markedly stable, precise quantitative results were easily obtained using high-performance liquid chromatography. CAA derivatives of the five adenine compounds, i.e., 1,N 6-etheno-adenine, -adenosine, -adenosine 5′-monophosphate, -adenosine 5′-diphosphate and -adenosine 5′-triphosphate were separated. The detection limits for the 1,N 6-etheno derivatives were 0.5—1.7 pmol per 10-μl injection. Due to its simplicity, speed, sensitivity and selectivity, this procedure is recommended for use in studies on the metabolism and biology of adenine compounds.