Direct injection of blood samples into a high-performance liquid chromatographic adenine analyser to measure adenine, adenosine and the adenine nucleotides with fluorescence detection

Abstract

Adenine (Ade), adenosine (Ado) and its nucleotides such as AMP, cAMP, ADP and ATP in blood or plasma were determined by a high-performance liquid chromatographic (HPLC) adenine analyser with fluorescence detection. In order to inject samples directly into the HPLC system without pretreatment except dilution, the analyser consisted of two systems each, having three columns (pre-, mini- and analytical). A precolumn with an inlet filter of pore size 40 μm was common to both systems and packed with Butyl-Toyopearl 650-M to remove hydrophobic compounds and blood cell membranes. In the system for analysis of the nucleotides, a mini-column of Hitachi anion-exchange gel 3013-N was used for adsorbing AMP, cAMP, ADP and ATP. The adsorbed nucleotides were separated by the Hitachi gel 3013-N analytical column. In the other system for analysis of Ado and Ade, they were adsorbed on a Develosil ODS-5 mini-column and separated by an Asahipak GS-320H size-exclusion analytical column. The adenine compounds in each eluate were derivatized online in a 15-m reaction coil at 115°C with bromoacetaldehyde as the fluorescent reagent in each mobile phase for the analytical column, and detected by spectrofluorimetry. ATP, ADP and AMP were accurately determined by the direct injection of hamster, rat and human whole blood. Authentic Ade and Ado were well separated and Ado in human plasma was determined, but it was difficult to determine it in rat plasma owing to interference from an unknown compound.