High-performance liquid chromatographic technique for detection of a fluorescent analogue of ADP-ribose in isolated blood vessel preparations

Abstract

Analysis of endogenous nucleotides in biologic media is hampered by rapid degradation and low final concentrations that are difficult to detect. A reversed-phase high-performance liquid chromatographic (HPLC) technique is described that efficiently detects a stable fluorescence derivative of adenosine 5'-diphosphoribose (ADPR), 1,N6-etheno-ADPR (epsilon-ADPR), at low femtomolar concentration range in vascular tissue superfusates. epsilon-ADPR was formed by the reaction of ADPR with chloroacetaldehyde at 80 degrees C and pH 4.0. Gradient elution with 0.1 M KH2PO4 (pH 6.0), increasing methanol (0-35% over 18 min), and a 25-cm by 4.5-mm (5 microm) silica ODS-AM column were employed. epsilon-ADPR was detected by fluorescence at an excitation wavelength of 230 nm and an emission wavelength of 410 nm. The detection sensitivity for epsilon-ADPR was approximately 10 fmol. Linearity of the HPLC detection method was demonstrated in the range from 0.0125 to 1 pmol epsilon-ADPR. The method was validated in terms of within-day and between-day reproducibility of retention times and peak areas of standard nucleotide. Matrix-assisted laser desorption/ionization mass spectrometry measurements confirmed the presence of an etheno ring after reaction of ADPR with chloroacetaldehyde. The method was applied to quantitate the overflow of ADPR upon electrical field stimulation (8 Hz, 0.3 ms, 15 V, 1-2 min) of both canine and guinea-pig isolated mesenteric artery segments.