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High-Performance Liquid Chromatographic Technique for Detection of a Fluorescent Analogue of ADP-Ribose in Isolated Blood Vessel Preparations by Janette Bobalova; Pavel Bobal; Violeta N. Mutafova-Yambolieva is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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## Analysis of endogenous nucleotides in biologic me dia is hampered by rapid degradation and low final concentrations that are difficult to detect. A reversedphase high-performance liquid chromatographic (HPLC) technique is described that efficiently detects a stable fluorescence derivative of adenosine 5-diphosphoribose (ADPR), 1,N 6 -etheno-ADPR (⑀-ADPR), at low femtomolar concentration range in vascular tissue superfusates. ⑀-ADPR was formed by the reaction of ADPR with chloroacetaldehyde at 80°C and pH 4.0. Gradient elution with 0.1 M KH 2 PO 4 (pH 6.0), increasing methanol (0 -35% over 18 min), and a 25-cm by 4.5-mm (5 m) silica ODS-AM column were employed. ⑀-ADPR was detected by fluorescence at an excitation wavelength of 230 nm and an emission wavelength of 410 nm. The detection sensitivity for ⑀-ADPR was approximately 10 fmol. Linearity of the HPLC detection method was demonstrated in the range from 0.0125 to 1 pmol ⑀-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

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Author

Janette Bobalova; Pavel Bobal; Violeta N. Mutafova-Yambolieva

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2002

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)