Measurement of 6-cyanodopamine, 6-nitrodopa, 6-nitrodopamine and 6-nitroadrenaline by LC-MS/MS in Krebs-Henseleit solution. Assessment of basal release from rabbit isolated right atrium and ventricles.

Abstract

This study presents the validation of a sensitive method for the determination of 6-nitrodopa, 6-nitrodopamine, 6-nitroadrenaline and 6-cyanodopamine in Krebs-Henseleit solution by LC-MS/MS with ESI+ . HRMS was used to precisely characterize the structures of the fragment ions. The method was applied to investigate the catecholamine basal release from rabbit isolated atria and ventricles. The atria and ventricles were suspended separately in a 5ml organ bath containing Krebs-Henseleit solution with ascorbic acid (3mM), gassed (95%O2 /5%CO2 ) at 37°C for 30 min. Strata-X 33μm SPE cartridges were employed for the extraction of the catecholamines and the internal standard 6-nitrodopamine-d4 . The catecholamines were separated employing a 150 ×3mm Shim-pack GIST C18-AQ (3mm particle size), placed in an oven at 40°C and perfused by 65% of mobile phase A (MeCN/H2 O; 90/10, v/v) +0.4% CH3 COOH and 35% mobile phase B (deionized H2 O) +0.2% CH2 O2 at 320μl/min in isocratic mode. The method was linear at 0.1-20 ng/ml. The method was used to identify for the first-time basal release of the three nitrocatecholamines mentioned above and a member of a novel class of catecholamines, the cyanocatecholamines.