Rapid and sensitive assay of dobutamine in plasma by high-performance liquid chromatography and electrochemical...

A sensitive and specific high-performance liquid chromatographic method with electrochemical detection was developed for measuring dobutamine in human plasma samples. Following an external standard method, 0.1 ml of EDTA-glutathione plasma was diluted on ice with 0.2 ml of a 5% trichloracetic acid solution. The mixture was centrifuged, filtered, and 30 microliters were injected. Assessment was done by electrochemical detection. The assay was linear from 1 to 400 ng/ml plasma. For determination of dobutamine we also used a liquid-liquid extraction method routinely applied for plasma catecholamines. Liquid-liquid extraction requires application of 100-1000 microliters of plasma. The standard curve was linear from 0.1 to 600 ng/ml. Absolute recovery of dobutamine was 90 +/- 3% with the liquid-liquid extraction procedure and 91 +/- 3% with the protein precipitation method. For both methods dobutamine was separated on Nova-Pak C18 columns. The mobile phase used was 0.1 molar phosphate buffer-acetonitrile (80:20, v/v) with 1-octanesulfonic acid and triethylamine as ion-pair reagents. The pH was adjusted to 2.7.