Rapid assay of the monoamine content in small volumes of rat plasma

Abstract

A method for the simultaneous measurement of serotonin catecholamines, and their metabolites in rat plasma by ultrafiltration and microbore liquid chromatography with electrochemical detection (LC-ED) in small volumes is established. Prior to the LC assay, sixteen plasma ultrafiltrates are readily prepared within 30 min in the present study. The present method, applying a dual-electrode detection technique, provides an additional reliable assignment or measurement of peaks by identifying the peaks on the basis of their redox ratios. In addition, the important early-eluting peaks and interfering peaks are eliminated in the cathodic chromatogram resulting in a reliable measurement of norepinephrine, epinephrine, and 3,4-dihydroxyphenylacetic acid. Isocratic separation of serotonin, catecholamines, and their metabolites by a microbore column is achieved within 15 min. Hence, theoretically, over 90 analyses can be performed in a working day. The limit of detection (signal-to-noise ratio = 3) of this method is ca. 0.2–0.5 pg per injection for all analytes. The required volume of the plasma samples can be less than 100 μl. Hence, the remainder of the plasma sample can be analysed for other substances. This rapid, simple, and sensitive method can thus be used as a research tool in the simultaneous measurement of rat plasma serotonin, catecholamines, and their metabolites.