Fluorometric determination of octopamine in tissue homogenates by high-performance liquid chromatography.

Abstract

A rapid, sensitive method for quantitative determination of octopamine (a biogenic amine found in both vertebrate and invertebrate nervous tissue) has been developed using reversed-phase high-performance liquid chromatography. The biogenic amine is extracted with perchloric acid from tissue homogenates and derivatized witho-phthalaldehyde (OPT) prior to chromatography. Separation of the fluorescent octopamine-OPT adduct from other biogenic amines was achieved using a μBondapak C18 reversed-phase column and isocratic elution with a methanol-(0.08 mol/liter) acetic acid (50∶50 by vol, pH 2.9) mobile phase. A variable wavelength fluorometer with an 8-μl flow cell was used for detection (excitation 340 nm, 418 nm secondary filter). Linearity ranged from 500 pg to 30 ng injected onto the column. Recovery of internal standard added to tissue homogenates averaged 65.4% with a standard deviation of 3.1%. The method has been used for the determination of octopamine in ganglia ofAplysia californica.