Liquid chromatographic procedures for the analysis of compounds in the serotonergic and octopamine pathways of lobster hemolymph.

High-performance liquid chromatography, with serial electrochemical and ultraviolet detectors, was used with a reduced activity catecholamine C18 column to separate and quantify compounds important in the serotonergic and octopamine pathways in lobster hemolymph. The chromatographic mobile phase was composed of potassium dihydrogenphosphate buffer, trichloroacetic acid, sodium dodecyl sulfate, the sodium salt of ethylenedinitrilotetraacetic acid and the organic solvents, acetonitrile and methanol. The compounds serotonin, 5-hydroxyindoleacetic acid, tryptophan, 5-hydroxytryptophan, tryptamine, melatonin, octopamine and tyrosine were well resolved within 13 min. Good electrode maintenance, the use of a silica gel precolumn and careful sample preparation were necessary to give a stable baseline, high resolution of these compounds and reproducibility of retention times and peak heights. The electrochemical detector extended the range of detection to the picogram level. Because of the instability of the solutes and of the chromatographic baseline, sample preparation procedures were investigated. Deproteinization with ammonium sulfate gave the best recovery of the compounds of interest and the most stable baseline with the electrochemical detector. Peaks in the hemolymph were characterized by addition of standards, dual detection (electrochemical and ultraviolet) and the enzyme peak shift technique. With this methodology, important endogenous neurohormones in the hemolymph of lobsters can be quantitatively determined with respect to the molt cycle.