Reversed-phase high-performance liquid chromatography of catecholamines and indoleamines using a simple gradient solvent system and native fluorescence detection

A reversed-phase HPLC method using a C18 column and a two-mobile-phase gradient elution system containing only volatile components has been developed for separation of norepinephrine, octopamine, epinephrine, dopamine, dihydroxyphenylalanine, tyramine, tyrosine, serotonin, 5-hydroxytryptophan, N-acetyl-serotonin and tryptophan. Mobile phase A contains 0.05% aqueous trifluoroacetic acid and methanol (97.5:2.5, v/v) and mobile phase B contains 0.05% aqueous trifluoracetic acid and methanol (40:60, v/v). This method has the advantage that the mobile phase can be removed completely, without salt residues, from the eluted fractions thus simplifying further analytical procedures on isolated fractions. The elution profile of standards is related to structural characteristics allowing prediction of retention times of known compounds and insight into possible structural characteristics of unknown components in a mixture. Detection is via native fluorescence using excitation at 220 nm and emission at 320 nm and under the conditions described has a sensitivity range from 2.5 to 25 pmol, although the sensitivity range can be extended depending on the emission wavelength used.