Rapid and simple analysis of DOPA and 5-HTP using high performance liquid chromatography with electrochemical...

A rapid and simple assay using high performance liquid chromatography with electrochemical detection (HPLC-EC) has been developed for the concurrent measurement of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) extracted from brain tissue. With a one-step process using strong cationic exchange resin this method circumvented conventional solvent extraction and other preparative separations. Extraction efficiencies from tissue were 95.7 +/- 3.5% (n = 12) for DOPA and 96.0 +/- 7.0% (N = 6) for 5-HTP (numbers indicate the mean R/- S.E.M.). The limit of detection following injection of 30 microliter of standard solution was 500 pg for DOPA and 50 pg for 5-HTP. Additionally, the effects of apomorphine and parachlorophenylalanine on DOPA and 5-HTP accumulation were measured following inhibition of DA neuronal firing and aromatic L-amino acid decarboxylase. This method has been proven to be sensitive and selective enough to estimate changes of DA and 5-HT synthesis in discrete brain regions such as caudate nucleus and olfactory tubercle of rat brain.