Fluorometric determination of 1,2,3,4-tetrahydro-6,7-dihydroxyisoquinoline in biological materials by HPLC

In the belief that endogenous 1,2,3,4-tetrahydro-6,7-dihydroxyisoquinoline (DA-Fp) could be a potential marker involved in the etiology of various diseases such as Parkinsonism, we attempted to develop a fluorescence method for DA-Fp. It was synthesized by condensation of dopamine with formaldehyde according to an established method. Periodate was identified by screening from various oxidation reagents as a fluorescence reagent to DA-Fp. Optimal reaction conditions were obtained with 0.25 mM NaIO4 in 0.1 M phosphate buffer (pH 8.0) at 37 degrees C for 15 min. The fluorescence spectrum of the derivative showed that we had found a new reaction specific for DA-Fp. This reaction was coupled on-line to high performance liquid chromatography (HPLC), which enabled us to achieve a highly sensitive method for determining DA-Fp. A working curve was linear from 2 to 800 pmol of DA-Fp per injection. To determine DA-Fp in biological materials, the pretreatment before HPLC was optimized by hydrolysis of its conjugate and suppression of the artifact with l-phenylephrine. Urinary excretion of DA-Fp in men was measured by this new present method. The urinary excretion of endogenous DA-Fp increased in a rabbit given L-DOPA. The DA-Fp concentration was determined in rat brain. The significance of DA-Fp in these biological materials is discussed and evaluated. We conclude that the present method will be useful for studying tetrahydroisoquinolines involved in many diseases.