Simultaneous determination of 3,4-dihydroxyphenylglycol, 4-hydroxy-3-methoxymandelic acid, 4-hydroxy-3-methoxy-phenylglycol and their precursors in rat-brain tissue by high-performance liquid chromatography with electrochemical detection

A method was developed for the quantification of 3,4-dihydroxyphenylglycol (DHPG), 4-hydroxy-3-methoxymandelic acid (VMA), 4-hydroxy-3-methoxyphenylglycol (MHPG). The separation and determination of these compounds in biological samples were previously thought to be very difficult. In this work the separation has been well achieved by high-performance liquid chromatography on a reversed-phase column using three mobile phases with a step-wise gradient elution mode. The condition for coulometric detection has been developed for an effective determination of these compounds. In the analysis of the rat-brain tissue, DHPG, VMA and MHPG were separated and determined successfully. The average levels of DHPG, VMA, and MHPG in nine different rat-brain tissue samples were 11.8+/-0.57, 2.13+/-0.13 and 6.77+/-1.84 ng/g wet brain, respectively. Simultaneously, their precursors DOPA, DA, NE, DOPAC, HVA, 3MT and NMN, and indolamine 5HT and its metabolite 5HIAA may also be determined by the same chromatographic run.