Optimization of the hydrolysis of conjugated L-DOPA, dopamine and dihydroxyphenylacetic acid in human urine for assay by high-performance liquid chromatography with electrochemical detection.

Abstract

Conjugates of the catechol compounds, L-dihydroxyphenylalanine (L-DOPA), dopamine and dihydroxyphenylacetic acid in human urine were analysed using the isocratic ion-pair reversed-phase HPLC method with electrochemical detection. Acid hydrolysis, using 4 mol/l HCl for 60 min, was more effective than treatment with sulphatase for the generation of free catechols. Free (non-conjugated) catechols already present, as well as those produced by either of the hydrolysis procedures, were adsorbed onto aluminium oxide and extracted in acid solution. The repeatability of the technique for within and between-batch urine analysis was less than 2% and 8%, respectively. Free urinary dopamine (and dihydroxyphenylacetic acid) concentrations were much higher in the urine of patients treated with L-DOPA for Parkinson's disease than in healthy volunteers. At high dopamine (and dihydroxyphenylacetic acid) levels the conjugation capacity was apparently exceeded, since the overall percent conjugation of L-DOPA, dopamine and dihydroxyphenylacetic acid was decreased "concentration dependently" where the concentrations of free catechols were increased. Both in the control group and L-DOPA-treated groups, enzymatic hydrolysis was much less effective than acid hydrolysis in generating free catechols. This indicated that there were other, non-sulphated conjugates in the urine, accounting for between 66 and 100% of total conjugates.