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

Conjugates of the catechol compounds, L-dihydroxyphenylalanine (L-DOPA ), dopamine and dihydroxyphenylacetic acid in human urine were analyse d using the isocratic ion-pair reversed-phase HPLC method with electro chemical detection. Acid hydrolysis, using 4 mol/l HCl for 60 min, was more effective than treatment with sulphatase for the generation of f ree catechols. Free (non-conjugated) catechols already present, as wel l as those produced by either of the hydrolysis procedures, were adsor bed onto aluminium oxide and extracted in acid solution. The repeatabi lity of the technique for within and between-batch urine analysis was less than 2% and 8%, respectively. Free urinary dopamine (and dihydrox yphenylacetic acid) concentrations were much higher in the urine of pa tients treated with L-DOPA for Parkinson's disease than in healthy vol unteers. At high dopamine (and dihydroxyphenylacetic acid) levels the conjugation capacity was apparently exceeded, since the overall percen t conjugation of L-DOPA, dopamine and dihydroxyphenylacetic acid was d ecreased ''concentration dependently'' when the concentrations of free catechols were increased. Both in the control group and L-DOPA-treate d groups, enzymatic hydrolysis was much less effective than acid hydro lysis in generating free catechols. This indicated that there were oth er, non-sulphated conjugates in the urine, accounting for between 66 a nd 100% of total conjugates.