Adenosine and dopamine simultaneously determined in urine by reversed-phase HPLC, with on-line measurement of ultraviolet absorbance and electrochemical detection.

Abstract

Abstract This assay method allows the simultaneous determination of adenosine and dopamine in 0.5 mL of human urine within approximately 35 min, by using isocratic, reversed-phase, high-performance liquid chromatography coupled with an on-line detector for ultraviolet absorbance and electrochemical activity. The analytes were rapidly separated on an affinity column packed with phenylboronate-bonded silica. Mean recoveries for adenosine, 2-chloroadenosine (internal standard), dopamine, and (+/-)-isoproterenol (also an internal standard) were 91%, 99%, 88%, and 104%, respectively. The calibration curves for adenosine and dopamine were linear (r = 0.999, P less than 0.01) over the respective concentration ranges of 0.10 to 2.00 mg/L and 0.05 to 1.00 mg/L. Analytical precisions, assessed by the within-day and the day-to-day CVs, were 4.5% and 6.5% for adenosine, and 5.1% and 6.6% for dopamine, respectively. The mean (+/- SD) concentrations of adenosine (1.27 +/- 0.73 mg/L) and dopamine concentrations (0.38 +/- 0.25 mg/L) measured in urine from 19 healthy subjects agreed well with those previously reported.