Analysis of catecholamines by high-performance liquid chromatography with polarographic detection by use of catalytic maximum wave

A new polarographic detection system employing catalytic maximum wave in reversed phase high-performance liquid chromatography (HPLC) has been studied for determinat ion of catecholamines. In normal pulse polarography with a dropping mercury electrode, a well defined "catalytic maximum wave" of catecholamine has been observed in the potential range from -0.7 to -1.0 V vs. Ag/AgCL in 0.8 mol.dm-3 ammonia/ammonium nitrate buffer (pH 8.2) containing 0.3 mol.dm-3 sodium bromate and 5 mmol.dm-3 molybdenum (VI). The peak currents of the catecholamines such as epinephrine (E), norepinephrine (NE), dopamine (DA)and 3, 4-dihydroxybenzylamine (DBA) are much larger than those of the other cate chol compounds (Table 1). In HPLC with the catalytic polarographic detection, 0.3 mol.dm-3 ammonium nitrate solution adjusted to pH 3 with nitric acid as the eluent and 1.4 mol.dm-3 ammonia/ammonium buffer solution (pH 8.35) containing 10 mmoldm-3 Mo(VI) and 0.6 mol.dm-3 NaBrO3 as the postcolumn reagent were used, respectively. The chromatographic peak heights are proportional to the amount of each of catecholamines in the range from 10 to 100 ng. The calculated detection limits of E, NE, DA and DBA were 2.7 ng (16 pmol), 1.4ng (7.7 pmol), 2.0 ng (13 pmol) and 1.2 ng (8.6 pmol), respectively, with a signal-to-noise ratio of 2. Also, the obtained chromatograms were smoothed by a personal com puter. When the chromatogram was smoothed by an adaptive smoothing method developed by Kawata and Minami using 25 data points, no distortion in the smoothed chromatographic peaks was found, and noise in the baseline region was reduced remarkably. Thus, the signal-to-noise ratio of the smoothed chromatogram was improved by a factor of ca.1.5 in comparison with that of the original one without smoothing.