Sensitive determination of (-)-isoproterenol and (-)-(R)-1-(3,4- dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino] ethanol (T-0509), a cardiotonic agent, in rat plasma utilizing a fully automated catecholamine analyser.

(-)-Isoproterenol (ISO), used as bronchodilator, and (-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol (T-0509), a new cardiotonic agent, were determined in rat plasma (only 25 microL) using a fully automated catecholamine (CA) analyser that includes in-line pre-extraction of CAs, coupled with an HPLC-ethylenediamine condensation reaction and a peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). The chromatographic conditions were: precolumn, SERUMOUT-CEX; buffer for delivering CAs in the precolumn, 10 mM potassium phosphate buffer (pH 7.5)/ethanol (92 + 8, v/v) (1 mL/min); precolumn clean-up solution, 4% phosphoric acid/acetonitrile (50 + 50, v/v) (1 mL/min); analytical column, TSKgel ODS-80Ts, 250 x 4.6 mm i.d.; eluent, 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (83.6 + 4.4 + 12, v/v/v for ISO and 76 + 4 + 20 v/v/v for T-0509) (1 mL/min); fluorogenic reagent solution, 105 mM ED and 175 mM imidazole in acetonitrile/ethanol (90 + 10, v/v) (0.25 mL/min); chemiluminogenic reaction solution, 0.25 mM TDPO, 150 mM hydrogen peroxide and 110 mM TFA in dioxane/ethyl acetate (50 + 50, v/v) (1.4 mL/min). Colterol, (+/-)-1-(3,4-dihydroxyphenyl)-2-[(1,1-dimethyl)ethylamino]ethanol, (+/-)-N-t-butylnorepinephrine was used as an internal standard (IS) for ISO, and (+/-)-1-(3,4-dihydroxyphenyl)-2-[(3,4,5- trimethoxyphenethyl)amino]ethanol, (+/-)-(3,4,5-trimethoxyphenethyl- aminomethyl)-3,4-dihydroxybenzylalcohol (T-1583) for T-0509. The detection limits for ISO and T-0509 were 1.3 and 0.9 fmol on injection, respectively.