A new enzyme immunoassay for aconitine and its application to quantitative determination of aconitine levels in plasma.

ABSTRACT

A reliable enzyme immunoassay (EIA) method was developed for quantitative determination of aconitine with high sensitivity and specificity. The bovine serum albumin (BSA)- and beta-galactosidase (beta-Gal) conjugates as immunogens and enzyme-labeled antigens were prepared by coupling of their proteins with succinic acid (short chain length; n=2, where n represents the number of methylene units) and hexadecanedioic acid (long chain length; n=14) hemiesters of benzoylaconine through the respective N-hydroxysuccinimide esters as intermediates. Two types of the BSA-conjugates with short and long chains were repeatedly injected into rabbits to obtain anti-aconitine antisera (As1 and As2, respectively). All combinations of beta-Gal-labeled antigens LAg1 (n=2) and LAg2 (n=14) with antisera As1 (n=2) and As2 (n=14) showed high sensitivity to aconitine in a range of 0.1-1.0 ng. Although the combination of LAg2 (n=14) with antiserum As1 (n=2) showed high specificity to aconitine, the combination of LAg2 (n=14) and As2 (n=14) was highly specific to both aconitine and mesaconitine. When aconitine was intravenously administered to rats, the aconitine concentration in their plasma remarkably decreased within the first 60 min, and then gradually declined, suggesting a two-compartment pharmacokinetic model in (V(c) 0.41+/-0.09 l/kg, V(dss) 1.7+/-0.4 l/kg, CL(tot) 10+/-2 ml/min x kg, AUC(0-4800) 2055+/-294.3 ng x min/ml). Following oral administration of aconitine to rats at two doses of 0.1 and 1.0 mg/kg b.w., the maximum plasma concentrations (C(max)) were 0.73+/-0.08 and 3.3+/-0.6 ng/ml at times of 45+/-9 and 150+/-52 min, respectively, and the AUC(0-1440) values were 130+/-4 and 1600+/-270 ng x min/ml. The bioavailability (F) of aconitine was determined to be 0.013, where only 1.3% of the aconitine administered orally was absorbed into the body fluid.