Hapten synthesis and immunoassay development for the detection of pyrrolizidine alkaloids in vitro

A two-step enzyme-linked immunosorbent assay (ELISA) was developed for the determination of retronecine in the part-per-billion range. An I{dollar}sb{lcub}50{rcub}{dollar} value of 11 {dollar}pm{dollar} 3 ppb was obtained, with detection limits of 1.0 ppb to 100 ppb. Indirect detection of monocrotaline, retrorsine and senecionine was achieved by hydrolytic digestion of these pyrrolizidine alkaloids (PA's), demonstrating the utility of the assay as a class specific detection method.; Polyclonal ELISA's were developed for the detection of retrorsine, monocrotaline, and retronecine in the part-per-billion (ppb) range. A set of three bifunctional linking arms were synthesized. By N-alkylation of pyrrolizidine alkaloids (PA's) retrorsine, monocrotaline, and retronecine acetonide, six haptens were synthesized and useful rabbit sera obtained. The resulting anti-retrorsine antiserum gave a fifty percent inhibition (I{dollar}sb{lcub}50{rcub}{dollar}) value of 0.9 {dollar}pm{dollar} 0.2 ppb for retrorsine with detection limits of 0.5 to 10 ppb. The same ELISA system also detected isatidine with an I{dollar}sb{lcub}50{rcub}{dollar} of 1 ppb and senecionine with an I{dollar}sb{lcub}50{rcub}{dollar} of 100 ppb. A second, monocrotaline-based ELISA detected monocrotaline with an I{dollar}sb{lcub}50{rcub}{dollar} of 36 {dollar}pm{dollar} 9 ppb with detection limits of 5 to 500 ppb and shows no cross-reactivity with retrorsine or senecionine; this ELISA demonstrates the potential for a substrate specific detection method. A third, retronecine-based ELISA detects retronecine with I{dollar}sb{lcub}50{rcub}{dollar} of 3000 {dollar}pm{dollar} 600 ppb (3 {dollar}pm{dollar} 0.6 ppm) retronecine with detection limits of 600 to 10 000 ppb.; None of these ELISA's cross-react with swainsonine or lupinine. PAs were detected in extracts of Senecio vulgaris and Crotalaria retusa, but not in Lupinus spp., as a demonstration of the ELISAs' usefulness.; Ethyl 2,3-dihydro-1H-pyrrolizine-7-carboxylate was prepared from L-proline yielding side products ethyl 2,3-dihydro-1H-pyrrolizine-6-carboxylate and ethyl 2,3-dihydro-5-methyl-1H-pyrrolizine-7-carboxylate. Ethyl 2,3-dihydro-1H-pyrrolizine-7-carboxylate was oxidized in the peroxyester reaction giving products cis- and trans-ethyl 1,3-dibenzoyl-2,3-dihydro-1H-pyrrolizine-7-carboxylates.