SEPARATION, DETECTION, AND QUANTIFICATION OF IMINE-LINKED OPINES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Protocols for reversed-phase high-performance liquid chromatography (R P-HPLC) with fluorescence detection were developed for the separation, detection and quantification of imine-linked opines, following 4-fluo ro-7-nitrobenzoxadiazole (NBD-F) derivatization, Both natural opines ( mannopine, mannopinic acid, cucumopine, octopine, octopinic acid, and nopaline) and opine analogs (nor-mannopine, galactopine, glucopine) we re included in the study. The on-column detection limits for NBD-opine s varied from 0.1 pmol to 5 pmol. These methods were applied to quanti fy mannopine on the leaf surfaces of transgenic tobacco (Nicotiana tab acum) plants expressing mannityl opine synthesis (mas) genes from Agro bacterium tumefaciens. (C) 1998 Elsevier Science B.V. All rights reser ved.