Use of immunoaffinity chromatography as a simplified cleanup technique for the liquid chromatographic determination of phenylurea herbicides in plant material

Abstract

Immobilized polyclonal antibodies were evaluated for the cleanup of extracts of several food samples (carrots, celery, corn, grapes, onions, potatoes and strawberries). The antibodies were generated in the rabbit after inoculations with an antigen prepared from the urea herbicide, isoproturon. The antibodies were immobilized onto the surface of activated silica particles and packed into disposable plastic syringe barrels and used in the same general manner as cartridges for standard solid-phase extraction. They displayed substantial cross-reactivity with six other urea herbicides which permitted them to be used as a multi-urea herbicide cleanup procedure. Reversed-phase liquid chromatography with UV detection at 244 nm was the only equipment required for the quantitation. Methanolic extracts of the plant tissue samples were concentrated and then diluted with water before passage through the immunoaffinity (IA) cartridge. The cartridge was washed and the herbicides eluted with 70% methanol in water for analysis by HPLC. The cleanup provided by the IA cartridge enabled the direct detection and quantitation of the herbicides at a concentration level of 25 ng/g in potatoes and carrots. An additional cleanup step using a strong anion-exchange solid-phase extraction cartridge (SPE-SAX) was required for determination of the herbicides in grape, onion, celery, corn and strawberries at levels of 25 ng/g. With the combined SPE-SAX and IA cleanup, the detection limits in the plant material examined were about 2–5 ng/g depending upon the herbicide. At 25 ng/g spiking levels, recoveries through the complete procedure for monuron, chlortoluron, isoproturon and durion averaged 103±10% ( n=6 for each herbicide); for chloroxuron (80±5%, n=6); chlorbromuron (65±12%, n=6); linuron (37±15%, n=6). The only organic solvent used was methanol mainly for the initial sample extraction and in the LC mobile phase. No organic-aqueous partitions or adsorption chromatography employing organic solvents were required.