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Abstract
A class selective enzyme-linked immunosorbent assay (ELISA) was developed for the detection of benzodiazepines, such as nitrazepam, clonazepam, 7-aminonitrazepam, 7-amidoclonzepam, chlordiazepoxide, delorazepam and nordazepam. Class selective artificial antigens were prepared by haptens of 7-aminonitrazepam and 7-aminoclonazepam conjugating to the carrier protein bovine serum albumin (BSA). Class selective polyclonal antibodies (PcAbs) were generated by immunising male New Zealand white rabbits with the class selective immunogens. Characterisation studies of the PcAbs showed that PcAbs raised by 7-aminonitrazepam–BSA had high affinity and specificity to the six relative benzodiazepines, while PcAbs raised by 7-amidoclonazepam–BSA had high affinity and specificity to five. So, PcAbs raised by 7-aminonitrazepam–BSA were selected to establish an indirect competitive ELISA for multi-residue determination. The IC50s of the optimised immunoassay were 2.50 µg/l, 3.13 µg/l, 1.92 µg/l, 2.78 µg/l, 3.13 µg/l and 4.55 µg/l for nitrazepam, clonazepam, 7-aminonitrazepam, 7-aminoclonazepam, delorazepam and nordazepam, respectively. Assay performance was validated using spiked pig urine by adding nirazepam. Recoveries were between 75 and 103%, and the coefficient variation was below 14%. This assay can be applied to detect multi-residues of benzodiazepines in urine.