The development of a non-competitive immunoenzymometric assay of cocaine

A non-competitive immunoenzymometric assay (IEMA) of cocaine has been developed. An excess of alkaline phosphatase (ALP) labelled polyclonal antibody (pAb) is added to cocaine in solution. After short-time incubation, the free antibody-ALP conjugate is separated from the cocaine–pAb–ALP complex by passing an affinity column POROS 50 OH derivatized with an immobilized cocaine derivative (benzoylecgonine-1,8-diamino-3,4-dioxaoctane or ecgonine-1,8-diamino-3,4-dioxaoctane). The cocaine–pAb–ALP complex is collected after passing the column. The cocaine concentration is quantified spectrophotometrically by measuring the ALP activity using p-nitrophenyl phosphate as substrate. The analytical response was proportional to the cocaine concentration in the sample with a detection limit of 0.5 nM (0.15 ng ml−1) cocaine. The combined approach developed with an amperometric bienzyme electrode, and the potential for on-line cocaine monitoring are discussed.