Bioluminescent assay for heroin and its metabolites

Illicit heroin is trafficked as a solid particulate drug, while heroin abuse is monitored by testing urine samples for its principal metabolites, morphine and morphine-3-glucuronide. Two novel bacterial enzymes were used in the development of a linked-enzyme assay for heroin and its metabolites: heroin esterase, which converts heroin to morphine, and morphine dehydrogenase, which oxidizes morphine to morphinone with the concomitant reduction of NADP+. A bioluminescent assay involving heroin esterase, morphine dehydrogenase, and the bacterial luciferase from Vibrio harveyi was developed and shown to be sensitive to 89 ng/mL heroin and 2.0 ng/mL morphine. Excellent correlation with the results from 83 authentic samples submitted for urine drug screening at a hospital laboratory was obtained. The bioluminescent assay exhibited greater specificity and speed than current immunological screening methods. A novel format of the bioluminescent assay involving immobilized enzymes was sensitive to 101 ng (250 pmol) of heroin and responded well to particulate heroin. This form of the test was sensitive enough to respond to one or two typical particles of illicit heroin.