Immunoassay screen for qualitative detection of methadone in urine.

Methadone, a synthetic opioid, has been used in the treatment of heroin addiction. Methadone compliance is essential and can be effectively monitored by urine screening for methadone or its metabolite. When methadone is ingested, it is rapidly metabolized in the liver. The primary methadone metabolite is formed by N-demethylation to normethadone. However, normethadone is rarely detected as it readily dehydrates to form 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidine, commonly known as EDDP. Both TLC and GC methods are laborious and subject to interference. 

The methadone screening assay uses specific antibodies, which can detect methadone in urine. The assay is based on the competition of an enzyme glucose-6-phosphate dehydrogenase (G6PDH) labeled drug and the drug from the urine sample for a fixed amount of specific antibody binding sites. In the absence of drug from the sample, the specific antibody binds to the drug labeled with G6PDH and the enzyme activity is inhibited. This phenomenon creates a direct relationship between the drug concentration in the urine and the enzyme activity. The enzyme G6PDH activity is determined spectrophotometrically at 340 nm by measuring its ability to convert nicotinamide adenine dinucleotide (NAD) to NADH.