Immunoassay screen for qualitative detection of fentanyl in urine.

Fentanyl [N-(1-(2-phenylethyl)-4-piperidinyl)-N-phenylpropanamide] is a synthetic opioid narcotic analgesic similar to morphine. Fentanyl is 50-100 times more potent than morphine and is prescribed for patients with chronic pain and is used to manage pain after surgery or for treatment of breakthrough pain in cancer patients. Fentanyl is prescribed in various forms: by injection (intravenous or intramuscular), transdermal patch, and orally (transmucosal lozenge or film).

Fentanyl such as the transdermal system can be abused in a manner like other opioid agonists, legal or illicit. Fentanyl has high potency and short duration of action, and it is abused for its intense euphoric effects. It is very dangerous when substituted illicitly for other opioids because of its potency and overdoses can lead to respiratory depression and death. It is a Schedule II substance under the U.S. Controlled Substances Act. The test is not intended to differentiate between drugs of abuse and prescription use of fentanyl. There are no uniformly recognized drug levels for fentanyl in urine.

The primary metabolism of fentanyl leads to the time-dependent urinary excretion of fentanyl and norfentanyl and the half-life of fentanyl may range 3 - 12 hours. Fentanyl is exclusively metabolized by N-dealkylation and hydroxylation. More than 90% of the dose is eliminated as norfentanyl and hydroxylated metabolites. Less than 7% of the dose is excreted unchanged in the urine. The fentanyl screening assay is based on competition between the drug in the specimen and the drug labeled with recombinant glucose-6-phosphate dehydrogenase (rG6PDH) for antibody binding sites. As the latter binds antibody, enzyme activity decreases.

In the presence of drug from the specimen, enzyme activity increases and is directly related to the drug concentration. Active enzyme converts nicotinamide adenine dinucleotide (NAD) to NADH in the presence of glucose-6-phosphate (G6P), resulting in an absorbance change that is measured spectrophotometrically. Endogenous G6PDH does not interfere because the coenzyme NAD functions only with the bacterial enzyme used in the assay.