Immunoassay screen for qualitative detection of amphetamine in urine.

Amphetamines are synthetic derivatives of ephedrine. The most common amphetamines include d-amphetamine, d-methamphetamine, and d-l-amphetamine. They act as stimulants for the central nervous system. When the amphetamine is ingested, it is either rapidly deactivated in the liver or excreted unchanged into the urine. Other ephedrine derivatives such as methamphetamine can be metabolized and excreted in urine as amphetamine.

The screening assay uses specific antibodies, which can detect amphetamine and/or methamphetamine in urine with minimal cross-reactivity to various over-the-counter structurally unrelated compounds. 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 free drug from the sample, the specific antibody binds the drug labeled with G6PDH and causes a decrease in enzyme activity.

In the presence of free drug, the free drug occupies the antibody binding sites, allowing the drug labeled G6PDH to interact with the substrate, resulting in enzyme activity. This phenomenon creates a direct relationship between drug concentration in urine and the enzyme activity. The enzyme activity is determined spectrophotometrically at 340 nm by measuring its ability to convert nicotinamide adenine dinucleotide (NAD) to NADH.