Urine drug testing is used in many applications, including preemployment screening, human performance testing, neonatal care, probation monitoring, and emergency room care. Rapid, cost-effective immunochemical techniques such as enzyme immunoassays are effective tools for excluding the presence of various drugs; however, they may lack the specificity necessary for accurate identification. Confirmatory techniques such as HPLC, gas chromatography–mass spectrometry (GC–MS), and thin-layer chromatography are often used to validate presumptive immunochemical findings. Immunochemical cross-reactivity causes obvious problems for the clinical laboratory. Eliminating false positive screening results would increase accuracy, efficiency, and cost effectiveness and improve patient management. The current study was designed to isolate, identify, and quantitate the compound(s) responsible for false positive methadone results, using enzyme immunoassay reagents from Diagnostic Reagents, Inc. Many screening panels include a test for methadone, a narcotic agonist used in narcotic maintenance treatment, especially for heroin addiction. Methadone is extensively metabolized by hydroxylation and dealkylation of the nitrogen methyl groups; these dealkylated metabolites are unstable and spontaneously cyclize to form 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), which may further N-demethylate (1). In maintenance subjects, urine concentrations for methadone and EDDP range from 1 to 50 mg/L (2). Verapamil, a calcium channel-blocking agent used for the treatment of hypertension and cardiac arrhythmias, is also extensively metabolized. Metabolic pathways include N-demethylation, O-demethylation, and cleavage of the C—N—C bonds (3). Chemical structures of methadone, verapamil, and several verapamil metabolites are presented in Fig. 1⇓ (3). Enzyme immunoassay reagents were purchased from Diagnostic Reagents, Inc. (lot nos. 5E059, 5M112, 6M174, and 6E083, cat. no. 0597). Verapamil and norverapamil were gifts from Searle; the other metabolites were gifts from Knoll AG. Methadone, sodium phosphate, sodium hydroxide, sodium carbonate, and 1-heptanesulfonic acid were acquired from Sigma Chemical. Methanol, acetic acid, isopropanol, phosphoric acid, acetonitrile, and dichloromethane were purchased from Fisher Scientific; deuterium-labeled internal …