Introduction: The increasing abuse of amphetamine-like compounds presents a challenge for clinicians and clinical laboratories. Although these compounds may be identified by mass spectrometry-based assays, most clinical laboratories use amphetamine immunoassays that have unknown cross-reactivity with novel amphetamine-like drugs. To date, there has been a little systematic study of amphetamine immunoassay cross-reactivity with structurally diverse amphetamine-like drugs or of computational tools to predict cross-reactivity.
Methods: Cross-reactivities of 42 amphetamines and amphetamine-like drugs with three amphetamines screening immunoassays (AxSYM(®) Amphetamine/Methamphetamine II, CEDIA(®) amphetamine/Ecstasy, and EMIT(®) II Plus Amphetamines) were determined. Two- and three-dimensional molecular similarity and modeling approaches were evaluated for the ability to predict cross-reactivity using receiver-operator characteristic curve analysis.
Results: Overall, 34%-46% of the drugs tested positive on the immunoassay screens using a concentration of 20,000 ng/mL. The three immunoassays showed differential detection of the various classes of amphetamine-like drugs. Only the CEDIA assay detected piperazines well, while only the EMIT assay cross-reacted with the 2C class. All three immunoassays detected 4-substituted amphetamines. For the AxSYM and EMIT assays, two-dimensional molecular similarity methods that combined similarity to amphetamine/methamphetamine and 3,4-methylenedioxymethampetamine most accurately predicted cross-reactivity. For the CEDIA assay, three-dimensional pharmacophore methods performed best in predicting cross-reactivity. Using the best performing models, cross-reactivities of an additional 261 amphetamine-like compounds were predicted.
Conclusions: Existing amphetamines immunoassays unevenly detect amphetamine-like drugs, particularly in the 2C, piperazine, and β-keto classes. Computational similarity methods perform well in predicting cross-reactivity and can help prioritize testing of additional compounds in the future.