Detection of Fentanyl and its Analogs by Enzyme-Linked Immunosorbent Assay*

A prototype enzyme-linked immunosorbent assay (ELISA) for fentanyl, a potent synthetic narcotic analgesic, was used to detect the presence of fentanyl and its analogs in human urine. Human drug-free urine was spiked with 0.02 to 100 ng/mL of fentanyl or with 0.1 to 500 ng/mL of each analog and used as a sample in the ELISA. Sensitivity and precision of the assay were analyzed specifically for detection of fentanyl. Cross-reactivities of fentanyl and nine analogs, acetylfentanyl, p-methylfentanyl, p-fluorofentanyl, butyrylfentanyl, (+/-)trans-3-methylfentanyl, (+/-)cis-3-methylfentanyl, alpha-methylfentanyl, benzylfentanyl, and alfentanil for this assay were also determined. The results indicate that the ELISA was capable of detecting urinary fentanyl with a limit of detection of 0.25 ng/mL and a reasonable "working cutoff" of 0.5 ng/mL. The assay was moderately precise with within-run precision, determined at fentanyl concentrations of 10.0 and 2.5 ng/mL, showing coefficients of variation from 7.7 to 10.4% and between-run precision, determined at concentrations of 10.0 and 2.5 ng/mL, showing coefficients of variation from 14.2 to 23.7%. Acetylfentanyl, p-fluorofentanyl, p-methylfentanyl, and butyrylfentanyl show good cross-reactivity in the assay relative to fentanyl. The cross-reactivities equivalent to 2 ng/mL of fentanyl for acetylfentanyl, p-fluorofentanyl, p-methylfentanyl, and butyrylfentanyl were 111, 93, 87, and 77%, respectively. The (+/-)trans-3-methylfentanyl, alpha-methylfentanyl, and (+/-)cis-3-methylfentanyl show less cross-reactivity; their percent cross-reactivities equivalent to 2 ng/mL of fentanyl were 50, 19, and 3%, respectively. Benzylfentanyl and alfentanil, however, do not show any cross-reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)