OBJECTIVE: Fentanyl is an extremely potent synthetic opioid that is widely used for chronic pain treatment; it is highly addictive and prone to abuse. The objective is to develop a high throughput homogeneous enzyme immunoassay (HEIA) for the rapid detection of fentanyl in human urine. METHODS: The HEIA is based on an immunoassay format in which both the antibody and enzyme-drug conjugate are in ready-to-use solution. In the absence of the target analyte in the specimen, enzyme-labeled drug conjugate binds to the antibody and results in a decrease of the enzyme (G6PDH) activity; hence there is lower absorbance at 340 nm. If the target analyte is present in the specimen, it competes with the enzyme-labeled drug to bind to limited amount of specific antibody that result in more enzyme activity and yields an increased absorbance at 340 nm. A polyclonal "in-house" antibody was selected that is capable of measuring fentanyl at low concentrations thus the assay detection limit was determined to be 1 ng/mL. The assay was validated with clinical urine specimens that previously confirmed positively or negatively for fentanyl/norfentanyl by LC-MS/MS. RESULTS: The intra-day (n = 20) and inter-day (n = 100) precision of the assay was less than 1% CV. No interferences from structurally unrelated and commonly ingested drugs were observed at a concentration of 10,000 ng/mL. A total of 209 LC-MS/MS confirmed urine specimens (149 positive and 57 negative samples) were analyzed by HEIA. The sensitivity, specificity, and accuracy values were 99%, 95%, and 98% respectively. CONCLUSION: This paper describes the development of a highly sensitive homogenous enzyme immunoassay for detecting fentanyl in urine at a cut-off concentration of 2 ng/mL.
OBJECTIVE:Fentanyl is an extremely potent synthetic opioid that is widely used for chronic pain treatment; it is highly addictive and prone to abuse. The objective is to develop a high throughput homogeneous enzyme immunoassay (HEIA) for the rapid detection of fentanyl in human urine. METHODS: The HEIA is based on an immunoassay format in which both the antibody and enzyme-drug conjugate are in ready-to-use solution. In the absence of the target analyte in the specimen, enzyme-labeled drug conjugate binds to the antibody and results in a decrease of the enzyme (G6PDH) activity; hence there is lower absorbance at 340 nm. If the target analyte is present in the specimen, it competes with the enzyme-labeled drug to bind to limited amount of specific antibody that result in more enzyme activity and yields an increased absorbance at 340 nm. A polyclonal "in-house" antibody was selected that is capable of measuring fentanyl at low concentrations thus the assay detection limit was determined to be 1 ng/mL. The assay was validated with clinical urine specimens that previously confirmed positively or negatively for fentanyl/norfentanyl by LC-MS/MS. RESULTS: The intra-day (n = 20) and inter-day (n = 100) precision of the assay was less than 1% CV. No interferences from structurally unrelated and commonly ingested drugs were observed at a concentration of 10,000 ng/mL. A total of 209 LC-MS/MS confirmed urine specimens (149 positive and 57 negative samples) were analyzed by HEIA. The sensitivity, specificity, and accuracy values were 99%, 95%, and 98% respectively. CONCLUSION: This paper describes the development of a highly sensitive homogenous enzyme immunoassay for detecting fentanyl in urine at a cut-off concentration of 2 ng/mL.
Authors: Logan C Krajewski; Kenneth D Swanson; William A Bragg; Rebecca L Shaner; Craig Seymour; Melissa D Carter; Elizabeth I Hamelin; Rudolph C Johnson Journal: Toxicol Lett Date: 2019-12-05 Impact factor: 4.372
Authors: Rebecca L Shaner; Pearl Kaplan; Elizabeth I Hamelin; William A Bragg; Rudolph C Johnson Journal: J Chromatogr B Analyt Technol Biomed Life Sci Date: 2014-05-24 Impact factor: 3.205