Citation

Wang G, Huynh K, Barhate R, Rodrigues W, Moore C, Coulter C, Vincent M, Soares J. Forensic Sci. Int. 2011; 206(1-3): 127-131.

Affiliation

Immunalysis Corporation, 829 Towne Center Drive, Pomona, CA 91767, USA.

Copyright

(Copyright © 2011, Elsevier Publishing)

DOI

10.1016/j.forsciint.2010.07.022

PMID

20801588

Abstract

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 340nm. 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 340nm. 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 1ng/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 10,000ng/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 2ng/mL.

Language: en