Development of a homogeneous immunoassay for the detection of fentanyl in urine

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.

Abbreviations: HEIA, homogeneous enzyme immunoassay, % CV, percent coefficient of variation, LC-MS/MS, liquid chromatography mass-spectrometry/mass-spectrometry, GC/MS, gas chromatography mass spectrometry, ELISA, enzyme-linked immunosorbent assay, SOFT, Society of Forensic Toxicologists, G6PDH, glucose-6-phosphate dehydrogenase, RA, reagent antibody, RE, reagent enzyme, Tris–HCl, Tris–hydrochloric acid, NAD, nicotinamide adenine dinucleotide, NaCl, sodium chloride, BSA, bovine serum albumin, G6P, glucose-6-phosphate

Keywords: Homogeneous immunoassay, Fentanyl, Fentanyl metabolite, Urine screening, Antibody, Enzyme conjugate