A novel receptor binding based assay to screen for opiates and (synthetic) opioids in human urine samples

Abstract

Up to date there are, without knowledge of the compounds structure or utilization of antibodies, almost no comprehensive screening methods for the detection (synthetic) opioid uptake in urine samples available. Consequently, appearance of new opioids on the drug market require a tedious effort to update the present methods (e.g. ELISA or LC-MS/MS methods). Detection of the binding of a compound at the human μ-opioid receptor (MOR) offers a promising alternative analytical target as almost all of the opioids on the drug market take effect through binding at this receptor. Recently, we demonstrated that it is possible to accurately determine the binding of a compound (Ki value) at the MOR by LC MS/MS analysis rather than by radioactive detection methods. Here we set up the assay to allow for a detection of compounds that bind at the MOR in urine samples, thus allowing the assay to be utilised as a screening tool for opioid uptake. The assay is performed in a 96-well format and is based on the incubation of readily available cell membranes (prepared from CHO-K1 cells stably transfected with MOR) with the selective MOR-ligand DAMGO ([D-Ala2, N-MePhe4, Gly5-ol]-enkephalin) and 100 μL of urine for 60 minutes. After filtration and washing steps the amount of DAMGO in the eluate is analysed by a rapid and highly sensitive LC-MS/MS method on a QTRAP 6500+ system. Absence of DAMGO in the eluate corresponds to a competing MOR-ligand in the urine sample thus indicating opiate/opioid uptake by the suspect. Sensitivity and specificity was determined by the analysis 200 consecutive forensic routine casework urine samples (with known presence/absence of opiates and opioids determined by immunochemical assays, GC-MS and LC-Q-ToF screenings). DAMGO binding in the positive samples ranged from < 1% to 59% (Mean: 7%, Median: 3%). Twelve different opiates/opioids and/or there metabolites were present in 35 positive samples. A pronounced displacement (<10% DAMGO binding) was observed in 29 of the 35 samples. Detection of fentanyl uptake proved to be the most challenging analyte with 5 out of 10 positive samples showing an insufficient DAMGO displacement (14%–30% DAMGO binding, fentanyl concentration in these five sample: 1.0–5.7 ng/mL. Additionally, a urine sample containing 91 ng/mL tramadol and 42 ng/mL O-desmethyltramadol showed a poor displacement of DAMGO at the MOR. This can be explained by the low Ki values of the compounds at the MOR of 6014 and 40 nM, respectively. Among the nine false positive samples, no cluster of a particular compound was observed. Consequently, a cut-off value of 10% DAMGO binding would lead to a sensitivity of 83% and a specificity of 95% while a sensitivity of > 90% and a specificity of 88% would be achieved by applying a cut-off value of 17% DAMGO binding. The novel assay proved to be a suitable screening tool for opiate/opioid presence in urine samples. The non-targeted approach and possible automation of the assay make it a promising high throughput-capable alternative to conventional methods.