title: Validation of the Immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine.
abstract: The purpose of this study was to validate the Immunalysis Buprenorphine Microplate enzyme-linked immunosorbent assay (ELISA) for the detection of buprenorphine in urine samples. Sixty-nine urine samples were obtained from volunteers on the Subutex treatment program and from routine samples submitted to the laboratory for buprenorphine testing. For ELISA analysis, samples were diluted 1:10 with K(2)HPO(4) (0.1M, pH 7.0). The limit of detection was calculated as 0.5 ng/mL buprenorphine. The intra-assay and interday precision was 3.8% (n = 10) and 8.6% (n = 50) respectively at 1 ng/mL buprenorphine. At a low concentration of norbuprenorphine (1 ng/mL), the immunoassay demonstrated a cross-reactivity of 78%. A higher cross-reactivity of 116% was observed at a higher concentration of norbuprenorphine (10 ng/mL). Dihydrocodeine, codeine, tramadol, morphine, propoxyphene, methadone, and EDDP were tested at concentrations of 10 ng/mL and 10,000 ng/mL and demonstrated no cross-reactivity with the assay. For liquid chromatography-tandem mass spectrometry (LC-MS-MS), deuterated internal standard mixture, 1M acetate buffer (pH 5.0), and b-glucuronidase were added to the standards and samples, which were then incubated for 3 h at 60 degrees C. After incubation, 3 mL K(2)HPO(4) (0.1M, pH 6.0) was added and the pH altered to pH 6.0 using 1M KOH. Buprenorphine and norbuprenorphine were subsequently extracted by solid-phase. Twenty-one samples were confirmed positive and 48 samples were confirmed negative by LC-MS-MS. Using a cut-off value of 0.5 ng/mL buprenorphine, the immunoassay demonstrated a sensitivity and specificity of 100%.
authors.affiliation: Forensic Medicine and Science Department, University of Glasgow, University Place, Glasgow G12 8QQ, Scotland.
authors.forename: Eleanor I
authors.initials: EI
authors.lastname: Miller
authors.forename: Hazel J
authors.initials: HJ
authors.lastname: Torrance
authors.forename: John S
authors.initials: JS
authors.lastname: Oliver
mesh_terms: D002047:Buprenorphine / Q000031:analogs & derivatives* / Q000652:urine*; D004797:Enzyme-Linked Immunosorbent Assay; D005554:Forensic Medicine / Q000379:methods; D006801:Humans; D008832:Microchemistry / Q000379:methods; D009294:Narcotics / Q000652:urine*; D015203:Reproducibility of Results; D015813:Substance Abuse Detection / Q000379:methods*
chemical_list: D009294:Narcotics; D002047:Buprenorphine; C043585:norbuprenorphine