TECHNICAL NOTE: Validation of the Immunalysis® Microplate ELISA for the Detection of Buprenorphine and Its Metabolite Norbuprenorphine in Urine

Authors: Miller, Eleanor I.1; Torrance, Hazel J.1; Oliver, John S.1

Source: Journal of Analytical Toxicology, Volume 30, Number 2, March 2006 , pp. 115-119(5)

Publisher: Preston Publications

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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 K2HPO4 (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°C. After incubation, 3 mL K2HPO4 (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%.

Document Type: Research article

Affiliations: 1: Forensic Medicine and Science Department, University of Glasgow, University Place, Glasgow, G12 8QQ, Scotland

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