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

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

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

Publication date: 2006-03-01

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The Journal of Analytical Toxicology (JAT), established in 1977 and published 9 times a year, is the international source covering a broad range of clinical, forensic, and industrial laboratory topics regarding the isolation, identification, and quantitation of potentially toxic substances.

With an emphasis on practical application, JAT articles provide improved and novel techniques for use in clinical, forensic, workplace, sports testing (doping), and other toxicology laboratories. Articles describe newly developed methods in immunoassay testing, gas chromatography, liquid chromatography, mass spectrometry, atomic absorption spectrometry, solid and liquid phase extraction techniques, and other analytical approaches. Worldwide readership includes toxicologists, pathologists, chemists, clinicians, researchers, and educators working in medical examiner and law enforcement laboratories, hospitals, university and independent analytical laboratories, as well as the drug manufacturing industry.

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JAT, as determined by ISI Citation Index, is one of the two most referenced international journals in forensic science.
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