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Validation of the Immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine.
J Anal Toxicol. 2006 Mar; 30(2):115-9.JA

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+Show Affiliations

Forensic Medicine and Science Department, University of Glasgow, University Place, Glasgow G12 8QQ, Scotland.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Validation Study

Language

eng

PubMed ID

16620543

Citation

Miller, Eleanor I., et al. "Validation of the Immunalysis Microplate ELISA for the Detection of Buprenorphine and Its Metabolite Norbuprenorphine in Urine." Journal of Analytical Toxicology, vol. 30, no. 2, 2006, pp. 115-9.
Miller EI, Torrance HJ, Oliver JS. Validation of the Immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine. J Anal Toxicol. 2006;30(2):115-9.
Miller, E. I., Torrance, H. J., & Oliver, J. S. (2006). Validation of the Immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine. Journal of Analytical Toxicology, 30(2), 115-9.
Miller EI, Torrance HJ, Oliver JS. Validation of the Immunalysis Microplate ELISA for the Detection of Buprenorphine and Its Metabolite Norbuprenorphine in Urine. J Anal Toxicol. 2006;30(2):115-9. PubMed PMID: 16620543.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Validation of the Immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine. AU - Miller,Eleanor I, AU - Torrance,Hazel J, AU - Oliver,John S, PY - 2006/4/20/pubmed PY - 2006/5/5/medline PY - 2006/4/20/entrez SP - 115 EP - 9 JF - Journal of analytical toxicology JO - J Anal Toxicol VL - 30 IS - 2 N2 - 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%. SN - 0146-4760 UR - https://www.unboundmedicine.com/medline/citation/16620543/Validation_of_the_Immunalysis_microplate_ELISA_for_the_detection_of_buprenorphine_and_its_metabolite_norbuprenorphine_in_urine_ L2 - https://academic.oup.com/jat/article-lookup/doi/10.1093/jat/30.2.115 DB - PRIME DP - Unbound Medicine ER -