title: Immunoassay detection of benzodiazepines and benzodiazepine metabolites in blood.
abstract: We evaluated the capability of commercially available benzodiazepine immunoassays from four vendors (Abuscreen radioimmunoassay [RIA-a] from Roche Diagnostic Systems and serum RIA-d from Diagnostic Products Corporation; X-systems serum and urine fluorescence polarization immunoassays [FPIA] from Abbott Laboratories; and EMIT TOX serum and EMIT urine enzyme immunoassays [EIA] from Syva) to detect 22 benzodiazepines and 16 benzodiazepine metabolites in blood samples. Drug-free bovine blood was fortified with the different compounds at concentrations relevant to blood analysis: 7, 30, and 70 ng/mL. One-milliliter samples were extracted with n-butyl chloride, dried, and reconstituted with 0.3 ml. of buffer. Results from the two urine-specific kits (EIA-u and FPIA-u) produced either equivalent or reduced cross-reactivity compared with their serum kit counterparts; these results are presented but not discussed further. Among the 38 compounds, the following were detected by all four assays at concentrations of 70 ng/mL or less: alprazolam, alpha-hydroxy-alprazolam, norclobazam, diazepam, estazolam, flunitrazepam, norflunitrazepam, miclazolam, 4-hydroxy-midazolam, nitrazepam, noradinazolam, oxazepam, prazepam, 3-hydroxy-prazepam, temazepam, and triazolam. 7-Amino-clonazepam, 7-acetamido-clonazepam, 3-hydroxy-7-acetamidoclonazepam, haloxazepam, loprazolam, 7-amino-nitrazepam, 7-acetamido-nitrazepam, and oxazolam were not detected by any of the assays at concentrations up to 70 ng/mL. A number of benzodiazepines or their metabolites were detectable by some but not all of the six assays: bromazepam, chlordiazepoxide, demoxepam, and 4-hydroxy-alprazolam (both RIAs and EIA); adinazolam (RIA-a, EIA, and FPIA); norchlordiazepoxide (both RIAs); clonazepam (RIA-a and EIA); bentazepam, clotiaze-pam, and mexazolam (EIA and FPIA); 7-amino-flunitrazepam and quazepam (EIA); and alpha-hydroxy-triazolam and medazepam (RIA-a).
authors.affiliation: Department of Pharmacology and Toxicology, University of Utah, Salt Lake City 84112, USA.
authors.forename: W
authors.initials: W
authors.lastname: Huang
authors.forename: D E
authors.initials: DE
authors.lastname: Moody
mesh_terms: D000818:Animals; D001569:Benzodiazepines / Q000097:blood* / Q000378:metabolism* / Q000652:urine; D002417:Cattle; D003429:Cross Reactions; D005454:Fluorescence Polarization / Q000379:methods; D007124:Immunoenzyme Techniques; D000432:Methanol / Q000737:chemistry; D015394:Molecular Structure; D011863:Radioimmunoassay / Q000379:methods*; D012015:Reference Standards
chemical_list: D001569:Benzodiazepines; D000432:Methanol