IMMUNOASSAY DETECTION OF NORDIAZEPAM, TRIAZOLAM, LORAZEPAM, AND ALPRAZOLAM IN BLOOD

The ability of commercial benzodiazepine immunoassays to detect nordiazepam, lorazepam, alprazolam, and triazolam in blood samples was investigated. Two radioimmunoassays (RIA) (Abuscreen [RIA-a] and the Diagnostic Products Corporation serum kit [RIA-d]), two enzyme immunoassays (EIA) (Emit d.a.u. [EIA-u] and Emit TOX serum assay [EIA-a]), and two fluorescence polarization immunoassays (FPIA) (X-systems urine [FPIA-u] and X-systems serum assay [FPIA-s]) were evaluated for their ability to detect benzodiazepines in fortified drug-free human or bovine blood. Prior extraction of the blood was necessary for analysis on the equipment used for EIA and FPIA. Extraction with an organic solvent, such as butyl chloride used in this study, was preferable to precipitation with methanol or zinc sulfate. For all these assays, extraction eliminated matrix effects and offered the possibility of increased sensitivity by reconstitution of the extract in a smaller volume. Extraction was necessary in bovine blood, as there is some nonextractable substance(s) in this matrix that increases non-specific binding. Using extraction with reconstitution in one-half the original volume, the apparent limit of detection for nordiazepam in blood ranged from 3 ng/mL, with RIA-d, to 30 ng/mL, with EIA-s and FPIA-s. These limits of detection were improved by further reduction of the reconstitution volume. RIA-a, EIA-s, and FPIA-s had cross-reactivity for alprazolam that was equivalent to or slightly better than nordiazepam. RIA-d had enhanced cross-reactivity for alprazolam at 30–300 ng/mL. The crose-reactivity for lorazepam at 70 ng/mL was 24, 7, 38, and 42% for RIA-a, RIA-d, EIA-s, and FPIA-s, respectively. For triazolam at 70 ng/mL, it was 37, 13, 66, and 71% for the same assays. Detection by any of these assays was not affected by aging benzodiazepine-fortified human blood at room temperature for up to five weeks. Commercial immunoassays can be used to detect benzodiazepines in blood. When the blood is extracted with an oganic solvent and reconstituted in a reduced volume, benzodiazepines can be detected at low concentrations. © 1993 Oxford University Press.