False-negative results for bromazepam use observed by the EMIT II Plus benzodiazepine assays

Abstract

During our extensive routine analyses of drugs of abuse and other drugs such as benzodiazepines using both immunoassays and gas chromatography-mass spectrometry (GC-MS), we have noticed that the EMIT II benzodiazepine assays for urine samples sometimes gave false-negative results for bromazepam use or abuse. The negative benzodiazepine immunoassays and positive GC-MS results for bromazepam in urine could be explained by the absence (or below the detection limit) of 3-hydroxybromazepam (3HB) and the presence of 2-amino-3-hydroxy-5-bromobenzoylpyridine (AHBBP), which are the two major metabolites of bromazepam. 3HB cross-reacted with the antibody contained in the EMIT II, but AHBBP did not, due to their structural characteristics. To avoid such false-negative results for bromazepam, AHBBP screening by GC-MS is recommended. The false-negative results observed for bromazepam are probably not limited to the EMIT II kit; the same phenomena may be also true for other commercially available kits that are based on immunochemical reactions.