A comparison of five commercial immunoassays for the detection of flunitrazepam and other benzodiazepines in urine

Abstract

Five commercially available immunoassay test kits (SYVA EMIT(R) d.a.u(TM), SYVA EMIT(R) II assay, Abbott FPIA, Cozart Auto-Lyte(R) and Roche Abuscreen(R) Online(TM), all used for the benzodiazepine group of drugs) were evaluated for their ability to detect flunitrazepam, its major urinary metabolite, 7-aminoflunitrazepam, and several other benzodiazepines at serial dilutions (final concentration 25-1000 ng/ml) in drug-free urine and in urines following oral administration of flunitrazepam (1-3 mg). For comparison, gas chromatography/mass spectrometry was used to measure urinary levels of 7-aminoflunitrazepam. Levels of drug detected in the study were compared with the cross-reactivities presented by the manufacturers for each individual kit. One to three mg doses of flunitrazepam were taken by volunteers and levels excreted in urine analysed over several hours. A positive response was obtained in several samples from volunteers who had taken 2 mg or 3 mg doses, but not a 1 mg dose. Thirty-five clinical samples from the individuals suspected of benzodiazepine abuse were also examined. The results were not consistent among the kits evaluated.We conclude that the test kits evaluated in this study do not detect flunitrazepam reliably, due primarily to their poor sensitivities.

Detection of Flunitrazepam in Biological Fluids and Beverages:A Review

“…Recently, considerable research efforts are dedicated to enzyme‐linked immunosorbent assay (ELISA), Micro‐plate enzyme and kit [31–35] . Negrusz and coworkers designed a micro‐plate enzyme immunoassay method to determine FLU and 7AF in hair and urine sample.…”

“…[29,30] Recently, considerable research efforts are dedicated to enzyme-linked immunosorbent assay (ELISA), Micro-plate enzyme and kit. [31][32][33][34][35] Negrusz and coworkers designed a microplate enzyme immunoassay method to determine FLU and 7AF in hair and urine sample. Authors used two different organic solvents: methanol and glacial acetic acid for sample preparation.…”

Multi-residue detection of benzodiazepines by ELISA based on class selective antibodies

“…With the advantages of sensitivity, specificity, rapidity, simplicity and cost-effectiveness, immunochemical methods, especially enzyme-linked immunosorbent assay (ELISA), is becoming more and more useful for screening benzodiazepines in toxicological and clinical samples. Many immunoassay methods for benzodiazepines have been developed in the past two decades (Barrett et al, 1999;Borrey et al, 2002;Elian, 2003;Huang & Moody, 1995;Kemp, Sneed, Kupiec, & Spiehler, 2002;Li et al, 2008;Meatherall & Fraser, 1998;Miller, Wylie, & Oliver, 2006;Peng et al, 2008a,b; . Various authors (Elian, 2003;Huang & Moody, 1995) have tested commercial immunoassays for their specificity against benzodiazepines, benzodiazepine metabolites, clonazepam and 7-aminoclonazepam, however, these assay either did not detect all of the compounds or had low cross-reactivities (CRs) with the compounds (Liu & Xu, 2007;Wang, Xu, & Peng, 2006;Xu et al, 2006a,b,c,d).…”