Serum and urine concentrations of flunitrazepam and metabolites, after a single oral dose, by immunoassay and GC-MS.

A clinical study was conducted to assess the ability of commercially available immunoassays to detect flunitrazepam (FNP) in plasma and urine samples and to compare the results with those obtained by gas chromatography-mass spectrometry (GC-MS). The clinical study consisted of four individuals (two male and two female) who had taken a single 2-mg dose of FNP. Serum was collected over a 48-h period and urine was collected over a 72-h period. The serum and urine samples were analyzed by the COBAS INTEGRA Serum Benzodiazepines assay (SBENZ), the TDx serum and urine Benzodiazepines assay, and GC-MS. The GC-MS procedure was developed for analysis of FNP and metabolites in plasma and urine using an acid hydrolysis step resulting in the formation of specific benzophenones corresponding to FNP and its metabolites. The relative sensitivities of the assays for the detection of FNP and metabolites in serum and urine were GC-MS > SBENZ > TDx. The immunoassay results for serum samples showed peak concentrations of FNP metabolites at 8 h after FNP ingestion for three individuals and at about 1 h for the fourth individual. The GC-MS, SBENZ, and TDx urine immunoassays detected drug above the stated limit of detection (LOD) in 44, 41, and 35 serial FNP urine samples, respectively. FNP metabolites were detected in urine samples with all three assays for up to 72 h after a 2-mg dose. The improved detection rate with the SBENZ assay as compared to the TDx assay is likely explained by its higher cross-reactivity with the major metabolite, 7-amino-flunitrazepam (7-amino-FNP), and its lower LOD.

New fluorescence polarization immunoassays for analysis of barbiturates and benzodiazepines in serum and urine: performance characteristics.

The performance of the new fluorescence polarization immunoassay reagents Cassette COBAS INTEGRA Serum Benzodiazepines assay (SBENZ) and Cassette Serum Barbiturates assay (SBARB) was evaluated as compared to other immunoassays (Abbott TDx Serum Benzodiazepines, Abbott TDx Urine Benzodiazepines, Behring EMIT Serum Benzodiazepines, Abbott ADx Serum Barbiturates, Behring EMIT Serum Barbiturates, and the COBAS INTEGRA Barbiturates (BARB) urine assay) and gas chromatography-mass spectrometry (GC-MS). Recoveries of nordiazepam and secobarbital using the SBENZ and SBARB assays, respectively, were equivalent for serum, plasma, and urine. Cross-reactivities of structurally related benzodiazepines, barbiturates, and their metabolites were very similar in serum and urine for the SBENZ and SBARB assays. Precision was within 5.4% for SBENZ serum and within 11% from 10 to 100 ng/mL for urine. Precision was within 5% for SBARB serum and within 7% from 136 to 277 ng/mL for the urine application. The standard curves for SBENZ and SBARB were stable for at least 16 weeks with the reagents stored open on the COBAS INTEGRA analyzer. Clinical comparison of the SBENZ serum assay indicated an increased pickup rate, as confirmed by GC-MS, compared to TDx and EMIT. The diagnostic sensitivities of the SBENZ serum application, TDx, and EMIT versus GC-MS were 100%, 89%, and 36%, respectively. The diagnostic specificities were 71%, 79%, and 100%, respectively. The diagnostic sensitivities of the SBENZ urine application and TDx versus GC-MS were 100% and the diagnostic specificities were 88%. The increased positive pick-up of the SBENZ assay compared to the other immunoassays is most probably due to the difference in the limit of detection (LOD) and the increased cross-reactivity for the low-dose benzodiazepines. Clinical comparison of the SBARB serum assay indicated an increased positive pick-up rate, as confirmed by GC-MS. The diagnostic sensitivities of the SBARB serum application, ADx, and EMIT versus GC-MS were 96%, 65%, and 35%, respectively. The diagnostic specificities were all 100%. The diagnostic sensitivities for the SBARB urine application and BARB versus GC-MS were all 100%, and the diagnostic specificities were all 91%. The SBENZ and SBARB kits demonstrated increased sensitivity for the detection of benzodiazepines and barbiturates in both serum and urine compared to the other immunoassays.