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.

Analysis of lidocaine on the COBAS INTEGRA.

A new fluorescence polarization (FP) immunoassay was developed for the COBAS INTEGRA to quantitate lidocaine in serum and sodium heparin derived plasma. The COBAS INTEGRA, a random and continuous access clinical analyzer, performs the new lidocaine assay at a throughput of 300 tests per hour. Comparison studies with the Abbott TDx Lidocaine assay yielded a correlation coefficient of 0.99 and a regression value of INTEGRA = -0.01 + 0.95 TDx, n = 69. The assay was found to be linear throughout the concentration range of 0 to 10 micrograms/ml using CASCO STANDARDS's DOCUMENT TDM 1 Linearity Test Set and the COBAS-FP TDM Calibration Verification Test Set. The new lidocaine assay exhibited good precision as determined by the National Committee for Clinical Laboratory Standards (NCCLS) protocol EP5-T2 with total percent coefficient of variation (%CV) values of less than 4 percent across the assay range. Interference was less than 10 percent for abnormal levels of total protein (2.1 to 10 g/dl), lipid (up to 2200 mg/dl triglyceride), hemoglobin (up to 10 g/dl), and icteric samples (up to 17.5 mg/dl bilirubin). Finally, the standard curve was stable for greater than 25 weeks which was attributable to the on-board reagent cooling and a unique cassette closure system.

Automated fluorescence polarization immunoassay for monitoring streptomycin.

The fluorescence polarization immunoassay technique has been used previously for the assay of vancomycin and the aminoglycoside antibiotics (gentamicin, tobramycin, and amikacin). We extended this technique to assay streptomycin. Fluorescein-labeled streptomycin was used as the tracer, and antiserum specific for streptomycin was raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum were combined, and the polarization of tracer fluorescence was determined in a specially designed fluorometer (Abbott TDx). Because of the instrument design, the possibility of fluorescent interferences was eliminated. The coefficient of variation within run was 4% (n = 5), and between run it was 5% (n = 5). We compared the fluorescence polarization immunoassay technique (TDx streptomycin) with a conventional bioassay, using Bacillus subtilis for clinical specimens (n = 39). A least-squares linear regression analysis gave a slope of 1.16, an intercept of 2.41 mg/liter, and a correlation coefficient of 0.885. Repeat analyses by both techniques showed that the largest discrepancies could be explained by the imprecision of the bioassay.

Automated fluorescence polarization immunoassay for monitoring vancomycin.

We have extended fluorescence polarization immunoassay (FPIA) technology for the measurement of drugs to include the complex amphoteric glycopeptide antibiotic vancomycin (molecular weight, 1,449). Fluorescein-labeled vancomycin was employed as a tracer, and antisera specific for vancomycin were raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum were combined, and the polarization of tracer fluorescence is determined in a specially designed fluorometer (Abbott TDx). Because of instrument design, the possibility of fluorescent interferences is minimized. The assay can measure as little as 0.6 mg/L of vancomycin and is free of interferences from hemolysis, lipemia, bilirubin, and changes in protein concentration. The coefficient of variation within assay was 3% (n = 5) and between assays was 5% (n = 5). The FPIA assay (TDx Vancomycin) was compared to a liquid chromatographic (LC) assay for vancomycin and to a commercially available radioimmunoassay (RIA) for 98 clinical specimens. A linear least-squares regression analysis gave a correlation coefficient for LC of 0.980 from the equation FPIA = 1.09 LC + 3.04, and a correlation coefficient for RIA of 0.957 from the equation FPIA = 1.036 RIA + 1.66.

Fluorescence polarization immunoassay. iii. an automated system for therapeutic drug determination.

A fully automated system for performing fluorescence polarization immunoassay has been developed. Reagents for each assay are contained in coded reagent packs, and no reagent reconstitution is required. A common buffer is used for all assays, minimizing changeover and set-up times for each assay. A single sample may be assayed in 5 min, or 20 samples in 10 min. A single-tube blank subtraction for each sample results in highly precise polarization values and obviates sample interferences. We have used this method for assays of gentamicin, theophylline, phenytoin, and phenobarbital. CVs are 1-4%, and the results correlate well with those by other methods. Because of the instrument design and the stability of the reagents, daily calibration is not required; samples may therefore be run immediately upon receipt or batched as desired.

Fluorescence polarization immunoassay. I. Monitoring aminoglycoside antibiotics in serum and plasma.

Fluorescence polarization immunoassays of the aminoglycoside antibiotics gentamicin, tobramycin, and amikacin in plasma and serum are described and shown to be clinically useful. The aminoglycoside tracers were prepared by reacting the parent compounds with 5-[(4,6-dichlorotriazin-2-yl)-amino] fluorescein. Antisera specific for the compounds were raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum are combined and, after a 15-min incubation at ambient temperature, the polarization of the fluorescence of the tracer is determined in a specially designed fluorometer. The assays are designed to give accurate trough (i.e., minimum during therapy) values and to be free of matrix effects. Severely icteric samples may interfere, but this can be overcome by blank subtraction. The performance of the assays with clinical specimens compared favorably with that of some commercially available assays.