Flunitrazepam excretion patterns using the Abuscreen OnTrak and OnLine immunoassays: comparison with GC-MS.

A study was conducted to compare the performance of the OnLine and OnTrak immunoassays for benzodiazepines with gas chromatographic-mass spectrometric (GC-MS) analysis in detecting flunitrazepam (FNP) and its metabolites in human urine. Urine was collected over a 72-h period from six individuals (four male and two female) who had taken a single oral dose of either 1 or 4 mg of FNP. The OnTrak assay was run at a 100-ng/mL cutoff of nordiazepam (NDP), and the OnLine assay was run with a standard curve from zero to 200 ng/mL of NDP with and without beta-glucuronidase treatment. Each sample was analyzed by GC-MS using FNP, 7-amino-FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP as standards with beta-glucuronidase treatment. The specimens from the 1-mg dose did not yield a positive result by immunoassay over the 72-h collection period. Specimens from the 4-mg dose did yield positive results in both immunoassays. The time of the first positive result ranged from 4 to 12 h, and the time to the last positive result ranged from 18 to 60 h. Treatment of the samples with beta-glucuronidase increased the OnLine values between 20 and 60%, but it did not appreciably increase the detection time. GC-MS analysis showed no detectable levels of FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP. However, all samples collected past time zero showed detectable levels of 7-amino-FNP (> 2 ng/mL) with peak concentrations at 12-36 h. The peak levels of 7-amino-FNP by GC-MS paralleled the peak levels of the immunoassay response. The amount of 7-amino-FNP metabolite quantitated by GC-MS, however, accounted for only 15-20% of the total immunoassay crossreactive FNP metabolites.

Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo.

IL-12 is a heterodimeric cytokine that has been shown to enhance natural killer (NK) and cytotoxic T lymphocyte (CTL) responses, and to induce IFN-gamma production in vitro. In this study, we have examined the effects in vivo of administering purified murine rIL-12 to normal mice. Daily injections of rIL-12 i.p. (1 ng to 10 micrograms/day) caused dose-dependent enhancement of NK cell lytic activity in the spleens and livers of treated mice. Histologic examination of the livers of IL-12-treated mice revealed focal mononuclear cell infiltrates, and flow cytometry studies indicated that the livers of IL-12-treated mice contained increased numbers of NK cells, CD8+ T cells, and monocytes. Liver and splenic lymphoid cells from IL-12-treated mice, unlike liver and splenic lymphoid cells from control mice, spontaneously secreted IFN-gamma in vitro, suggesting that they had been induced by IL-12 to produce IFN-gamma in vivo. Consistent with this, IFN-gamma could be detected in the serum of IL-12-treated mice. In mice which had been immunized by footpad injection of allogeneic splenocytes, daily administration of rIL-12 i.p. was shown to enhance the specific CTL response in the draining lymph nodes. Thus, these studies demonstrate that IL-12 can enhance NK and CTL activity and induce IFN-gamma production in vivo, as well as in vitro, and suggest possible mechanisms by which IL-12 may exert therapeutic effects in the treatment of some tumors and infectious diseases.