Solid-phase extraction of methadone enantiomers and benzodiazepines in biological fluids by two polymeric cartridges for liquid chromatographic analysis.

The aim of this work was to present the advantages of two polymeric cartridges (Oasis HLB from Waters and Abselut Nexus from Varian) for the solid-phase extraction of methadone enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and of some benzodiazepines (diazepam, flunitrazepam, nitrazepam, oxazepam) in serum and urine in comparison with classical C18-bonded-silica cartridges or liquid extraction. After addition of serum or urine samples, these two cartridges were washed with a water-methanol mixture (95:5, v/v) and eluted with diethylether. After rapid evaporation, the residue was regenerated with mobile phase and injected either in a chiral column (Cyclobond I-2000 RSP) for methadone enantiomers and its metabolite or in a reversed-phase column (Symmetry Shield RP8) for benzodiazepines. The results showed that the chromatograms of blank serum and urine were cleaner than those obtained from classical solid-phase extraction or liquid extraction. The recoveries from these two polymeric cartridges were higher (95-102%) than those obtained by the two previous classical methods and the total time for extraction and solvent evaporation was also shorter (about 6-7 min). For methadone and benzodiazepine extraction, the use of acidic or alkaline buffer was not necessary.

Modulation of the primary and the secondary antibody response by tobacco smoke condensates.

Cigarette smoke condensate administered to C57BL/6 mice led to a decrease in the primary antibody response to OVA (hen egg albumin) antigen. Selenium (Se)-supplementation allowed to relieve significantly this inhibition. Moreover, even being not supplemented with Se, a preparation was found devoid of inhibitory effects. Furthermore, the presence of Se-supplemented tobacco smoke condensate at the time of antigen priming, contributed to an enhanced secondary antibody response.

In vitro and in vivo immunotoxic and immunomodulatory effects of nonsupplemented and selenium-supplemented cigarette smoke condensate.

Cigarette smoke condensate has been evaluated for its in vitro and in vivo immunotoxic and immunomodulatory properties. It was found that cigarette smoke condensate used in vitro at concentration from 6.6 to 20 microg/ml exerted pronounced inhibitory effects upon cell surface antigen-presenting major histocompatibility complex class I (MHC-I) expression and immunoglobulin (Ig) synthesis. In vivo, i.p. administration of cigarette smoke condensate to C57BL/6 mice before challenging with ovalbumin (OVA) antigen, has led to a decrease of anti-OVA specific antibody response. This inhibition affected more Ig protein synthesis than membrane bound MHC-I expression. Supplementation with selenium (Se) significantly reduced the inhibitory effects both in vitro and in vivo.

Separation of oxazepam, lorazepam, and temazepam enantiomers by HPLC on a derivatized cyclodextrin-bonded phase: application to the determination of oxazepam in plasma.

The enantioselective high-performance liquid chromatography (HPLC) of three racemic 3-hydroxybenzodiazepines, oxazepam (Oxa), lorazepam (Lor), and temazepam (Tem), is a difficult operation because of the spontaneous chiral inversion in polar solvent. To solve this problem, we have developed an HPLC method based on a chiral Cyclobond I-2000 RSP column, maintained at 12 degrees C, and a reversed mobile phase (acetonitrile in 1% triethylamine acetate buffer, TEAA) at a flow rate of 0.4 ml/min. Peaks were detected by a photodiode-array detector at 230 nm for quantification and by an optical rotation detector for identification of (+) and (-) enantiomers. The results showed that peak resolutions of Oxa, Lor, and Tem enantiomers, analyzed under the same conditions, were 3.2, 2.0, and 1.8, respectively. For the determination of Oxa enantiomers in plasma of rabbits, extraction with diethyl ether at pH 1.5, a polar organic mobile phase, and a Cyclobond I-2000 SP column were used. Other analytical conditions were the same as previously described. Blood samples were immediately cooled at 4 degrees C and centrifuged at 0 degrees C for the collection of plasma. The results showed a difference in plasma S(+)- and R(-)-oxazepam concentrations in rabbits. No racemization of S(+)- or R(-)-Oxa enantiomers, added alone to blank plasma, was observed after extraction and enantioselective HPLC analysis.