Preparation of a novel sorptive stir bar based on vinylpyrrolidone-ethylene glycol dimethacrylate monolithic polymer for the simultaneous extraction of diazepam and nordazepam from human plasma.

A new monolithic coating based on vinylpyrrolidone-ethylene glycol dimethacrylate polymer was introduced for stir bar sorptive extraction. The polymerization step was performed using different contents of monomer, cross-linker and porogenic solvent, and the best formulation was selected. The quality of the prepared vinylpyrrolidone-ethylene glycol dimethacrylate stir bars was satisfactory, demonstrating good repeatability within batch (relative standard deviation < 3.5%) and acceptable reproducibility between batches (relative standard deviation < 6.0%). The prepared stir bar was utilized in combination with ultrasound-assisted liquid desorption, followed by high-performance liquid chromatography with ultraviolet detection for the simultaneous determination of diazepam and nordazepam in human plasma samples. To optimize the extraction step, a three-level, four-factor, three-block Box-Behnken design was applied. Under the optimum conditions, the analytical performance of the proposed method displayed excellent linear dynamic ranges for diazepam (36-1200 ng/mL) and nordazepam (25-1200 ng/mL), with correlation coefficients of 0.9986 and 0.9968 and detection limits of 12 and 10 ng/mL, respectively. The intra- and interday recovery ranged from 93 to 106%, and the relative standard deviations were less than 6%. Finally, the proposed method was successfully applied to the analysis of diazepam and nordazepam at their therapeutic levels in human plasma. The novelty of this study is the improved polarity of the stir bar coating and its application for the simultaneous extraction of diazepam and its active metabolite, nordazepam in human plasma sample. The method was more rapid than previously reported stir bar sorptive extraction techniques based on monolithic coatings, and exhibited lower detection limits in comparison with similar methods for the determination of diazepam and nordazepam in biological fluids.

Simultaneous analysis of diazepam and its metabolites in rat plasma and brain tissue by HPLC-UV and SPE.

Diazepam is frequently used as an adjuvant during antidepressant therapy. Recently, some studies have suggested that the treatment with benzodiazepines could have different efficacy in depressed patients as opposed to non-depressed ones. To clarify the matter, a study is currently underway, regarding the drug metabolism in rats. In order to obtain a more complete and significant set of data, the main diazepam metabolites have also been considered, namely: nordiazepam, temazepam and oxazepam. A feasible and reliable HPLC method has been developed for the simultaneous determination of these compounds in plasma and brain tissue of rats. The method has been applied to "normal" rats and to genetic rat models of depression in order to estimate drug metabolism in different breeds. Analyte separation was achieved on a C8 reversed phase column using an acidic phosphate buffer/acetonitrile mixture as the mobile phase. The detection wavelength was 238 nm. An original sample pre-treatment, based on solid-phase extraction (SPE) was developed in order to eliminate endogenous interference, using only 250 microL of matrix (brain homogenate or plasma) for a complete analysis. The method has been validated with good results in terms of precision, extraction yield, sensitivity, selectivity and accuracy on both matrices and has been successfully applied to samples from some rats subjected to the preliminary study. The obtained data will hopefully contribute to the clarification of possible differences between depressed and non-depressed subjects with respect to benzodiazepine biotransformation.

Detection of desmethyldiazepam and diazepam in brain of different species and plants.

Recent data suggest that desmethyldiazepam (DD), a major metabolite of several benzodiazepines (BZD), might be of natural origin. Therefore we tried to quantify DD and diazepam (D) in animals during maturation (e.g. hen, chicken, eggs), in brain of species at different evolutionary stages e.g. salmon, frog, monitor/reptile, rat, cat, dog, deer, bovine) including newborn and adult humans. Since low concentrations of DD (range 0.01-0.04 ng/g wet wt) and D (range 0.005-0.02 ng/g) could be measured in different species by sensitive and specific mass spectrometry (GC-MS), we analysed also several plants (e.g. maize corn, lentils, potatoes, soybeans, rice, mushrooms). Again, DD and D could be detected in low amounts (0.005-0.05 ng/g) in some plant products. This would suggest that DD and D might be of natural origin and incorporated via the foodchain into the animal and human body. The biological role or clinical relevance of these intriguing findings need still to be elucidated.

Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.

An endogenous brain substance that binds to the central-type benzodiazepine receptors with agonist properties is present in both rat and bovine brains. This substance has been purified to homogeneity from bovine brain by immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reversed-phase HPLC steps. The purified substance was characterized as the benzodiazepine N-desmethyldiazepam (nordiazepam). The techniques used for the identification were mass spectrometry, HPLC, spectrophotometry, benzodiazepine receptor binding, and immunological techniques. Benzodiazepine-like immunoreactivity was also found in all the human brains tested, including six brains that had been stored in paraffin since 1940, fifteen years before the first synthesis of benzodiazepines. These results show that benzodiazepine-like molecules of natural origin--and possibly benzodiazepines themselves--are present in human and other mammalian brains.

N-desmethyldiazepam: a new metabolite of chlordiazepoxide in man.

Following administration of chlordiazepoxide HCl to man, N-desmethyldiazepam, a known metabolite of diazepam (Valium), was identified in plasma. The metabolite was identified on the basis of its thin-layer chromatographic (TLC) mobility, electron-capture gas-chromatographic (EC-GC) retention time, and mass spectrum relative to authentic N-desmethyldiazepam. Plasma levels of N-desmethyldiazepam in subjects receiving both single and chronic doses of chlordiazepoxide were determined by an EC-GC method with a limit of sensitivity of 10 ng/ml using 2-ml samples and by a radioimmunoassay procedure which had a limit of sensitivity of 20 ng/ml using a 0.1-ml sample. Both assay methods gave good agreement for the levels of N-desmethyldiazepam. In subjects receiving a single 30-mg oral or intravenous dose of chlordiazepoxide, measurable levels of N-desmethyldiazepam in plasma (10 to 60 ng/ml) were obtained 24 to 72 hr after administration. In 5 subjects receiving 10 mg of chlordiazepoxide three times a day, steady-state levels of N-desmethyldiazepam in plasma were reached after about 1 wk of administration. The mean maximum and minimum steady-state levels of N-desmethyldiazepam were 260 and 220 ng/ml of plasma, respectively. Similar steady-state levels were observed on treatment with 30 mg of chlordiazepoxide over 24 hr.