Determination of clorazepate and its major metabolites in blood and urine by electron capture gas-liquid chromatography.

A sensitive and specific blood level method employing differential extraction was developed for the determination of clorazepate and its N-desmethyldiazepam metabolite by electron capture gas-liquid chromatography (GLC-ECD). The assay requires the initial extraction of N-desmethyldiazepam, the major metabolite, into benzene-methylene chloride (90:10) from the biological sample made alkaline with 0.1 N NaOH. The samples is then acidified with 2 N HCl to decarboxylate clorazepate to N-desmethyldiazepam, which is then extracted into benzene-methylene chloride (90:10) after adjusting the pH to 12.8 with NaOH. The two extracts are evaporated and the residues are dissolved in benzene which contains griseofulvin as the reference standard. These solutions are assayed by GLC-ECD. The overall recovery and sensitivity limit of the assay for clorazepate is 60+/-5% (S.D.) and 4.0 ng/ml blood, respectively, while that for N-desmethyldiazepam is 95+/-5% (S.D.) and 4.0 ng/ml blood, respectively. The urinary excretion of clorazepate was determined by the measurement of the levels of N-desmethyldiazepam and oxazepam, the major urinary metabolites of clorazepate, both prior to and after enzymatic deconjugation. These methods were applied to the measurement of clorazepate and its metabolites in blood and urine following a single 15-mg dose of clorazepate dipotassium.     

Comparative study of several algorithms for flexible ligand docking

We have performed a comparative assessment of several programs for flexible molecular docking: DOCK 4.0, FlexX 1.8, AutoDock 3.0, GOLD 1.2 and ICM 2.8. This was accomplished using two different studies: docking experiments on a data set of 37 protein-ligand complexes and screening a library containing 10,037 entries against 11 different proteins. The docking accuracy of the methods was judged based on the corresponding rank-one solutions. We have found that the fraction of molecules docked with acceptable accuracy is 0.47, 0.31, 0.35, 0.52 and 0.93 for, respectively, AutoDock, DOCK, FlexX, GOLD and ICM. Thus ICM provided the highest accuracy in ligand docking against these receptors. The results from the other programs are found to be less accurate and of approximately the same quality. A speed comparison demonstrated that FlexX was the fastest and AutoDock was the slowest among the tested docking programs. The database screening was performed using DOCK, FlexX and ICM. ICM was able to identify the original ligands within the top 1% of the total library in 17 cases. The corresponding number for DOCK and FlexX was 7 and 8, respectively. We have estimated that in virtual database screening, 50% of the potentially active compounds will be found among approximately 1.5% of the top scoring solutions found with ICM and among approximately 9% of the top scoring solutions produced by DOCK and FlexX.     

In vitro bioactivity of wollastonite–titania materials obtained by sol–gel method or solid state reaction

In order to obtain materials for bone tissue replacement and regeneration that show an appropriate bioactivity, antibacterial behavior and higher mechanical properties than those of the existing bioactive systems, titania–wollastonite materials were obtained by both solid state reaction and sol–gel methods. In the solid state reaction process, titania and wollastonite powders were mixed and sintered. Two different types of materials were obtained by sol–gel: (i) using titanium butoxide, calcium nitrate tetrahydrate and tetraethyl orthosilicate as precursors or (ii) mixing titanium butoxide with wollastonite powder. The in vitro bioactivity was assessed by immersing samples in simulated body fluids for different periods of time. Alamar blue assays and osteoblast-like cells were used for testing citotoxicity. A higher bioactivity was observed on the samples synthesized by sol–gel. However, a higher cell proliferation was observed on the samples obtained by solid state reaction.     

Comparison of Approaches to Calibrate a Surface Complexation Model for U(VI) Sorption to Weathered Saprolite

A surface complexation model describing the sorption of uranyl ions and uranyl carbonate on weak and strong sites was used to analyze experiments conducted on pH-dependent U(VI) sorption to weathered shale/limestone saprolite. Sorption data were collected at two different solid to solution ratios. Various methods of estimating equilibrium reaction coefficients and site densities were investigated. As a first approximation, extractable iron oxides were assumed to behave as ferrihydrite with reaction coefficients as reported by Waite (Geochim Cosmochim Acta 58:5465–5478, 1994). A generalized composite (GC) approach was then employed with coefficients estimated by an inverse modeling method applied both in a stepwise fashion and simultaneously to whole data set. Uncertainty in model parameters and predictions was lowest using the simultaneous inverse method, but results from the stepwise method were very similar. The generalized reaction network accurately described pH-dependent U(VI) sorption on weathered saprolite between pH 4 and 9.