Quantification of mephenytoin and its metabolites 4'-hydroxymephenytoin and nirvanol in human urine using a simple sample processing method

A reliable and easy to use liquid chromatography/tandem mass spectrometry (LC/MS/MS) method without the use of sample extraction was developed for the simultaneous quantification of urinary concentrations of mephenytoin, a standard phenotyping substrate for the cytochrome P450 enzyme CYP2C19, and its phase I metabolites 4'-hydroxymephenytoin and nirvanol. Fifty microL of urine were diluted with a buffered beta-glucuronidase solution and incubated at 37 degrees C for 6 h followed by addition of methanol, containing the internal standard 4'-methoxymephenytoin. The chromatographic separation was achieved using a 100 x 3 mm, 5 micro Thermo Electron Aquasil C18 column with a gradient flow, increasing the organic fraction (acetonitrile/methanol 50:50) of the mobile phase from 10 to 90%. Quantification by triple-stage mass spectrometry (TSQ Quantum, Thermo Electron) was accomplished by negative electrospray ionization in the selected reaction monitoring mode. Linearity was observed for all substances in the concentration range 15-10 000 ng/mL. The lower limit of quantification (LLOQ) was 20 ng/mL for 4'-hydroxymephenytoin and 30 ng/mL for nirvanol and mephenytoin, respectively. Intra- and inter-day inaccuracy did not exceed 9.5% for all substances from LLOQ to 10 000 ng/mL. Intra- and inter-day precision were in the range of 0.8-10.5%. The method was validated according to international ICH and FDA guidelines and successfully applied for phenotyping of Caucasian male volunteers who received an oral dose of 50 mg mephenytoin.     

Use of 19F NMR spectroscopy to screen chemical libraries for ligands that bind to proteins

Identification of compounds from chemical libraries that bind to macromolecules by use of NMR spectroscopy has gained increasing importance during recent years. A simple methodology based on (19)F NMR spectroscopy for the screening of ligands that bind to proteins, which also provides qualitative information about relative binding strengths and the presence of multiple binding sites, is presented here. A library of fluorinated compounds was assembled and investigated for binding to the two bacterial chaperones PapD and FimC, and also to human serum albumin (HSA). It was found that library members which are bound to a target protein could be identified directly from line broadening and/or induced chemical shifts in a single, one-dimensional (19)F NMR spectrum. The results obtained for binding to PapD using (19)F NMR spectroscopy agreed well with independent studies based on surface plasmon resonance, providing support for the versatility and accuracy of the technique. When the library was titrated to a solution of PapD chemical shift and linewidth changes were observed with increasing ligand concentration, which indicated the presence of several binding sites on PapD and enabled the assessment of relative binding strengths for the different ligands. Screening by (19)F NMR spectroscopy should thus be a valuable addition to existing NMR techniques for evaluation of chemical libraries in bioorganic and medicinal chemistry.     

Biharmonic map heat flow into manifolds of nonpositive curvature

Let M ^m and N be two compact Riemannian manifolds without boundary. We consider the L ² gradient flow for the energy . If and N has nonpositive sectional curvature we show that the biharmonic map heat flow exists for all time, and that the solution subconverges to a smooth harmonic map as time goes to infinity. This reproves the celebrated theorem of Eells and Sampson [6] on the existence of harmonic maps in homotopy classes for domain manifolds with dimension less than or equal to 4.Received: 27 March 2003, Accepted: 5 April 2004, Published online: 16 July 2004Mathematics Subject Classification (2000): 58E20, 58J35     

Non-destructive testing of tubes using a time reverse numerical simulation (TRNS) method

A method for the detection of defects in cylindrical structures and the determination of their positions and orientations is presented in this paper. The scattered field, which is generated by the interaction of excited guided waves with a defect, is evaluated with an approach named time reverse numerical simulation method (TRNS). Since the excited waves and the scattered field propagate along the sample, the time-consuming scanning of the whole tube can be eliminated. The scattered displacement field is measured in three dimensions over time with a laser vibrometer at different locations distributed equally around the circumference at a fixed axial coordinate far away from the defect. Instead of analyzing the complicated time signals directly, they are played back in time. If the recorded displacement histories of the scattered field are reversed in time and played back in an identical structure, the waves travel back the same path and interfere to a maximum at their origin. The result is an amplitude increase at the position of the defect where the scattered field was generated. Instead of playing back the recorded time signals in an experiment, this step is replaced by a numerical simulation. Only this enables the visualization and detection of the amplitude increase. As long as the simulation is of high accuracy, the position of the maximum interference corresponds exactly to the location of the defect in the experiment, although no defect is implemented in the simulation.     

Interaction of C60 with carbon nanotubes and graphite

The interaction of C60 with single-wall carbon nanotubes (SWNTs) and graphite is studied experimentally by thermal desorption spectroscopy and theoretically by molecular-mechanics and molecular-dynamics calculations. The van der Waals parameters and force field for C60-graphene and C60-SWNT interactions are derived from the low-coverage C60 binding energy to the graphite surface. We use these to compare the efficiency of different mechanisms by which C60 can be encapsulated into SWNTs.     

Nearest-neighbor configuration in (GaIn)(NAs) probed by x-ray absorption spectroscopy

Ga(1-x)In(x)N(y)As(1-y) is a promising material system for the fabrication of inexpensive "last-mile" optoelectronic components. However, details of its atomic arrangement and the relationship to observed optical properties is not fully known. Particularly, a blueshift of emission wavelength is observed after annealing. In this work, we use x-ray absorption fine structure to study the chemical environment around N atoms in the material before and after annealing. We find that as-grown molecular beam epitaxy material consists of a nearly random distribution of atoms, while postannealed material shows segregation of In toward N. Ab initio simulations show that this short-range ordering creates a more thermodynamically stable alloy and is responsible for blueshifting the emission.     

Hydrogen bond dynamics probed with ultrafast infrared heterodyne-detected multidimensional vibrational stimulated echoes

Hydrogen bond dynamics are explicated with exceptional detail using multidimensional infrared vibrational echo correlation spectroscopy with full phase information. Probing the hydroxyl stretch of methanol-OD oligomers in CCl4, the dynamics of the evolving hydrogen bonded network are measured with ultrashort (<50 fs) pulses. The data along with detailed model calculations demonstrate that vibrational relaxation leads to selective hydrogen bond breaking on the red side of the spectrum (strongest hydrogen bonds) and the production of singly hydrogen bonded photoproducts.     

New pyrazolium-carboxylates as structural analogues of the pseudo-cross-conjugated betainic alkaloid nigellicine

Pyrazolium-3-carboxylates were examined as relatives of the betainic alkaloid Nigellicine and as new examples of the sparsely populated class 16 of heterocyclic pseudo-cross-conjugated mesomeric betaines (PCCMB). The title compounds were prepared in a 4-step procedure starting from beta-diketo compounds 8 which were cyclized with substituted hydrazines. The resulting isomeric pyrazole esters 9 and 10 were separated and subsequently quaternized with dimethyl sulfate in the presence of nitrobenzene to pyrazolium esters 11 and 12. Saponification was best accomplished in diluted sulfuric acid, which resulted in the formation of the pseudo-cross-conjugated mesomeric betaines 13 and 14 in one step. Protonation to the corresponding carboxylic acids required the treatment of the betaines with tetrafluoroboric acid in dichloromethane. The effect of negative solvatochromism proves the charge separation in the ground state of the molecules. X-ray crystallographic analyses, semiempirical calculations, and ESI mass spectrometric measurements were performed to gain knowledge about the phenomenon of pseudo-cross-conjugation.     

Elastic stability of DNA configurations. II. Supercoiled plasmids with self-contact

Configurations of protein-free DNA miniplasmids are calculated with the effects of impenetrability and self-contact forces taken into account by using exact solutions of Kirchhoff's equations of equilibrium for elastic rods of circular cross section. Bifurcation diagrams are presented as graphs of excess link, DeltaL, versus writhe, W, and the stability criteria derived in paper I of this series are employed in a search for regions of such diagrams that correspond to configurations that are stable, in the sense that they give local minima to elastic energy. Primary bifurcation branches that originate at circular configurations are composed of configurations with D(m) symmetry (m=2,3,...). Among the results obtained are the following. (i) There are configurations with C2 symmetry forming secondary bifurcation branches which emerge from the primary branch with m=3, and bifurcation of such secondary branches gives rise to tertiary branches of configurations without symmetry. (ii) Whether or not self-contact occurs, a noncircular configuration in the primary branch with m=2, called branch alpha, is stable when for it the derivative dDeltaL/dW, computed along that branch, is strictly positive. (iii) For configurations not in alpha, the condition dDeltaL/dW>0 is not sufficient for stability; in fact, each nonplanar contact-free configuration that is in a branch other than alpha is unstable. A rule relating the number of points of self-contact and the occurrence of intervals of such contact to the magnitude of DeltaL, which in paper I was found to hold for segments of DNA subject to strong anchoring end conditions, is here observed to hold for computed configurations of protein-free miniplasmids.     

Elastic stability of DNA configurations. I. General theory

Results are presented in the theory of the elastic rod model for DNA, among which are criteria enabling one to determine whether a calculated equilibrium configuration of a DNA segment is stable in the sense that it gives a local minimum to the sum of the segment's elastic energy and the potential of forces acting on it. The derived stability criteria are applicable to plasmids and to linear segments subject to strong anchoring end conditions. Their utility is illustrated with an example from the theory of configurations of the extranucleosomal loop of a DNA miniplasmid in a mononucleosome, with emphasis placed on the influence that nicking and ligation on one hand, and changes in the ratio of elastic coefficients on the other, have on the stability of equilibrium configurations. In that example, the configurations studied are calculated using an extension of the method of explicit solutions to cases in which the elastic rod modeling a DNA segment is considered impenetrable, and hence excluded volume effects and forces arising from self-contact are taken into account.