On-line coupling of sequential injection extraction with restricted-access materials for sample clean-up and analysis of drugs in biological matrix

In this contribution, the on-line coupling of solid phase extraction (SPE), based on a restricted-access material (RAM), with sequential injection technique (SIA) for the analysis of biological samples, is described. The SIA-RAM system was tested with a new potential antileucotrienic drug (VUFB-19363 (Quinlukast)) for serum analysis. The method is based on SPE with the novel internal-surface reversed-phase column packing material-alkyl-diol silica (ADS). The supports tolerate direct and repetitive injection of proteinaceous fluids (plasma, serum) and allow reversed-phase partitioning at the internal surface. A column packed with a 25 microm C18 alkyl-diol support was used for direct serum injection. Using a 6-port selection valve and the system of three mobile phases, the polar matrix compounds and metabolites are removed by sequentially aspirated mobile phases with lower content of the organic part (methanol-water (2:98) and following acetonitrile-water (20:80)) to the waste, and then, the analyte enriched on the column is eluted by a strong mobile phase (acetonitrile-methanol-water (40:20:40)) to the UV detector without transfer loss. With the fully automated SIA system, a total analysis time of less than 10 min was achieved. The only off-line sample pre-treatment step required to remove particulate matter was centrifugation. The studies showed a range of linearity (2-40 microg ml(-1)) and a high recovery (93.6-96.8%) of drug from the biological matrix with coefficients of variation (RSD) less than 5.0% (n = 6). This paper introduces a new, simple and robust analytical technique suitable for screening determination and direct analysis of drugs in biological materials.     

The electronic structure of benzvalene : Photoelectronspectroscopic studies

The photoelectron(PE)spectrum of tricyclo[3.1.0.0^2,6] hex-2-ene(benzvalene 1) has been recorded. The first four bands in the PE spectrum of 1 can be assigned to transitions to ²B₂, ²A₁, ²A₂ and ²B₁ states of 1¹. This assignment is discussed in terms of the results of semiempirical and ab initio calculations on 1. Furthermore the highest occupied MO's of 1 are derived qualitatively from an interaction diagram between a distorted bicyclobutane and an ethylene moiety.     

A fluorimetric assay for cortisol

A simple, rapid and sensitive fluorimetric assay for the quantitative determination of cortisol is reported. The assay is based on the formation of a fluorescent dye when cortisol is incubated with a mixture of sulfuric acid and acetic acid. The fluorescence spectrum recorded for the resulting dye shows a maximum extinction at 475 nm and a maximum emission at 525 nm. The solvent 2-methyl-4-pentanone was used for extraction and was found to act as a fluorescence amplifier. A limit of detection of 2.7 μM was achieved, making it possible to forego solvent evaporation. The assay suffers minor interference from 11-deoxycortisol which exhibits low fluorescence at λ _ex: 460 nm; λ _em: 505 nm. Typical standard deviations were below 4%. We validated the assay using a biotransformation with recombinant Schizosaccharomyces pombe which regioselectively hydroxylates 11-deoxycortisol to cortisol. The method described herein is suitable for preliminary screening of microorganisms capable of steroid hydroxylation.     

A novel technique to specifically analyze the secretome of cells and tissues

The secretome of cells and tissues may reflect a broad variety of pathological conditions and thus represents a rich source of biomarkers. The identity of secreted proteins, usually isolated from cell supernatants or body fluids, is hardly accessible by direct proteome analysis, because these proteins are often masked by high amounts of proteins actually not secreted by the investigated cells. Here, we present a novel method for the specific detection of proteins secreted by human tissue specimen as well as cultured cells and chose liver as a model. The method is based on the metabolic labelling of proteins synthesized during a limited incubation period. Then, the cell supernatant is filtered, precipitated, and subjected to two-dimensional gel electrophoresis. Whereas fluorography detected a large number of proteins derived from residual plasma and dead cells, the autoradiographs selectively displayed genuinely secreted proteins. We demonstrate the feasibility of this approach by means of the secretomes of the hepatocellular carcinoma-derived cell line HepG2 and human liver slices. The selective identification of cell- and tissue-specific protein secretion profiles may help to identify novel sets of biomarkers for wide clinical applications.     

α,ω-Diisocyanatocarbodiimides, -Polycarbodiimides,and Their Derivatives

Research in the field of low-molecular weight, oligomeric and polymeric α,ω-diisocyanatocarbodiimides and -polycarbodiimides has been fruitful, not only in connection with these compounds themselves, but also—as so often happens in chemistry—with quite different problems. Novel synthetic methods, discoveries concerning the properties of low-molecular weight carbodiimides and phosphane imide derivatives, as well as results on the fragmentation reactions of four-membered heterocyclic compounds containing oxygen, phosphorus, and nitrogen, and a better understanding of the diisocyanate polyaddition process are among the many by-products of this research. The “high- and low-temperature formation” of polycarbodiimides and the homogeneous and heterogeneous catalysis of this process are described, and the fundamental importance of four-membered ring fragmentation mechanisms resulting in the formation of phosphane imide derivatives is outlined. Interesting building blocks for the diisocyanate polyaddition and polycondensation processes can be synthesized by many derivatization reactions of oligomeric and high-molecular weight polycarbodiimides and polyuretonimines. The in situ production of polycarbodiimides via matrix reactions in flexible polyurethane foams leads to a cellular arrangement of the material due to the pronounced symmetrical growth processes. Combination-foams with increased carbonation tendencies are formed in this way. Attention is drawn to several industrial applications of α,ω-diisocyanatopolycarbodiimides, of high-molecular weight cross-linked polyuretonimines, and of polycarbodiimide foams.     

Acid-base reactions of 1,3,4-thiadiazolines and thiocarbonyl ylides; 1,3,4-thiadiazoline-2-spiro-2′-adamantane

The surprising formation of C₂₂H₃₂N₂S₂ from the title compound 1 at 45°C involves the interaction of the basic adamantanethione S-methylide ($\text{2}$) with its acidic precursor $\text{1}$, in the course of which the anion $\text{6}$ undergoes electrocyclic ring opening; the acid and base functions offer the clue to a prolific chemistry of the thiadiazoline $\text{1}$ and the thiocarbonyl ylide $\text{2}$.     

The preparation of a cabbage reference material for environmental monitoring and food analysis

 Cabbage is frequently used in environmental monitoring and food control, and, hence, cabbage reference materials (RMs) are required for ensuring quality assurance. A cabbage RM was prepared in view of certification of specific elements from the "black list" of high toxicological interest and nutritive importance. All tasks of the RM production (production of the plant material, cutting and freeze-drying, determination of the residual water content, preparation of the RM, homogeneity testing, stability testing, certification measurements) are described in detail. Received: 12 November 1999 / Accepted: 29 January 2000     

Orbital Theory of Heterolytic Fragmentation and Remote Effects on Nitrogen Inversion Equilibria

From a molecular orbital study of model systems we derive the electronic requirements for the Grob fragmentation. The necessary condition for an allowed fragmentation in an X-C₁-C₂-C₃-N system, or the amino cation ⁺C₁-C₂-C₃-N is the level ordering A below S . This in turn is set by maximal through-bond coupling of the empty cation orbital and the nitrogen lone pair. The conformational dependence of through-bond coupling is exactly that derived by Grob, namely parallel orientation of the cation orbital (or the C-X bond), the C₂-C₃-σ-bond, and the N-lone-pair. When the C₁-C₂-C₃ and C₂-C₃-N angles are small, the through-space interaction dominates, reversing the level ordering to S below A , and consequently makes the fragmentation forbidden even though the conformational requirements for it are met. Ring closure becomes allowed. Some examples exploiting this result are presented, as well as procedures for enhancing through-bond coupling and thus fragmentation. The through-bond-effect has also kinetic consequences, allowing the definition of a new type of remote neighbouring group participation operative through bonds and not by direct overlap. The position of equilibria in nitrogen inversion processes should also be influenced by remote substituents which are π-acceptors or donors.     

Isolierung und Aminosäuresequenz von Substanz P aus Pferdedarm

The isolation and amino acid sequence of substance P from horse intestine is reported. The sequence, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂ is identical with the sequence reported for substance P isolated from bovine hypothalami.     

Synthesis, structure, and dynamics of six-membered metallacoronands and metallodendrimers of iron and indium

In the reaction of the N-substituted diethanolamines (H(2)L(1-3)) (1-3) with calcium hydride followed by addition of iron(III) or indium(III) chloride, the iron wheels [Fe(6)Cl(6)(L(1))(6)] (4) and [Fe(6)Cl(6)(L(2))(6)] (6) or indium wheels [In(6)Cl(6)(L(1))(6)] (5), [In(6)Cl(6)(L(2))(6)] (8) and [In(6)Cl(6)(L(3))(6)] (9) were formed in excellent yields. Exchange of the chloride ions of 6 by thiocyanate ions afforded [Fe(6)(SCN)(6)(L(2))(6)] (7). Whereas the structures of 4, 5 and 7 were determined unequivocally by single-crystal X-ray analyses, complexes 8 and 9 were characterised by NMR spectroscopy. Contrary to what is normally presumed, the scaffolds of six-membered metallic wheels are not generally rigid, but rather undergo nondissociative topomerisation processes. This was shown by variable temperature (VT) (1)H NMR spectroscopy for the indium wheel [In(6)Cl(6)(L(1))(6)] (5) and is highlighted for the enantiotopomerisation of one indium centre [ 1/6[S(6)-5]<==>[1/6[S(6)-5']]. The self-assembly of metallic wheels, starting from diethanolamine dendrons, is an efficient strategy for the convergent synthesis of metallodendrimers.