A micellar electrokinetic capillary chromatography method for monitoring mycophenolic acid in serum of transplant recipients.

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive agent mycophenolate mofetil (MMF), was for the first time quantified in the serum of transplant recipients using micellar electrokinetic capillary chromatography (MEKC). Sample preparation was carried out with solid phase extraction (SPE) using octadecyl-modified endcapped silica (C18 EC) as sorbent. Extremely varying recovery rates in preliminary experiments showed both the importance of pH monitoring during the single SPE steps and the necessity of an internal standard. MPA carboxy butyl ether (CBE), a specifically developed reference standard, was employed. Furthermore, optimisation of the MEKC parameters detection wavelength and injection time was of primary importance in order to enable the quantitation of therapeutic trough serum levels of MPA in the range lower than 5 microg x mL(-1). Under optimised conditions, a limit of quantitation of 1.0 microg x mL(-1) was achieved allowing the determination of MPA in the serum of patients.

Synthesis and in vitro efficacy of transferrin conjugates of the anticancer drug chlorambucil.

One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity.

Residue mass plot and abundance plot: detection of isobaric interferences in DE-MALDI-TOF-mass spectra of complex polymer mixtures.

Mass spectra of complex polymer mixtures often disturbed by overlapping homologue peak series have been interpreted by means of the novel techniques of the residue mass plot and the abundance plot. The model substance used for the investigations is so far poorly characterized non-ionic emulsifier Cremophor EL (polyoxyl 35 castor oil) (CrEL), a heterogeneous polyethoxylate mixture. Because of its high amount of hydrophobic and hydrophilic components, CrEL was separated into two fractions, aqueous and methanolic, by cation exchange (CCaEx) chromatography. CrEL was then subjected to delayed extraction matrix-assisted laser desorption/ionization time of flight mass spectrometry (DE-MALDI-TOF-MS). Evaluation of the mass spectra was performed by comparing the residue masses of the homologue peak series with the calculated residue masses of potential components of the excipient cationized with Na+ and K+. A number of these series are overlapping because they differ in their theoretical residue masses by about 0.05 Da. The detection of these isobaric interferences was the basic requirement for our analysis method. This goal was achieved by high mass accuracy of the measurements (obtained by internal calibration) in combination with two newly developed evaluation methods, the residue mass plot and the abundance plot. Using this combined technique, generally applicable for complex polymer mixtures, it was shown that the aqueous CCaEx fraction contains hydrophobic components such as di- and triesters of ricinoleic acid and polyethylene glycol as well as glycerol polyoxyethylene di- and triricinoleates, whereas the methanolic fraction contains hydrophilic components, mainly polyethylene glycol (PEG) and glycerol polyoxyethylene ether. Moreover, free PEG was shown to consist of PEG 800 in contrast to the value of 600 Da described so far in literature.

Spirocyclic nortriterpenes from the bulbs of Veltheimia viridifolia.

A new spirocyclic nortriterpene, 22-acetoxy-15-deoxo-eucosterol, was isolated together with a corresponding pentaglycoside from the bulbs of Veltheimia viridifolia. The structures of the isolated compounds were elucidated by spectroscopic methods including 1D- and 2D-1H NMR, 13C NMR, 1D-INEPT, 1D- and 2D-TOCSY, HSQC, HMBC, ROESY experiments, FAB- and HRESI-mass spectrometry.

P2-receptor antagonists: IV. Blockade of P2-receptor subtypes and ecto-nucleotidases by compounds related to reactive blue 2.

Effects of reactive blue 2 and twelve structurally related compounds were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP; mediated by P2X-receptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y-receptors), and the degradation of ATP by rat vas deferens tissue. All compounds, except acid blue 41 and acid blue 129 (at up to 100 microM), shifted the concentration-response curve of alpha,beta-MeATP in the rat vas deferens to the right. Most increased, but uniblue A greatly decreased, the maximum of the curve. In the case of cibacron blue 3GA and reactive blue 19, of which three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from unity. The apparent Kd values of the effective substances ranged between 0.7 and 111 microM. Most compounds increased the contraction of the rat vas deferens elicited by high K+. In the guinea-pig taenia coli, all compounds, except uniblue A and reactive blue 19 (at up to 100 microM), shifted the concentration-response curve of ADPbetaS to the right in a parallel manner. In the case of acid blue 129 and acid blue 80, of which three concentrations were tested, the slope of the Arunlakshana-Schild regression did not differ from unity. The apparent Kd values of the effective substances were between 0.7 and 69 microM. Most compounds also reduced the relaxation of the guinea-pig taenia coli elicited by noradrenaline. The removal of ATP from the medium by vas deferens tissue was decreased only by reactive blue 2, cibacron blue 3GA, uniblue A and reactive blue 19, with IC25% values between 17 and 62 microM. The structure-activity relationships for P2X- and P2Y-receptor blockade in this series are strikingly dissimilar. In reactive blue 2 and its isomers, for example, both the 1-amino-anthraquinone-2-sulphonate core and the 'side-chain' of the molecule are involved in P2X-receptor binding; P2Y-receptor affinity, in contrast, resides largely or totally in the anthraquinone core. The most promising antagonists are uniblue A which is P2X- versus P2Y-selective and acid blue 129 which is P2Y- versus P2X-selective, both with few, if any, non-P2-receptor effects at concentrations blocking the respective P2-subtype.

Enantiomeric and diastereomeric high-performance liquid chromatographic separation of cyclic beta-substituted alpha-amino acids on a teicoplanin chiral stationary phase.

High-performance liquid chromatographic (HPLC) separation of stereomeric cyclic beta-substituted or-quaternary alpha-amino acids was performed on a chiral stationary phase based on the glycopeptide antibiotic teicoplanin. The investigated amino acids are the 1-amino-2-methylcyclohexanecarboxylic acids, the 1-amino-2-hydroxycyclohexanecarboxylic acids, Ala, Cha, Phe and Tle. The effects of the mobile phase composition (type and content of organic modifier, pH) and of the temperature on the enantio- and diastereoselectivity were studied and the conditions were optimised to resolve the four stereomers of one amino acid in a single chromatographic run. The influence of the modifier concentration and the pH of the mobile phase reveal two enantiomeric and diastereomeric discrimination mechanisms based on different interactions with the stationary phase. For optimal separation of diastereomers the column has to be conditioned with an acidic eluent.

Temperature-induced inversion of elution order in the enantioseparation of sotalol on a cellobiohydrolase I-based stationary phase.

The effect of temperature on the resolution of (RS)-sotalol by immobilized cellobiohydrolase I (CBH I) was studied between 5 and 40 degrees C and Van 't Hoff plots of ln k versus 1/T were acquired at different pH values of the aqueous mobile phase and in the presence of varying organic cosolvents. The elution order of the enantiomers reverses in the range between 17 and 28 degrees C. Beyond this range, enantioseparations with comparatively high resolution factors are achieved either by decreasing or by increasing the temperature. The composition of the mobile phase influences the "crossover" temperature as well as the character of the global adsorption process of the (R)-(-)-enantiomer. Under certain conditions, (R)-(-)-sotalol exhibits an unusual endothermic adsorption behavior. Its retention time increases with increasing temperature. At room temperature (23 degrees C) the enantiomeric elution order can also be regulated by the solvent additive.

Enantiomeric and diastereomeric high-performance liquid chromatographic separation of cyclic beta-substituted alpha-amino acids on a copper(II)-D-penicillamine chiral stationary phase.

High-performance liquid chromatographic (HPLC) separation of stereoisomeric cyclic beta-substituted alpha-quaternary alpha-amino acids was performed by ligand-exchange on a copper(II)-D-penicillamine chiral stationary phase. The investigated amino acids are the 1-amino-2-methylcyclohexanecarboxylic acids, the 1-amino-2-hydroxycyclohexanecarboxylic acids, the 1-amino-2-methylcyclopentanecarboxylic acids and the trans-configured 1,2-diaminocyclohexanecarboxylic acids. The effects of the mobile phase composition (copper(II) concentration, type and content of organic modifier, pH) and the temperature on the enantio- and diastereoselectivity were studied and the conditions were optimised to resolve the four stereoisomers of each of the said amino acids in single chromatographic runs. A reversal of the elution order occurred for enantiomers of some of the amino acids in dependence on the acetonitrile content of the eluent. This phenomenon is explained by at least two different copper(II) complexes of the tridentate ligand penicillamine.

EPC syntheses and structure-activity relationships of hypoglycaemic semicyclic amidines.

A series of homochiral sterically hindered mono- and bicyclic amidines was prepared as hypoglycaemic agents by lethargic reaction of O-methylcaprolactim and 3-ethoxy-2-azabicyclo[2.2.2]oct-2-ene, respectively, with homochiral cis-2-substituted cyclopentane amines provided by asymmetrical reductive amination of racemic 2-substituted cyclopentanones. All compounds, except the cyclohexylmethyl-isoquinuclidone derivative which inhibited secretion at 100 microM, significantly stimulated insulin secretion 2-8-fold at 10 microM and 100 microM in INS-1 cells. The most potent activator was the 2-cyclopentyl-substituted caprolactam derivative 5e. The stimulatory effects on secretion increased with rising steric hindrance of both the amidine alpha-carbon and the bicyclic amidine moiety itself. Enantiomeric discrimination was observed for the 2-¿(cis-2-bulkysubstituted cyclopentyl)iminohexahydroazepine halides 5e and 5f and for the 3-¿(cis-2-substituted cyclopentyl)imino-2-azabicyclo¿2.2.2octane halides 6a and 6c. The amidines depolarized INS-1 cells and generated action potentials, accompanied by a decrease of membrane conductance. Simultaneously [Ca(2+)](i) increased, probably due to Ca(2+)-entry through voltage-dependent Ca(2+)-channels. At high concentrations, where inhibition of secretion was observed, ¿Ca(2+)(i) still rose upon application of the amidines, indicating an additional inhibitory pathway downstream to the elevation of ¿Ca(2+)(i). Even at high concentrations (100 microM), the amidines had no toxic effects on insulin secreting INS-1 cells.

Polyoxyethylene-Delta(9,11)-didehydrostearate and glycerol-polyoxyethylene-Delta(9,11)-didehydrostearate: two new components of the non-ionic emulsifier Cremophor EL.

The polyethoxylated heterogeneous components of the so far poorly characterised non-ionic emulsifier Cremophor EL (polyoxyl 35 castor oil) (CrEL) were fractionated by cyclodextrin modified micellar electrokinetic capillary chromatography (CD-MEKC) combined with indirect UV detection. The resulting peaks were assigned to the corresponding components by delayed extraction matrix-assisted laser desorption/ionization time of flight mass spectrometry (DE-MALDI-TOF-MS) as detection device. In order to combine CE and MS the fractionating robot Probot was employed which enables both the online fractionation of the CE eluate on a MALDI target during the electrophoretic separation and the simultaneous dosage of the MALDI matrix. The obtained mass spectra were evaluated by comparing the residue masses of the homologue peak series of the polyethoxylates with the calculated residue masses of potential CrEL-components. The overlapping of homologue peak series with isobaric residue masses was detected by using the residue mass plot, the newly developed evaluation method. Combining theses techniques, both the first detailed structure elucidation and a semiquantitative analysis of the polyethoxylated CrEL-components was achieved. Together with the polyethoxylate series of yet elucidated structures two additional series were observed the corresponding components of which could not be identified at the beginning. Systematic investigations showed that the elimination of water from ricinoleic acid during the synthesis of the emulsifier leads to the polyethoxylates glycerol POE-Delta(9,11)-didehydrostearate and POE-Delta(9,11)-didehydrostearate so far unknown in CrEL.

Determination of Cremophor EL in plasma after sample preparation with solid phase extraction and plasma protein precipitation.

The non-ionic emulsifier Cremophor EL can be quantified using a special potentiometric titration technique with barium chloride activation and precipitation with sodium tetraphenylborate. The end point of the titration is indicated by an ionsensitive coated wire electrode which responds to an excess of tetraphenylborate ions. Sample preparation is necessary to quantify the excipient in plasma of patients receiving ciclosporin formulations with Cremophor EL (Sandimmun), since plasma proteins cause disturbances of the titration. Solid phase extraction was tested with various sorbent materials. Although some of the sorbents yielded good extraction rates of Cremophor EL from aqueous solutions, the extraction rates from plasma were significantly lower. Therefore, plasma protein precipitation with acetonitrile has been examined as an alternative to SPE and has been proved the superior method. Using the precipitation technique, a recovery rate of above 90% was achieved. Furthermore, the limit of detection from plasma was found to be 30 microg, in analogy to the determination from aqueous solutions. The combination of the plasma protein precipitation with the potentiometric titration allows quantitation and thus pharmakokinetic investigations of Cremophor EL in patients treated with Sandimmun after kidney-transplantation.

Evaluation and optimisation of separation buffers for the determination of corticosteroids with micellar electrokinetic capillary chromatography (MECC).

Separation buffers for the determination of the corticosteroids cortisone, hydrocortisone, prednisone and prednisolone with micellar electrokinetic chromatography were developed with respect to separation efficacy and the migration times, depending on the type and the concentration of the organic modifier acetonitrile as well as on the addition of gamma-cyclodextrin. The buffer containing 50 mM SDS and 16% (v/v) acetonitrile enables the rapid profiling of prednisolone together with cortisone and prednisone. Addition of gamma-cyclodextrin alters the elution sequence, but does not further enhance resolution of the corticosteroids. Baseline separation at long migration times for cortisone, hydrocortisone, prednisone and prednisolone is achieved with a buffer containing 50 mM each of SDS, dehydrocholic acid sodium salt and glycodeoxycholic acid sodium salt.

Titrimetric determination of Cremophor EL in aqueous solutions and biofluids: part 2: ruggedness of the method with respect to biofluids.

A titration method for Cremophor EL, as a multicomponent mixture commonly used as non-ionic emulgent for manufacturing certain parenteralia, was developed for quantitative routine analysis in biofluids. A coated wire electrode is used as the end-point indicator in potentiometric titrations of Cremophor EL with sodium tetraphenylborate. The method tolerates a broad pH range, addition of alkanols and components of drug formulations and is sufficiently rugged. Reliable results are obtained at 20 degrees C. Disturbing ions from biofluid matrices can be masked or complexed by addition of formaldehyde, ethylenediaminetetraacetic acid and sodium fluoride. Sodium hydroxide is used for the required adjustment of the samples to pH 10. Cremophor EL spiked urine samples can be determined directly, whereas the true value of the emulgent content in the case of Cremophor EL spiked plasma samples is achieved by means of a conventional method.

Efficiency of haemoperfusion materials at removing the fungal toxin orellanine from human plasma.

Orellanine is the main toxin of various Cortinarius mushrooms and responsible for their nephrotoxicity. The present study was undertaken to estimate the value of haemoperfusion in Cortinarius intoxications. The efficiency of the haemoperfusion materials activated charcoal (DHP-1) and Amberlite XAD 4 resin at removing orellanine from plasma was tested in an in vitro model. Quantification of the toxin in plasma samples was carried out following a previously reported fluorodensitometric TLC method. Orellanine is sufficiently bound to both haemoperfusion materials. However, the rate of orellanine adsorption was four times higher on activated charcoal (DHP-1) compared to Amberlite XAD 4 resin.

Asymmetric synthesis and chromatographic resolution of all four stereomers of the alpha,beta-propanoleucine.

The paper describes the first synthesis of the enantiomerically pure cis-alpha,beta-propanoleucines 6c and 6d by means of asymmetric Strecker synthesis. Furthermore, an improved procedure for the preparation of the stereomeric trans compounds 6a and 6b is proposed. Finally, the four feasible stereomeric alpha,alpha-quaternary-alpha-amino acids are resolved on a penicillamine based chiral stationary phase allowing the determination of ee values ranging from 92.9% to > 98%.

Constituents of the Egyptian Centaurea scoparia; Chlorinated Guaianolides of the Aerial Parts.

A new chlorine-containing guaianolide, chloroscoparin, was isolated from the ethanol extract of the aerial parts of CENTAUREA SCOPARIA Sieb. together with three known chlorine-containing guaianolides. The structures of the isolated compounds were elucidated by spectroscopic methods including (1)H-NMR-, 2D (1)H- (1)H COSY-, (13)C-NMR-, APT-, DEPT-, HETCOR-, long-range-HETCOR-, 2D INADEQUATE-, and mass spectra. The relative configuration was determined by 2D-NOESY studies. New NMR data for the known compounds are reported.

Constituents of the Egyptian Centaurea scoparia; Part II. Guaianolides of the Aerial Parts.

Aerial parts of CENTAUREA SCOPARIA Sieb. afforded a new chlorinated guaianolide with an unusual isobutyl structural feature, diain ( 1), together with three known guaianolides, janerin ( 2), cynaropicrin ( 3), and deacylcynaropicrin ( 4). Structural assignments of the isolated compounds are based on spectroscopic methods including 1D- and 2D-NMR spectroscopy as well as mass spectroscopy. New and revised (1)H- and (13)C-NMR data are reported.

Synthesis and antimycobacterial activity of nitroxanthones. 1st communication: synthesis and differential scanning calorimetry analysis.

The syntheses of mono-; di-, tri- and tetranitroxanthones via intramolecular Friedel-Crafts acylation of nitrated 2-phenoxybenzoic acids as well as via selective stepwise nitration of xanthone and the nitroxanthones gained from the first route are described. The synthesized nitroxanthones investigated by DSC (differential scanning calorimetry) analysis show a surprisingly high thermodynamic stability which was confirmed by quantum chemical calculations.